Title: Phage display screening and expression in plants of peptide aptamers that bind to PthA
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Title: Phage display screening and expression in plants of peptide aptamers that bind to PthA
Physical Description: Book
Language: English
Creator: Zhao, Ge, 1967-
Publisher: University of Florida
Place of Publication: Gainesville Fla
Gainesville, Fla
Publication Date: 2001
Copyright Date: 2001
 Subjects
Subject: Plant Molecular and Cellular Biology thesis, M.S   ( lcsh )
Dissertations, Academic -- Plant Molecular and Cellular Biology -- UF   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )
 Notes
Summary: ABSTRACT: The bacterial genus Xanthomonas is comprised of plant-associated bacteria, many of which cause severe plant diseases. One of those is citrus canker disease, caused by Xanthomonas citri. X. citri causes citrus canker by injecting a protein signal molecule, PthA, into citrus cells, and it is thought that several other severe Xanthomonas diseases are similarly caused by other members of the avrBs3/pthA gene family. This work was to determine if peptide aptamers could be selected that bind to PthA. A Phage Display Peptide Library was screened to select 7-peptide aptamers that specifically bind to full-length PthA and also to a truncated PthA consisting of the C-terminal 200 aa. Three different aptamer sequences were identified that bound to both full-length PthA and truncated PthA protein. ELISA tests indicated that there were no significant differences in the binding affinities of the three aptamers. Based on the citrus codon usage preference table, four PCR primers were designed and used to clone the aptamer sequences into the plant expression vector pBI221. The aptamers were genetically engineered as leader sequences fused to GUS and driven by a CaMV 35S promoter. The engineered constructs were transferred into Agrobacterium tumefaciens strain GV2260 and inoculated onto California light-red kidney bean and sweet orange leaves. The results showed that one aptamer, of sequence YPASYMQ, had a strong effect to block the Hypersensitive Response (HR) normally elicited by pthA expressed in beans, and the pathogenic response normally elicited by pthA on citrus.
Summary: ABSTRACT (cont.): Another aptamer, of sequence HPYTFLN, had reduced, but significant similar effects. Neither aptamer affected the HR symptom elicited by expression of pthA in tomato cells. This result is in consistent with other published reports that the COOH-terminal region of other members of the pthA gene family are not important for elicitation of the HR in tomato. These results confirmed that the aptamers exerted their effect by binding to the COOH-terminal end of pthA, and indicated that aptamers might be used to control citrus canker disease and several other severe diseases caused by Xanthomonas.
Summary: KEYWORDS: citrus canker, pthA, phage display, aptamer, transient expression
Thesis: Thesis (M.S.)--University of Florida, 2001.
Bibliography: Includes bibliographical references (p. 109-115).
System Details: System requirements: World Wide Web browser and PDF reader.
System Details: Mode of access: World Wide Web.
Statement of Responsibility: by Ge Zhao.
General Note: Title from first page of PDF file.
General Note: Document formatted into pages; contains xii, 116 p.; also contains graphics.
General Note: Vita.
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Bibliographic ID: UF00100802
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 50743497
alephbibnum - 002729366
notis - ANK7130

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PHAGE DISPLAY SCREENING AND EXPRESSION IN PLANTS
OF PEPTIDE APTAMERS THAT BIND TO PTHA









By

GE ZHAO


A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE


UNIVERSITY OF FLORIDA


2001



























Copyright 2001



by



Ge Zhao






























TO MY PARENTS

















ACKNOWLEDGMENTS


I would like to thank my advisor Dr. Dean W. Gabriel, who instructed me and

always supported me. Also, I am greatly indebted to my graduate advisory committee

members, Dr. Robert R. Schmidt, Dr. Alice C. Harmon and later on Dr. L. Curtis

Hannah, who gave me a lot of valuable advice.

Thanks go to the people in the lab: Gary C. Marlow, who gave me a lot of

technical support; Dr. Yongping Duan, who gave me many good comments, and to the

graduate students in the lab, Adriana Castaiieda, Asha Huisden-Brunings and

Abdulwahid Al-Saadi.

Thanks go to the people outside of the lab: Mark Elliott helped me to do ELISA

microplate reading; the ICBR DNA Sequencing Core staff did all of the phage

sequencing and the Division of Plant Industry, Florida Department of Agriculture and

Consumer Services, provided the quarantine greenhouses for pathogenic inoculation of

citrus canker.

I would like to thank Prof Yingchuan Tian from the Institute of Microbiology,

Chinese Academy of Sciences, in Beijing, China. He showed me a lot of experimental

techniques during his visit to our lab and initiated some parts of this project.

Finally, great thanks go to my parents who always support me.

iv


















TABLE OF CONTENTS


Page

A CKN OW LED GM EN TS .................................................... ........ .. iv

LIST OF TABLES ................... .................. ................. .. .......... viii

LIST OF FIGURES ................... ................... .................. .. ......... ix

ABSTRACT ..................................... ....................................... xi

CHAPTERS

1 INTRODUCTION .................. ................... ................. 1

C itrus C anker H history ..................................................... ........... 1
Xanthomonas avr/pth Family ....................................................... 3
PthA Protein Function ................................................................. 6
Fusion Protein Construction, the pET-19b Expression
System and the GST Gene Fusion System ............... ................ 10
Phage Display Peptide Library Biopanning ....................................... 12
PCR-UDG Cloning Technique .......... ..................... .................. 16
Agrobacterium tumefaciens Mediated Transient
Expression in Plants .......... ................. ................. 17
Experimental Design .................................. .. ....................... 19

2 MATERIALS AND METHODS .......... ....................... ............ 21

Expression and Purification of PthA
Full-length Protein by His.Tag ............ .............................. 21
Expression and Purification of GST Fusion PthA
C-terminal 200 aa Truncated Protein ...................................... 23









Phage Display Peptide Library Biopanning ........................ ........... 25
In Vitro Binding Affinity of Aptamers ELISA ........... .................. 27
PCR-UDG Cloning of Aptamers .................. .............. ... ... ............. 28
Agrobacterium tumefaciens Mediated Transient
Expression of Aptamer Constructs in Bean,
Sweet Orange and Tomato ........... .......... .................. 31

3 RESULTS .......................... .................... ........ 34

Expression and Purification of PthA Protein by His.Tag ......................... 34
Expression and Purification of the PthA
COOH-terminal 200aa Truncated Protein
by GST Gene Fusion System ................ ......................... 37
Phage Display Peptide Library Biopanning .......................................... 39
In Vitro Binding Assay ELISA ................... ........................ 42
PCR-UDG Cloning of Aptamers ............ ....... ........... ............ 45
Agrobacterium tumefaciens Mediated Transient
Expression of Aptamers in Bean, Sweet
Orange and Tomato Leaves ...................................... 49

4 D ISC U SSIO N ..................... ....................................... .......... 54

5 SUMMARY AND CONCLUSIONS .................................. .......... 62

APPENDICES

A MEDIA AND STRAINS USED .................. ............... .............. 64

M edia ................... ........................................ .... ......... 64
Strains ................... ................... .................. ... ......... 66

B PURIFICATION OF HIS.TAG PROTEIN
(FULL-LENGTH PTHA) .............. ............... .................. 68

C PURIFICATION OF GST FUSION PROTEIN
(PTHA C-TERMINAL 200AA
TRUNCATED PROTEIN) ............ .................... 76

D PHAGE DISPLAY PEPTIDE LIBRARY
BIOPANNING SCREENING OF APTAMERS ......................... 80

Phage Display Biopanning ................................ ........... ............ 80
Phage Amplification .............. ........ ....... ................. ............ 83
Phage Titer ............ ... ....................... ................... .... ......... 84









Characterization of Binding Clones ........................................ 86

E IN VITRO BINDING AFFINITY OF
APTAMERS ELISA ........... ................................... 88

F PCR-UDG CLONING OF APTAMERS ...................................... 93

G AGROBACTERIUM TUMEFACIENS MEDIATED
TRANSIENT EXPRESSION OF APTAMERS
IN BEAN, SWEET ORANGE AND
TOMATO LEAVES .............................. ................ 102

R EFEREN CE S ................... .................. .. .................. ..... ........ 109

BIOGRAPHICAL SKETCH ................. ....................................... 116


















LIST OF TABLES



Table Page



1. The genetic code for phage display peptide library biopanning ................ 13

2. The codon preference table of Citrus sinensis ........... ............ .......... 20


















LIST OF FIGURES



Figure Page


1. Citrus canker symptoms on sweet orange ............................... ................ 2

2. The gene-for-gene "quadratic check"................ ....................... .............. 3

3. PthA full length peptide sequence .......................... ................ ........... 6

4. The predicted functional domains of PthA .................. ............. 7

5. The predicted peptide structure of PthA ......................... ....... .. ........ 8

6. N-terminal sequence of random, seven peptide
gill coat protein fusion ............ ............... ............... ............ 13

7. PCR-UDG cloning technique diagram ............................ ............. 15

8. M ap of pYY50.13 plasmid .................. ............... ...................... 22

9. Map of pGNLS3-2 plasmid .................. ......................... ............ 24

10. The design of PCR-UDG oligonucleotides to amplify
aptamers YPASYMQ and HPYTFLN .................................. ........ 29

11. Time course of PthA full length protein expression
induced by 0.1 m M IPTG ................................... ............ .......... 35

12. The purification of PthA full length protein by His.BindTM
affinity column chromatography ..... .............. ........................... 36









13. The expression of PthA COOH-terminal 200 aa truncated
protein induced by 0.1 mM IPTG at different times ......... .......... 37

14. Electro-elution of the COOH-terminal end of PthA,
fused to G ST ................... ......................................... ...... 38

15. Characterization of M13 PthA-binding clones ............ ............. 40

16. The DNA sequences of the M13 PthA-binding clones .......... ............. 41

17. In vitro Binding Assay of Aptamers .............. ...... ..... ........ ... ........... 43

18. The in vitro binding affinity of aptamers P7 (HPYTFLN),
N4 (YPASYM Q) and P10 (HPHTFLN) ............................................. 44

19. PCR amplification of aptamers P7 and N4 ................................. ......... 46

20. Uracil DNA glycosylase treatment of PCR products ................................. 47

21. 15.0% polyacrylamide gel electrophoresis of apatmers ...................... 47

22. Plasmid maps of pGZ7.5 and pGZ7.6 ......... ....................... .............. 48

23. Aptamers P7 and N4 cloned in Agrobacterium binary
vector pYD40.1 and pYD40.2 ............................ ................ 50

24. The plasmid maps of pGZ8.1, pGZ8.2, pGZ8.3
and pGZ8.4 ............. .. ...................... .......... ........ 51

25. Inoculation of Agrobacterium tumefaciens strain GV2260
constructs to the leaves of bean (panel A), citrus
(panel B) and tomato (panel C) .............. ................................... 52

26. The predicted Antigenicity Index of PthA ................ ...... ........ 60

27. The predicted KD Hydrophilicity and KD
Hydrophobicity of PthA ............. .......................... ............ 60

28. The predicted Surface Probability of PthA ................ ............. 61


















Abstract of Thesis Presented to the Graduate School of the University of Florida
in Partial Fulfilment of the Requirement for the Degree of Master of Science


PHAGE DISPLAY SCREENING AND EXPRESSION IN PLANTS
OF PEPTIDE APTAMERS THAT BIND TO PTHA


By

Ge Zhao

May 2001


Chairperson: Dean W. Gabriel
Major Department: Plant Molecular and Cellular Biology



The bacterial genus Xanthomonas is comprised of plant-associated bacteria, many

of which cause severe plant diseases. One of those is citrus canker disease, caused by

Xanthomonas citri. X citri causes citrus canker by injecting a protein signal molecule,

PthA, into citrus cells, and it is thought that several other severe Xanthomonas diseases

are similarly caused by other members of the avrBs3/pthA gene family. This work was to

determine if peptide aptamers could be selected that bind to PthA. A Phage Display

Peptide Library was screened to select 7-peptide aptamers that specifically bind to full-

length PthA and also to a truncated PthA consisting of the C-terminal 200 aa. Three

xi









different aptamer sequences were identified that bound to both full-length PthA and

truncated PthA protein. ELISA tests indicated that there were no significant differences

in the binding affinities of the three aptamers. Based on the citrus codon usage

preference table, four PCR primers were designed and used to clone the aptamer

sequences into the plant expression vector pBI221. The aptamers were genetically

engineered as leader sequences fused to GUS and driven by a CaMV 35S promoter. The

engineered constructs were transferred into Agrobacterium tumefaciens strain GV2260

and inoculated onto California light-red kidney bean and sweet orange leaves. The

results showed that one aptamer, of sequence YPASYMQ, had a strong effect to block

the Hypersensitive Response (HR) normally elicited by pthA expressed in beans, and the

pathogenic response normally elicited by pthA on citrus. Another aptamer, of sequence

HPYTFLN, had reduced, but significant similar effects. Neither aptamer affected the

HR symptom elicited by expression of pthA in tomato cells. This result is in consistent

with other published reports that the COOH-terminal region of other members of the

pthA gene family are not important for elicitation of the HR in tomato. These results

confirmed that the aptamers exerted their effect by binding to the COOH-terminal end of

pthA, and indicated that aptamers might be used to control citrus canker disease and

several other severe diseases caused by Xanthomonas.
















CHAPTER 1
INTRODUCTION


Citrus Canker History

According to the Florida Citrus Outlook 1998-99 Season Report (The Economic

and Market Research Department, Florida Department of Citrus, 1998), total Florida

citrus production, including round orange, specialty-citrus and grape fruit is expected to

be 248.8 million 90-lb boxes. The total 1998-1999 earnings are projected to be $971.5

million to $1,221.2 million. The Florida citrus industry is the largest among all US states

- about 3 times more than the total citrus production in California, the second largest

citrus provider in the US.

This important industry to the state of Florida is now threatened by citrus canker -

a world-wide citrus disease, widely distributed in Southeast Asia, Japan, the Middle East,

Africa and South America (Swarup et al., 1991). Last year, Asiatic citrus canker

infected large areas of southern Florida, including Hendry, Dade and Broward counties.

About $200 million this year was set aside for canker eradication in Florida.

A tree infected by citrus canker will drop fruit prematurely and exhibit lesions on

leaves, stems and fruit (De Feyter et al., 1993) (Figure 1). The bacteria responsible for

the canker, Xanthomonas citri (Gabriel et al., 1989), is spread primarily by wind-blown









2

rain, but also can be spread by contaminated tools and equipment, by people and by

animals. There is no effective resistance in grapefruit and little resistance in sweet

orange. Control by chemical sprays is only partially effective. Control by eradication

has been effective and X citri is a quarantined pathogen, subject to eradication. Both

infected and exposed trees are destroyed as part of a typical eradication program. If

citrus canker cannot be contained in Florida, losses of $8.55 billion in revenue is

expected according to the Florida Citrus Outlook 1998-99 Season Report. As many as

the 121,000 people could be unemployed, and 845,260 acres of citrus trees could be

destroyed. In 1985, an earlier outbreak of citrus canker in Florida forced the destruction

of 20 million citrus trees.


Figure 1. Citrus canker symptoms on sweet orange.









Due to this situation, researchers at USDA, the University of Florida, are working
to find a way to stop citrus canker. The primary purpose of this research was to try to
find a molecular biological approach to cure citrus canker disease and a possible way to
genetically engineer "immune" or "resistant" citrus trees.



Host Cultivar With


Pathogen R&- ri

avri +



0 ++


Figure 2. The gene-for-gene "quadratic check".



Xanthomonas avr/pth Family

The genus Xanthomonas is unique among bacterial plant pathogens in exhibiting
a high degree of host-range specificity. The basis for the specifications is not clear.
Based on the classic "The gene-for-gene quadratic check" (Figure 2) (Ellingboe, 1976),
avirulence genes (avr) are considered to contribute to host range specificity in
Xanthomonas (Gabriel, 1997). Resistance in plants or avirulence in pathogens is usually











assayed by inoculation of pathogen into plants and is usually seen as a hypersensitive

response (HR) of the plant (Alfano and Collmer, 1996, Leach and White, 1996).

Surprisingly, most of the X campestris pv. malvacearum avr genes are also pathogenicity

(pth) genes (Yang et al., 1996). Even more surprisingly, these genes are members of the

same avr/pth gene family as the genes of X citri that are known to cause citrus canker

(Swarup et al., 1992, Duan et al., 1999). Most of the members of this avr/pth gene

family were originally isolated as avr genes and there was no evidence ofpth functions.

Similarly, pth genes were cloned by screening of pathogenecity, without evidence of avr

functions. Currently, 17 members of this gene family has been published, including X

campestris pv. malvacearum (cotton blight) (Yang et al., 1996), X citri (Asiatic citrus

canker) (Swarup et al., 1991), X campestris pv. aurantifolii (false citrus canker and

Mexican lime cancarosis) and X oryzae (rice blight) (Leach et al., 1996). The genes

cloned include avrBn (Gabriel et al., 1986), avrb6 (water soaking on cotton), avrB4,

avrb7 (water soaking on cotton), avrBIn (water soaking on cotton), avrBlO1 (water

soaking on cotton), avrB102 (water soaking on cotton) (De Feyter et al., 1993), avrB103,

avrB104 (water soaking on cotton), avrB5 (water soaking on cotton) (Yang et al., 1996),

pthN (water soaking on cotton), pthN2 (water soaking on cotton) (Chakrabarty et al.,

1997), avrBs3 (Bonas et al., 1989), pthA (canker on citrus) (Swarup et al., 1992), pthB

(canker on citrus), pthC (canker on citrus) (Yuan and Gabriel, unpublished data), avrXa5,

avrXa7 (elongated lesion on rice) and avrXalO (Hopkins et al., 1992).

The avr/pth gene encode signals affecting plant cell programs (Gabriel, 1999),

including "programmed death" (Dangl et al., 1996). All genes of the avr/pth family











found until today are from biotrophs which produce few degrading enzymes that could

assist releasing nutrient from living plant cells but not kill them (Vivian and Gibbon,

1997). A type III protein secretion system encoded by hrp genes (the name came from

affecting both hypersensitive response and pathogenicity of the bacterial strains) is

essential to deliver the avr/pth signals into plant cells (Van Gijsegem et al., 1995).

Mutation of this system abolish pathogenecity in biotrophic plant pathogenic Erwinia,

Pseudomonas and Xanthomonas (Alfano and Collmer, 1996). All of these bacterial

pathogens have very similar hrp genes (Galan, 1996).

Only a few molecules have been determined to be secreted by the hrp system.

One of these is a class of toxin-like molecules called harpins (Hoyos et al., 1996).

Harpins directly elicit plant symptoms on both host and nonhost plants. Harpins are

glycine rich proteins that seems to cause direct nutrient leakage from plant cells by

alkalization of the apoplast.

Another group of secreted molecules are the avr/pth gene family (Mecsas and

Strauss, 1996). Functional nuclear localization signals (NLSs) were found in all

members of Xanthomonas avr/pth gene family (Yang and Gabriel, 1995). This is

evidence that the signaling protein is directly targeted to plant cell nuclei. Site-directed

mutagenesis of NLS sequences abolished the pathogenicity phenotypes expressed by

these genes (Gabriel et al., 1996).















PthA Protein Function



Citrus canker disease has been historically described as having different "forms",


including Asiatic citrus canker (X. citri or "A form"), false citrus canker (X. campestris


pv. aurantifolii form B or "B form") and Mexican lime cancrosis (X. campestris pv.


aurantifolii form C or "C form"). A pathogenicity gene, pthA, was first cloned by on


pZit45 and confirmed necessary to for Xanthomonas citri to cause citrus canker disease,


the hyperplasia of citrus tissue (Swamp et al., 1991). The full length peptide sequence of


mdpirsrtps

spafsagsfs
mrvavtaarp
ealvghgfth
lltvagelrg

iggkqaletv
vvaiasnigg
ahgltpaqvv

rllpvlcqah
kqaletvqrl
aiasngggkq

gltpeqvvai
Ipvlcqahgl
aletvqrllp

acngggkqal
ltndhlvala


parellpgpq

dllrqfdpsl
prakpaprrr
ahivalsqhp

pplqldtgql
grllpvlcqa
kqaletvqrl
aiasniggkq

gltpdqvvai
Ipvlcqahgl
aletvqrllp

asngggkqal
tpeqvvaias
vlcqahgltp

etvqrllpvl

clggrpalda


961 qchshpaqaf ddamtqfgms


pdgvqptadr
fntslfdslp
aaqpsdaspa
aalgtvavky
lkiakrggvt

hgltpeqvva
Ipvlcqahgl
aletvqrllp

ashdggkqal
tpeqvvaias
vlcqahgltp

etvqrllpvl

nsggkqalet
eqvvaiashd

cqahgltpeq
vkkglphapa
rhgllqlfrr


rakpsptstq tpdqaslhaf adslerdlda
qsfevrapeq rdalhlplsw rvkrprtsig
gaaddfpafn eeelawlmel Ipq


gvsppaggpl
pfgahhteaa
aqvdlrtlgy
qdmiaalpea
aveavhawrn

iasnggkqal
tpeqvvaias
vlcqahgltp

etvqrllpvl

hdggkqalet
eqvvaiasng

cqahgltldq
vqrllpvlcq

ggkqaletvq
waiasnggg
likrtnrrip
vgvtelears

pspthegdqr

gglpdpgtpt


dglparrtms

tgewdevqsg

sqqqqekikp
theaivgvgk
altgaplnlt

etvqrllpvl

niggkqalet
eqvvaiasng

cqahgltpqq
vqrllpvlcq

ggkqaletvq
vvaiasnggg
ahgltpdqvv
rllpvlcqah

rpalesivaq
ertshrvadh

gtlppasqrw
rassrkrsrs

aadlaasstv


rtrlpsppap

Iraadapppt
kvrstvaqhh
qwsgaralea
peqvvaiasn

cqahgltpeq
vqrllpvlcq

ggkqaletvq
waiasnggg
ahgltpeqvv
rllpvlcqah

kqaletvqrl
aiashdggkq

gltpeqvvai
Isrpdpalaa
aqvvrvlgff
drilqasgmk

dravtgpsaq
mreqdedpfa


Figure 3. PthA full length peptide sequence (1163 amino acids in total, the carboxyl
terminal 200 aa truncated protein is indicated by bold-italic).


1021

1081
1141
























Figure 4. The predicted functional domains of PthA.





PthA is given in Figure 3 (Swamp et al., 1992). There are 1163 amino acids. pthA,

avrb6 and avrBs3 are members ofXathomonas avr/pth family (Yang et al., 1994). The

complete DNA sequence of pthA is 97% identical to avrb6 and avrBs3. The central

regions of the predicted PthA protein is comprised of 17.5 tandem repeats each 34 amino

acids in length (Figure 4). These repeats are leucine-rich, indicating a potential protein-

protein interaction. Also, leucine zipper-like heptad repeats are present, that may also

indicate protein-protein or protein-DNA interactions. Site-directed mutagenesis of such

repeats proved that they are critical for the function of cultivar specific avirulence and

species-specific pathogenicity (Yang et al., 1994). The repeat regions of pthA and avrb6

determined hyperplasia of citrus and water soaking of cotton, respectively. Fusion

experiments using pthA and avrb6 repeat regions showed that the direct tandem repeats

determine both phenotypes, and are host-specific. The coding sequences of all members

of the gene family are flanked by nearly identical 62bp terminal inverted repeats, and the



















































Figure 5. The predicted peptide structure of PthA (including KD Hydrophilicity, Surface
Probability, Antigenic Index, CF Turns, Alpha Helices, Beta Sheets and Glycosyl Sites).











terminal 38bp of both inverted repeats are highly similar to the 38bp consensus terminal

sequence of the Tn3 family of transposons (De Feyter et al., 1993). Therefore, these

genes may have the capability to transpose.

The carboxyl terminal portion of PthA encodes three nuclear localization signals

(K-R/K-X-R/K) that are critical for PthA function and for localization to the host cell

nucleus (Yang and Gabriel, 1995). These three putative nuclear localization sequences

are at positions 1020-1024 (K-R-A-K-P) (nlsA), 1065-1069 (R-K-R-S-R) (nlsB), and

1101-1106 (R-V-K-R-P-R) (nlsC) in PthA. Site-directed mutagenesis of the nuclear

localization signal reduced the pthA localized in onion cell nuclei and also reduced the

pathogenicity of citrus canker symptoms (Gabriel et al., 1996). Mutation of all three

NLSs reducedpthA localization to onion cell nuclei. Mutations of any two NLSs

abolished the ability the pthA to elicit cankers on citrus, but did not abolish its ability to

elicit a non-host HR on tomato. Another member of the gene family from X campestris

pv. vesicatoria, avrBs3-2, does not require the 3' end (encoding all three NLSs) to elicit a

HR on tomato. Because avrBs3-2 and pthA are in the same gene family, and the DNA

sequence are very similar, it is predicted that PthA C-terminal region is also not

important to elicit a HR on tomato (Gabriel et al., 1996). The DNA coding sequence for

the C-terminal regions of pthA was fused to a P-glucuronidase (GUS) reporter gene.

Figure 5 shows the predicted structure of PthA full-length protein including KD

hydrophilicity, surface probability, antigen index, CF turns, alpha helics, beta sheets, and

glycosyl sites, etc. Both the N-terminus and Carboxyl terminus of PthA are likely to be











exposed outside of the naturally folded protein, thus, the COOH-terminus of PthA may

have the chance to interact with other protein or DNA molecules.




Fusion Protein Construction, the pET-19b Expression System and the GST Gene Fusion
System


The pET system used in the expression of recombinant proteins in E. coli was

originally constructed by Studier and colleagues (Studier and Moffatt, 1986, Studier et

al., 1990). The pET19b vector provides a translational N-terminal fusion of a cloned

gene to a cleavable His.Tag sequence for rapid affinity purification of the protein

product. His.Tag is an oligohistidine domain that allows convenient, economical

purification by His.Bind affinity resin. The target gene is cloned in the pET plasmids

under the control of a T7 promoter which is induced by T7 RNA polymerase in the host

cells (Dubendorff and Studier, 1991). T7 RNA polymerase is highly selective, which

converts almost all of the cell's protein production to target gene expression (Derman et

al., 1993). The expected yield of target protein can be more than 50% of the total protein

production after induction. Target genes are initially cloned in E. coli hosts that do not

contain the T7 RNA polymerase gene, i.e., the target genes are silent in the non-

expression host. When these plasmids are transferred into the expression host, E. coli

BL21(DE3)pLysS, which contains a chromosomal copy of the T7 RNA polymerase gene,

under lacUV5 control, expression of the target gene is strongly induced by the addition of

IPTG (Doherty et al., 1995). Since some proteins naturally occurred in E. coli host cell











having Histidine rich sequences, for some His.Tag constructs, they are not quiet efficient

to bind to His.Bind affinity resin or have very high background which create the

difficulties for the further purification of His.Tag proteins. Also, the very short peptides

are not very efficiently purified by His.Tag approaches (Studier et al., 1990).

The Schistosomajaponicum Glutathione S-transferase (GST) gene encodes a

26kDa protein that is expressed in E. coli with full enzymatic activity (Parker et al.,

1990). Recombinant genes are constructed in pGEX vectors, designed for inducible,

high-level intracellular expression of genes in fusion with GST (Maru et al., 1996).

GST fusion proteins have GST enzymatic activity (Ji et al., 1992). Gene expression is

under the control of the tac promoter, which is induced using the lactose analog IPTG

(Kaelin et al., 1992). After lysis of bacterial cells, the lysates are incubated with

Glutathione Sepharose 4B affinity resin. The glutathione group of GST is attached to

Sepharose 4B by coupling to the oxirane group using epoxy-activation. The GST fusion

protein can be 90% recovered using Glutathione Sepharose 4B in a single

chromatographic step. Fusion proteins are recovered from the matrix under mild elution

conditions (10mM glutathione) that preserve antigenicity and functionality of the

proteins. Specific protease cleavage sites allow separation of the GST and the fusion

protein to yield separate products. The GST fusion protein system proved to be very

efficient for short peptide expression (McTigue et al., 1995).











Phage Display Peptide Library Biopanning

There are several rapid and efficient methods using combinatorial biology for

screening random peptide epitopes binding to specific protein targets (Scott and Smith,

1990). The Ph.D.-7 Phage Display Peptide Library (New England BioLabs) is based on

a library of random 7-mer peptides fused to a minor coat protein (pIII) of the filamentous

coliphage M13. The Ph.D-7 peptide library is comprised of 2 x 109 sequences. By

comparison, theoretical numbers of possible 7-mers in a combination of 20 different

amino acids is 207 = 1.28 x 109 possible 7-residue sequences. Each amplification cycle

yields about 100 copies of each amplified sequence in 10pl of the supplied phage. The

displayed hetapeptides are expressed directly at the N-terminus of the pIII coat protein

when the fusion is expressed with a leader sequence removed during secretion, therefore,

the first residue of the mature protein is the first randomized position (Figure 6). The

peptide is followed by a short spacer (Gly-Gly-Gly-Ser) and then the wild type pIII

sequence. The genetic code of the Ph.D. phage display library is reduced, using only 32

codons encoding 20 different amino acids (Table 1).

Biopanning is a in vitro selection process that allows rapid identification of

peptide ligands for a variety of target molecules (such as antibodies, enzymes, cell-

surface receptors, etc) (Schumacher et al., 1996, Goodson et al., 1994, Barry et al., 1996,

Parmley and Smith, 1988). Biopanning is carried out by incubating a large random

library of unselected phage-displayed peptides with a polystyrene plate coated with the

target. After washing away the unbound phage, some peptides stick to the target and









13



Table 1. The genetic code for phage display peptide library biopanning.


pIII leader sequence KpnI

5'- ... TTA TTC GCA ATT CCT TTA GTG GTA CCT TTC
3'- ... AAT AAG CGT TAA GGA AAT CAC CAT GGA AAG

... Leu Phe Ala Ile Pro Leu Val Val Pro Phe


Start of mature 7 peptide gIII fusion EagI

NNK NNK NNK NNK NNK NNK NNK GGT GGA GGT TCG GCC
NNM NNM NNM NNM NNM NNM NNM CCA CCT CCA AGC CGG

Xxx Xxx Xxx Xxx Xxx Xxx Xxx Gly Gly Gly Ser Ala


AGT TGT TTA GCA AAA TCC CAT ACA GAA AAT TCA TTT
TCA ACA AAT CGT TTT AGG GTA TGT CTT TTA AGT AAA

Ser Cys Leu Ala Lys Ser His Thr Glu Asn Ser Phe

- -28gIII sequencing primer

K = G or T; M = A or C


GAA ACT
CTT TGA

Glu Thr


ACT AAC ... -3'
TGA TTG ... -5'

Thr Asn ...


Figure 6. N-terminal sequence of random, seven peptide gill coat protein fusion
(The mature protein starting site is indicated by bold).


2nd T 2nd C 2ndA 2nd G

1st T Phe (F) Ser (S) Tyr (Y) Cys (C) 3rd T

Leu (L) Ser (S) Gin (Q) Trp (W) 3rd G

1st C Leu (L) Pro (P) His (H) Arg (R) 3rd T

Leu (L) Pro (P) Gin (Q) Arg (R) 3rd G

1st A Ile (I) Thr (T) Asn (N) Ser (S) 3rd T

Met (M) Thr (T) Lys (K) Arg (R) 3rd G

1st G Val (V) Ala (A) Asp (D) Gly (G) 3rd T

Val (V) Ala (A) Glu (E) Gly (G) 3rd G











remain bound, some with high affinity. Specifically-bound phage remains and is then

released under elution conditions. The eluted phage is then amplified in E. coli, the

enriched phage is collected, and then used to run additional binding/amplification cycles

to enrich the pool in a favor of binding sequences. After 3-4 rounds of enrichment, only

a few clones remain, the those are characterized by DNA sequencing.

The phage clones are also used for in vitro binding affinity assay with the original

target. Enzyme-linked immunosorbent assay (ELISA) is widely used for detection of

protein binding affinity in the phage display library screening, and the detection level can

be as low as 10-9 g of protein (Hoess et al., 1994). The method is to fix the specific target

protein on the supporting surface and add the selected binding peptide ligands, i.e., the

phage. After short incubating and washing, the unbound phage are washed off. At this

point, HRP conjugated anti-M13 antibody is added. The HRP converted colorless OPD

substrate into fluorescent yellow-orange color. Only the bound phage can react to anti-

M13 antibody and change the substrate color. The color level of the ELISA plate can be

read at 492nm.

Short peptide libraries displayed on phage have been used in a number of

applications, such as selection of phage-display peptides that bind to cucumber mosaic

virus coat protein (Gough et al., 1999), differentiating insecticidal activity of soybean

cystatins (Koiwa et al., 1998), and characterizing acquired resistance in lesion-mimic

transgenic potato expressing bacterio-opsin (Abad et al., 1997).
























































Figure 7. PCR-UDG cloning technique diagram.











PCR-UDG Cloning Technique

A novel ligation-independent method of site-directed mutagenesis of DNA

sequences using PCR amplification followed by uracil DNA glycosylase treatment

(termed UDG cloning) has proved to be very efficient and flexible (Rashtchian, 1995).

The method is to use two nucleotide primers that contain the desired DNA sequences and

which overlap at their 5' ends (Figure 7). The Thymine (dT) residues in the overlap

region are substituted with deoxyuracil (dU) in the primer nucleotide sequences. The

primers are designed to PCR amplify the entire plasmid to form a linear DNA fragment.

The desired base changes are in the primer. The two primers should be complementary

to each other (Smith et al., 1993). The presence of dU in the PCR products results the 5'

end of the amplified DNA fragments very susceptible to Uracil DNA glycosylase (UDG).

The UDG treatment of PCR products leads to excision of dU residues to produce 3'

protruding sticky ends. Because the two set of primers are overlapped, the two 3'

protruding ends are complementary each other and result in annealing of the PCR

products, i.e., the entire plasmid and the mutation sequence of the primer. The circular

PCR products are transformed to competent E. coli cells and any nicks of circularized

plasmid are filled by the E. coli in vivo repair system. The new plasmids are identical to

the wild-type parental plasmid except for the desired site-directed mutations designed on

the primers (Rashtchian et al., 1992).

One research group applied UDG cloning techniques to insert a chloramphenicol-

resistance gene into a SacI vector site and three segments of the lacZ PCR gene into a











correct orientation (Watson and Bennett, 1997). Another group designed primers with an

11 base overhang as an adaptor to PCR amplify the vector, while the PCR products were

treated by UDG to produce overhangs to yield a large numbers of inserts from human

cDNA cosmids. This method approved to be a highly efficient way to characterize genes

for shotgun sequencing of multiple shorter fragments in a large scale (Andersson et al.,

1994). Aptamer sequences selected from phage display peptide library biopanning only

comprise 21 base pairs (encoding 7 amino acids), and such a short DNA fragment is very

difficult to insert into a cloning vector and fuse with other stabilizing reporter genes in

the right orientation and in frame. UDG cloning techniques provide good approach to

genetic engineering of aptamers into the desired cloning vectors.




Agrobacterium tumefaciens Mediated Transient Expression in Plants

Agrobacterium tumefaciens can infect plants and introduce foreign genes into

plant cells (Hansen et al., 1994). At the site of infection, a crown gall (tumor tissue)

forms and synthesizes opines, which are novel amino acids that can only be metabolized

by Agrobacterium (Cho et al., 1997). The tumor is formed, and opines produced,

because of genes on the Ti plasmids (tumor-inducing plasmids) carried by

Agrobacterium. A small portion, usually only a 20kb segment, of the Ti plasmid, called

T-DNA (transferred DNA), carries the tumor-inducing and opine-producing genes and

integrates into the genome of the infected plant cell (Baron and Zambryski, 1996). The

bacteria can infect a susceptible plant only at the site of a wound, where plant cells secret











phenolic compounds that include acetosyringone and help activate the Ti plasmid genes

(Chumakov and Kurbanova, 1998). Scientists use molecular cloning techniques to insert

foreign DNA fragments between the left and right borders of the T-DNA and use

Agrobacterium Ti plasmids as a carrier to deliver cloned DNA into plant (Vaquero et al.,

1999). The ability of Agrobacterium T-DNA to integrate stably into its host's genome

gives plant molecular biologist a powerful tools to understand gene function. However,

transformation of plants take a lot of laboratory effort, and is not successful in every plant

species.

Even without stable transformation, Agrobacterium Ti plasmids can be used to

achieve transient expression of genes in plants. Non-integrated T-DNA copies transiently

stay in the nucleus, where they are transcribed (Kapila et al., 1997). The transient

expression rate is much higher (at least 1,000 times) than that of the stable expression rate

in petunia leaf discs (Janssen and Gardner, 1989). Transient gene expression systems are

widely used because gene expression can be measured very shortly after T-DNA

delivery, there is no position effect, and the gene expression can be tested without

regeneration of a transgenic plant (Tai et al., 1999). A number of other techniques are

used to transfer DNA into intact plant tissue to achieve transient expression, including

electroporation (Lindsey and Jones, 1987, Dekeyser et al., 1990), particle bombardment

(Russell and Fromm, 1995), microinjection (Bilang et al., 1993), syringe injection

(Robinette and Matthysse, 1990) and vacuum infiltration (Kapila et al., 1997). Several

Agrobacterium tumefaciens host strains such as GV2260 (Deblaere et al., 1985), AGL1











(Lazo et al., 1991) and LBA4404 (Ooms et al., 1982) are commonly used in plant

transformation, and in transient expression assays. I used Agrobacterium tumefaciens

GV2260 with binary vector constructs to inject DNA into citrus, tomato and bean leaves

to assay pthA gene function and the interactions between the PthA and selected apatmers.




Experimental Design

The first part of this thesis project was to translationally fusedpthA with His.Tag,

so that the recombinant PthA::His.Tag protein could be purified by His.Bind resin. The

COOH terminal 600bp end of pthA, encoding all three nuclear localizing signals (NLSs)

was translationally fused with GST and cloned into pGEX-4T-3. The recombinant GST

fusion protein was purified by Glutathione Sepharose 4B. The two purified recombinant

proteins, PthA full length protein and PthA COOH terminal 200 amino acid peptide, were

used as the targets to screen the Ph.D.-7 Phage Display Peptide Library to select 7-mer

amino acid aptamers by biopanning. The interesting M13 phage clones were DNA

sequenced and confirmed to bind PthA by in vitro binding affinity assay ELISA. The

aptamer clone sequences were fused to a GUS reporter gene by PCR-UDG cloning. The

codon of each amino acid was modified based on the codon preference table of citrus

(Table 2). The constructs were then cloned into an Agrobacterium binary vector with

and without the pthA gene and used to inoculate the Agrobacterium tumefaciens strains

on citrus, bean and tomato leaves. Transient expression assays were used to identity the

function of the aptamers in plants.








20




Table 2. The codon preference table of Citrus sinensis (the aptamer coding sequence
from phage display biopanning was translated into peptide sequence and replaced by
citrus preference codon).


Citrus sinensis [gbpln]: 17 CDS's (6141 codons)



fields: [triplet] [frequency: per thousand] ([number])

















CHAPTER 2
MATERIALS AND METHODS



Expression and Purification of PthA Full-length Protein by His.Tag

Full length pthA was cloned into the BamHI site of pET-19b to form pYY50.13

(Yang et al., 1995) (Figure 8). pYY50.13/E. coli DHSa single colonies were inoculated

into LB medium (Bacto tryptone 10g/l, Bacto yeast extract 5g/l, NaCl 5g/l, pH 7.5) with

100 [ig/ml of ampicillin and grown overnight. DNA mini-preps were made (Sambrook et

al., 1989) and used to transform E. coli protein expression host strain BL21(DE3)pLysS

(Studier et al., 1990). Fresh pYY50.13/E. coli BL21(DE3)pLysS colonies were

inoculated into LB medium with 50[lg/ml of carbenicillin and 34[lg/ml chloramphenicol

at 370C and shaken on a rotary shaker at 220 rpm until the cell density reached

OD600=0.5. Protein expression was evaluated by using 0.1 mM, ImM and 100mM IPTG

at 280C with shaking at 220 rpm for 3 hours. The cell pellet was collected and suspended

in IX Binding Buffer (5 mM Imidazole, 0.5 M NaC1, 20 mM Tris-HC1, pH 7.9) plus

0.1% NP-40 and 1 mM PMSF. Cells were then sonicated 60 seconds on ice to disrupt the

membranes using a SONICATOR Model W-225 (%duty cycle = 50, out put control=6,

pulsed). His.BindTM resin columns were set up by washing with 3 bed volumes of sterile






































Figure 8. Map of pYY50.13 plasmid (pthA gene in pET-19b vector fused with His.Tag).




deionized water, charging with 5 bed volumes of 1X Charge Buffer (50mM NiSO4) and

equilibrating with 3 bed volumes of IX Binding Buffer. His.Tag fusionpthA protein

crude extract was loaded onto His.Bind resin column. The column was washed by 10

bed volumes of IX Binding Buffer, 6 bed volumes of IX Washing Buffer (60mM

Imidazole, 0.5 M NaC1, 20 mM Tris-HC1, pH 7.9). His.TagpthA protein was eluted with

lX Elute Buffer (1 M Imidazole, 0.5 M NaC1, 20 mM Tris-HC1, pH 7.9). The protein

elute was dialyzed against 50 mM NH4HCO3 at 40C overnight and lyophilized by











vacuum freeze drying. The freeze driedpthA protein was redissolved in 200 p1 1X

Treatment Buffer (0.125 M Tris.HC1, pH 6.8, 4% SDS, 20% glycerol, 0.2M DTT, 0.02%

Bromophenol Blue, pH 6.8) and examined for purity by SDS polyacrylamide gel

electrophoresis. The SDS-PAGE gel was soaked in 0.2 M ice cold KC1 and the 130 kDa

protein band excised with a sharp razor. The PthA protein was electro-eluted out of the

polyacrymide gel slice with Protein Elution Buffer (25 mM Tris base, 192 mM glycine,

0.1% SDS), using a setting of 10mA per tube for 8 hr. The Protein Elution Buffer was

exchanged for the same buffer without SDS and electro-elution was continued for

another 30 min to remove the SDS from the protein sample. The electro-eluted sample

was dialyzed against 50 mM NH4HCO3 overnight at 40C and freeze dried. Gel eluted

PthA was re-dissolved in 100 p of 0.1 M NaHCO3 (pH 8.6). The optical density of the

sample at 260 nm and 280 nm was measured and used to calculate the protein

concentration. The sample was stored at -200C until ready for use. Details are in

Appendix B.




Expression and Purification of GST Fusion PthA C-terminal 200 aa Truncated Protein

The DNA encoding the carboxyl terminal 200 amino acids of PthA was cloned

from pthA in pYY50.13 by PCR amplification of a 660 bp gene fragment into the pGEX-

4T-3 vector (Pharmacia) by Prof. Yingchuan Tian (Gabriel and Tian, unpublished). This

resulted in a fusion of the COOH-terminus of PthA to Glutathione S-transferase (GST).

The clone was named pGNLS3-2 (Figure 9). Single colonies of pGNLS3-2/E. coli

































Figure 9. Map of pGNLS3-2 plasmid (pthA gene COOH-terminal 660 bp fused with
GST in pGEX-4T-3 vector).


DH5ca were used to inoculate LB medium with ampicillin to a final concentration of

100[lg/ml and mini-preps of pGNLS3-2 plasmid DNA were used to transform E. coli

protein expression strain BL21(DE3)pLysS competent cells. pGNLS3-2/E. coli

BL21(DE3)pLysS cells were grown in a rotary shaker at 370C at 220 rpm in 2X YT

medium (Tryptone 16g/l, yeast extract 10g/l, NaCl 5g/l, pH 7.0) with 50[lg/ml

carbenicillin and 34[lg/ml chloramphenicol until growth reached the mid log phage

(OD600=0.5). Protein expression was induced by addition of 0.1 mM IPTG with

continued incubation at 300C at 220 rpm for 3 hours. Cells were collected by

centrifugation at 7,000 rpm at 4C for 10 min and suspended in 1X PBS (140 mM NaC1,











2.7 mM KC1, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) plus 0.1% NP-40 and 1 mM

PMSF. Cells were disrupted by sonication on ice. The lysate was centrifuged at 12,000

rpm for 20 min at 4C, the supernatant was collected and passed through a 0.45 micron

membrane filter. One ml of this crude protein extract was combined with 100 pl1

Glutathione Sepharose 4B (a w/v 50% slurry in IX PBS), and slowly inverted to mix

using a rotary platform shaker with gentle agitation at 4C for 30 min. The GST

(Glutathione S-transferase) fusion protein was eluted using 1 ml of Glutathione Elution

Buffer (10 mM reduced glutathione, 50mM Tris-HC1, pH 8.0) at 4C for 30 min. The

eluted protein mixture was centrifuged at 500 x g for 10 min at 4C to pellet the protein.

The protein pellet was lyophilized as described previously for PthA. The truncated PthA

COOH-terminal 200 aa GST fusion protein was redissolved into 100 p1 of 0.1 M

NaHCO3 (pH 8.6). Details are in Appendix C.




Phage Display Peptide Library Biopanning

The full length PthA (His.Tag fusion) and PthA COOH-terminal 200 amino acids

truncated protein (GST fusion) were diluted to 100 plg/ml in 0.1 M NaHCO3 (pH 8.6).

100 p[g/ml of Glutathione S-transferase in 0.1 M NaHCO3 (pH 8.6) was prepared as pre-

absorption blocking agent. Also, 100 [lg/ml of Bovine Serum Albumin (BSA) was used

as the background control. A 96-well microtiter plate was coated with 150 p|l of each

target per well, with gentle agitation at 40C overnight. Each well was rinsed with

Blocking Buffer (NaHCO3, pH 8.6, 0.1M, BSA, 5mg/ml, NaN3, 0.02%) for at least 1 hr











at 4C and then washed 6 times by IX TBST (Tris.HC1, pH 7.5, 50 mM, NaC1, 150 mM,

Tween-20 0.1% v/v) at room temperature. The original unselected library (New England

BioLabs, Ph.D.-7TM Phage Display Heptapeptide Library, 2 x 1013 pfu/ml) was diluted to

2 x 1011 phage in 1 ml of 1X TBST and incubated on the coated and blocked plates at

room temperature for 60 min with gentle agitation.

To reduce non-specific binding of GST fusion protein, the unselected phage

library was first incubated with GST coated and blocked plates at room temperature for

60 min, and then transferred to the PthA COOH-terminal 200 aa GST fusion protein

coated plates and incubated for another 60 min at room temperature. After incubation,

all plates were washed 10 times using IX TBST. Bound phage was eluted with 100 pfl

of 100 plg/ml full length PthA protein or PthA COOH-terminal 200 aa truncated GST

fusion protein in IX TBS (Tris.HC1, pH 7.5 50 mM, NaC1, 150 mM) at room temperature

for 60 min. The eluted phage was amplified until the titer was up to 2 x 1013 pfu/ml.

These biopanning steps were repeated three times. After the second round of selection,

the Tween-20 concentration was increased to 0.5% v/v in the washing steps. For the

GST fusion protein, each round of selection used plates that were pre-absorbed by GST.

For the PthA full length protein, four rounds of phage display biopanning were carried

out. A fourth round of selection was carried out using full length PthA protein on the

library previously selected using only the truncated GST fusion PthA COOH-terminal

200 aa protein. At least 10 clones from both screened libraries were picked at random for

DNA sequencing. The DNA sequencing primer was -28gIII (5'-











GTATGGGATTTTGCTAAACAAC-3'). The DNA sequencing data was used to find out

the consensus sequence of binding clones. Interesting clones were stored at -200C.

Details are in Appendix D.




In Vitro Binding Affinity of Aptamers ELISA

Representative interesting clones were used for in vitro binding affinity assays.

The putative phage clone was amplified to a titer of 1013 pfu/ml. As a control, 1 1l of

M13 phage from the original unselected library (New England BioLabs, Ph.D.-7TM Phage

Display Heptapeptide Library, 2 x 1013 pfu/ml) was amplified to a titer of 1013 pfu/ml.

Two targets were used to coat 0.5ml microcentrifuge tubes: 150 1l of 100 [lg/ml full

length PthA protein in 0.1 M NaHCO3 (pH 8.6) or 150 p1l of 100 plg/ml PthA COOH-

terminal 200 aa truncated GST fusion protein in 0.1 M NaHCO3 (pH 8.6). As a target

background control, 150 p1 of 100 [lg/ml ofBSA in 0.1 M NaHCO3 (pH 8.6) was used.

The coated microcentrifuge tubes were incubated with gentle agitation at 4C overnight.

Each tube was then blocked using 200p1 Blocking Buffer at 4C for 2 hr. Serial dilutions

of phage in IX TBST were prepared in a dilution range of 107 to 1012. Each tube was

washed 6 times with IX TBST at room temperature. To reduce non-specific binding, the

serial dilutions of phage were first incubated in microcentrifuge tubes which were only

rinsed by Blocking Buffer for 60 min at room temperature and then the pre-absorbed

phage was transferred into the blocked target protein tubes to continue incubating at room

temperature for another 60 min. Each tube was then washed 6 times with IX TBS with











0.5% (v/v) Tween-20. Horse Radish Peroxidase/Anti-M13 Monoclonal Conjugate

(Pharmacia) was diluted 1:5000 in IX Blocking Buffer and dispersed 200 pLl to each

phage-treated tube and incubated at room temperature for 60 min with gentle agitation.

Each tube was washed 6 times in 1X TBS with 0.5% (v/v) Tween-20. Sigma FAST

OPDTM (O-Phenylenediamine Dihydrochloride) substrate tablet was reconstituted by

adding 20 ml distilled water and 200 Ipl was added to each tube to incubate in the dark at

room temperature for 30 min. To stop the reaction, 50 Ipl of 3M HC1 was added to each

tube. 150 p1l of solution from each tube was transferred to a 96-well ELISA microtiter

plate for comparisons. A Microplate Autoreader (model EL 309) Bio-Tek Instruments

was used to read absorbance of each well at 492nm. Details are in Appendix E.




PCR-UDG Cloning of Aptamers

A PCR-UDG cloning strategy was designed to clone the aptamer, YPASYMQ

and HPYTFLN, respectively, as the leading sequence and in-frame fusion with GUS

reporter gene in a plant expression vector generated from pBI221 (Figure 10). Each

amino acid codon was modified based on the citrus codon preference (Table 2). Full

length pthA gene was cloned into the BamHI site of plant expression vector pBI221 to

create a tranlational fusion with GUS reporter gene on the vector by Dr. Yongping Duan

(Duan et al., 1999). The resulting clone was named pYD12.9F. E. coli DH5ca was

transformed with this vector and single colonies were inoculated into LB medium with

ampicillin at a concentration of 100[ig/ml and grown at 370C in a rotary shaker at 220





















































Figure 10. The design of PCR-UDG oligonucleotides to amplify aptamers YPASYMQ
and HPYTFLN (The aptamer sequences are indicated by italic on the oligo).











rpm overnight. Plasmid DNA was extracted and digested with BamHI. After

phenol/chloroform extraction and ethanol precipitation, pYD12.9F/BamHI fragments

were resuspended in TE (pH 8.0) with the final concentration of 5ng/|ll. Two sets of

oligonucleotides were designed for UDG cloning according to the amino acid sequences

of selected aptamers (YPASYMQ and HPYTFLN) and codon preference table of Citrus

sinensis (Figure 10 and Table 2). Each codon coding the amino acid sequence of

aptamers was specifically converted to codon usage appropriate for citrus and the

aptamer sequences were included in the synthetic oligonucleotides. Overlaps of 18

nucleotides were designed with 4 to 8 uracil molecules and after Uracil DNA glycosylase

treatment, the Uracil residuals dissolved and anneal the overlaps. The oligonucleotides

were synthesized by GIBCO BRL and reconstituted in TE (pH 8.0). PCR reaction

mixtures were set up as 2[pl of 5ng/pl pYD12.9F/BamHI, 15 pil of 10X PCR buffer, 4.5pl

of 50 mM MgC12, 15pl of 2.5mM dNTP, 82.75pl of distilled water. Oligonucleotides

were added in two sets: 15 ptl each of primers GZ1 (4.36 piM) and GZ2 (4.32 pIM) or 15

p1l each of primers GZ3 (4.73 [pM) and GZ4 (4.82 [pM). Details of PCR cycle conditions

are in Appendix F. After PCR amplification, Uracil DNA Glycosylase (UDG) treatment

was carried out by incubation of 5pAl of crude PCR product, 2pl of 10X PCR buffer, 1~1l

of 1U/pl Uracil DNA glycosylase (GIBCO BRL) and 12 p1l of distilled water at 370C for

30 min followed by denaturation at 65 C for 10 min. The UDG treated PCR products

were transformed to E. coli DH5a competent cells. Single colonies were inoculated into

LB medium with ampicillin at a concentration of 100p[g/ml in a rotary shaker at 220 rpm











and grown at 370C overnight. The plasmid DNA was extracted and then separately

digested by Xbal/BamHI and Xbal/Smal and examined by 15.0% polyacrylamide gel

electrophoresis. Interesting clones were saved as glycerol (50% v/v) stock cultures at -

700C. GUS activity assays were carried out by resuspending pellets from 1 ml overnight

cultures into 570 p1 distilled water and 330 p1 of 0.3 mg/m 5-bromo-4-chloro-3-indolyl-

P-D-glucuronide (X-gluc). The suspensions were incubated at room temperature

overnight. Putative clones showing blue color as compared to the control of E. coli

DH5a cells were examined further. Plasmid DNA of putative clones was purified using

QIAGEN Midi Kits and DNA sequencing was carried out with the DNA sequencing

primer DG39R (5'- CATAAGGGACTGACCA -3'). Details are in Appendix F.




Agrobacterium tumefaciens Mediated Transient Expression of Aptamer Constructs in
Bean, Sweet Orange and Tomato


Single colonies of above PCR-UDG putative clones were inoculated into LB

medium with ampicillin at a concentration of 100p[g/ml in a rotary shaker at 220 rpm and

grown at 370C overnight. Plasmid DNA was extracted and digested by EcoRI. The

digested insert DNA fragments were precipitated by ethanol and redissolved in TE (pH

8.0) to a final concentration of 1l[g/p[l. Full length pthA gene was cloned into pGZ6.4

binary vector by Dr. Yongping Duan, and the resulting clone was named pYD40.1 (Duan

et al., 1999). As a control, pthA was deleted by digesting with BamHI and religation,

generated a control clone named pYD40.2 (Duan et al., 1999). E. coli DH5a/pYD40.1











and DH5a/pYD40.2 were inoculated into LB medium with kanamycin at the

concentration of 50p[g/ml, respectively, and incubated at 370C in a rotary shaker at a

speed of 220 rpm overnight. Plasmid DNA was extracted and digested by EcoRI.

The digested DNA fragments of pYD40.1 and pYD40.2 were separately extracted

by phenol/chloroform, precipitated by ethanol, and redissolved in TE (pH 8.0). 20 ng of

each EcoRI digest were incubated with 5 p[l of 10X buffer and 1 l1 of Shrimp Alkaline

Phosphatase (1:1 fresh diluted by IX Dilution Buffer) in a total volume of 50pl at 37C

for 60 min followed by 65 C denaturation for 20 min. Ligation reactions were set up

with 4 pil of EcoRI digest of PCR-UDG putative clones, 2[pl of the pYD40.1 or pYD40.2

EcoRI digests, 2[il of 5X ligation buffer, 1 il of T4 DNA ligase in a total volume of 10 pl

at 160C overnight. 2 pAl of the crude ligation mixture was used to transform E. coli DH5a

competent cells and transformants were selected on LB agar plates containing 50p[g/ml of

carbenicillin and 50[lg/ml of kanamycin.

Single colonies were inoculated into LB medium containing 50[lg/ml of

carbenicillin and 50[lg/ml of kanamycin and incubated in a rotary shaker at 370C with a

speed of 220 rpm overnight. Plasmid DNA extracts were digested by EcoRI to confirm

the insertion. The orientation of interesting clones was tested by digestion of SstI.

Putative clones were mated into Agrobacterium tumefaciens GV2260 by tri-parental

matings and incubated on LB agar plate at 280C overnight. Selection for transconjugants

was on LB agar plates containing 50[lg/ml of carbenicillin, 50[lg/ml of kanamycin and

75p[g/ml rifomycin. As a control, pYD40.1 and pYD40.2 were also mated into











Agrobacterium tumefaciens GV2260 and selected on LB agar plates containing 50p[g/ml

kanamycin and 75p[g/ml rifomycin. Single colonies of Agrobacterium tumefaciens

GV2260 and transconjugants were inoculated into 20 ml YEB medium (Beef extract 5g/l,

yeast extract lg/1, peptone 5g/l, sucrose 5g/l, MgSO4 2mM, MES 10mM, acetosyringone

20piM, pH 5.6) with the same antibiotics as used in the mating selections and incubated

in a rotary shaker at 280C and a speed of 60 rpm until the OD600 reached 0.8. Bacterial

cells were then collected by micro-centrifugation at 4C and a speed of 5,000 rpm and

the pellets were resuspended in MMA medium (IX MS salt, MES 10mM, sucrose 20g/l,

acetosyringone 200piM, pH 5.6) until OD600=0.5. The suspension was kept at room

temperature for 1 hr before use.

Ten-day-old young leaves of California light red kidney bean were infiltrated by

Agrobacterium tumefaciens GV2260 suspension using vacuum infiltration at a pressure

of 0.5 mbar for 20 min. The infiltrated leaves were incubated in 100mm petri-dishes with

three-layers of 3MM paper moistured by distilled water and kept at 220C under light for

36 hr. Also, cell suspensions ofAgrobacterium tumefaciens GV2260 (OD600=0.3) were

injected using the blunt end of tubular syringe the abaxial side of tender sweet orange

leaves or young tomato leaves (Kapila et al., 1997). Inoculated sweet orange plants were

grown at 280C for four weeks in a quarantined green house. The inoculated tomato

plants grew under low light for 24 hr in growth chambers. Details are in Appendix G.
















CHAPTER 3
RESULTS



Expression and Purification of PthA Protein by His.Tag

Full-length PthA protein expression was induced by adding IPTG. Three

different IPTG concentrations (0.1mM ImM and 100mM), seven induction times (Ohr,

lhr, 2hr, 3hr, 4hr, 5hr and 6hr) and three induction temperatures (280C, 300C and 370C)

were tested. The optimum IPTG induction concentration was 0. mM and the induction

temperature was 280C. 10l of each sample was mixed with 10 1l of IX Treatment

Buffer (0.125 M Tris.HC1, pH6.8, 4% SDS, 20% glycerol, 0.2M DTT, 0.02%

bromophenol blue), boiled 10 min, and centrifuged at 10,000 rpm for 10 min.

Supernatant was loaded on 8% (w/v) polyacryamide separating gel with 4% (w/v)

stacking gels. Figure 11 shows the time course of full-length PthA protein expression

induced by 0. ImM IPTG. A new 130 kDa protein band was very clear after IPTG

induction for 1 hr (Figure 11, band D). Maximum induction was obtained after 3hr

(Figure 11, band F). Compared to pYY50.13/E.coli BL21(DE3)pLysS control (Figure

11, band A) and no IPTG induction control (Figure 11, band B), the results obtained that

0. mM IPTG was optimum to induce His.Tag fusion protein expression.











A B C D E F


Figure 11. Time course of PthA full length protein expression induced by 0.1 mM
IPTG.

A: pYY50.13/E. coli BL21(DE3)pLysS, B: no IPTG,
C: IPTG 0 hr, D: IPTG 1 hr, E: IPTG 2 hr, F: IPTG 3 hr,
G: IPTG 4 hr, H: IPTG 5 hr, I: IPTG 6 hr, J: MW marker.





After His.Bind affinity column chromatography, each fraction was collected and

10pl of each sample was mixed 1:1 with IX Treatment Buffer for SDS-PAGE. Figure 12

shows PthA His.Tag protein after His.Bind affinity column purification. Compared to

pYY50.13/E.coli BL21(DE3)pLysS control (Figure 12, band A), a good yield of full-

length PthA protein was achieved by 0. Im IPTG induction for 3 hr (Figure 12, band B).

After sonication, most PthA protein was found in the soluble protein fractions (Figure 12,


G H I











A B C D


Figure 12. The purification of PthA full length protein by His.BindTM
affinity column chromatography.


A: pYY50.13/BL21(DE3)pLysS no induction, B: 0.1 mM IPTG induction for 3 hr,
C: Soluble protein fraction after sonication, D: Soluble protein after His.Bind resin
binding, E: His.Bind column PthA protein elute, F: Electro gel elute PthA protein, G:
MW marker.





band C). After His.Bind affinity column chromatography, the absence of the 130 kDa

band in eluate indicated that most PthA protein was bound to the column (Figure 12,

band D). Most of the protein in the elute from the His.Bind column was PthA protein

(Figure 12, band E). After electro gel elution, the PthA full-length protein was very pure

and appeared as a single clear band at 130 kDa (Figure 12, band F).


E F G











Expression and Purification of the PthA COOH-terminal 200aa Truncated Protein by
GST Gene Fusion System



Similar experiments were carried out with the PthA COOH-terminal 200aa GST

fusion protein. Three different IPTG concentrations (0.1mM, ImM and 100mM), seven

induction times (Ohr, lhr, 2hr, 3hr, 4hr, 5hr and 6hr) and three induction temperatures

(280C, 300C and 370C) were tested. The optimum IPTG induction concentration was

0.1 mM and the induction temperature was 30C.



A B C D E F G H I J




















Figure 13. The expression of PthA COOH-terminal 200 aa truncated protein
induced by 0.1 mM IPTG at different times.

A: pGNLS3-2/E. coli BL21(DE3)pLysS, B: no IPTG, C: IPTG 0 hr,
D: IPTG 1 hr, E: IPTG 2 hr, F: IPTG 3 hr, G: IPTG 4 hr,
H: IPTG 5 hr, I: IPTG 6 hr, J: MW marker.














AB


S 55KDa







I



Figure 14. Electro-elution of the COOH-terminal end of PthA, fused to GST.
A: Electro gel elution of PthA C-terminal 200 aa truncated protein, B: MW marker.


Figure 13 shows the expression pattern of GST fusion proteins at 300C after
induction by adding 0. 1mM IPTG. After 1 hr of 0. 1mM IPTG induction, a new 55kDa
protein band appeared (Figure 13, band D) compared to the pGNLS3-2/E. coli
BL21(DE3)pLysS control (Figure 13, band A), expression of the 55kDa protein reached
its peak at 2 hr (Figure 13, band E). Longer induction did not increase the expression
level of GST fusion protein (Figure 13, band F-I).










Figure 14 shows the purity of electro-gel-eluted PthA COOH-terminal 200aa GST

fusion protein. The GST fusion PthA COOH-terminal 200 aa peptide was found at the

expected size of 55 kDa (Figure 14 band A).




Phage Display Peptide Library Biopanning

After the fourth round of selection, unamplified phage was titered. Single

plaques were picked up in plates with about 100 plaques. Single stranded M13 phage

DNA was prepared and dissolved in 30pil of TE (pH 8.0) (Appendix D). l1[l of each

phage prep was tested by 0.7% agarose gel electrophoresis (Figure 15). Figure 15 Panel

A shows 10 randomly selected clones following biopanning selection using PthA full

length protein as a target for all four rounds of selection. Figure 15 Panel B shows 10

clones using PthA COOH-terminal 200 aa truncated protein as a target for three rounds of

selection and then using PthA full length protein as a target in the 4th selection round.

10pl of each selected M13 phage DNA was sequenced and the result is

summarized in Figure 16. Of 11 random clones sequenced from the phage population

after four rounds of selection, a DNA sequence encoding HPYTFLN appeared 5 times

(45.5%), a sequence encoding HPHTFLN appeared twice (18.2%) and a sequence

encoding YPASYMQ appeared 4 times (36.4%) (Figure 16, panel A). Of 10 random

clones from the phage population after four rounds of selection, a DNA sequence

encoding HPYTFLN appeared twice (20.0%) and a sequence encoding YPASYMQ

appeared 7 times (70.0%) (Figure 16, panel B).











A 1 2 3 4 5 6 7 8 91011
















B 1 2 3 4 5 6 7 8 9 1011


















Figure 15. Characterization of M13 PthA-binding clones.

Al:1 Kb DNA Ladder; A2 Al 1:10 clones using PthA full length protein as target in
phage display biopanning of all four rounds of selection; B B10: 10 clones using PthA
COOH-terminal 200 aa truncated protein as target till 3rd round selection and cross
react to PthA full length protein in 4th round selection; B 11: 1 Kb DNA Ladder.













A. Phage display sequences of binding clones after 4th round selection (all four rounds of
selection against PthA full length protein):


(ATT
(ATT
(ATT
(ATT
(CTG
(ATT
(ATT
(CTG
(CTG
(ATT
(CTG


CAG
CAG
CAG
CAG
CAT
CAG
CAG
CAT
CAT
CAG
CAT


AAA
AAA
AAA
AAA
ATA
AAA
AAA
ATA
ATA
AAA
ATA


CGT
CGT
CGT
CGT
CGA
CGT
CGT
CGA
CGA
CGT
CGA


HPHTFLN
HPYTFLN
HPYTFLN
HPYTFLN
YPASYMQ
HPHTFLN
HPYTFLN
YPASYMQ
YPASYMQ
HPYTFLN
YPASYMQ

HPYTFLN
HPHTFLN
YPASYMQ


B. Phage display sequences of binding clones after 4th round cross-reaction against PthA
full length protein (previous selection against PthA C-terminal 200 aa truncated protein


till 3rd round)

N3.28g
N4.28g
N7.28g
N8.28g
N11.28g
N12.28g
N13.28g
N14.28g
N15.28g
N17.28g


YPASYMQ
YPASYMQ
HYGFPPP
YPASYMQ
YPASYMQ
HPYTFLN
YPASYMQ
YPASYMQ
HPYTFLN
YPASYMQ

HPYTFLN
YPASYMQ
Pseudo-


(CTG CAT ATA CGA AGC AGG ATA)
(CTG CAT ATA CGA AGC AGG ATA)
(probably bound to polystyrene well)
(CTG CAT ATA CGA AGC AGG ATA)
(CTG CAT ATA CGA AGC AGG ATA)
(ATT CAG AAA CGT ATA CGG ATG)
(CTG CAT ATA CGA AGC AGG ATA)
(CTG CAT ATA CGA AGC AGG ATA)
(ATT CAG AAA CGT ATA CGG ATG)
(CTG CAT ATA CGA AGC AGG ATA)


of 10,
of 10,
of 10,


20.0%)
70.0%)
10.0%)


Figure 16. The DNA sequences of the M13 PthA-binding clones (The conversion of
nucleotide sequence to peptide sequence is listed in Table 1).


P2.28g
P6.28g
P7.28g
P8.28g
P9.28g
P10.28g
P13.28g
P14.28g
P15.28g
P16.28g
P17.28g


(5 of 11, 45.5%)
(2 of 11, 18.2%)
(4 of 11, 36.4%)


ATG
ATA
ATA
ATA
AGC
ATG
ATA
AGC
AGC
ATA
AGC


CGG
CGG
CGG
CGG
AGG
CGG
CGG
AGG
AGG
CGG
AGG


ATG)
ATG)
ATG)
ATG)
ATA)
ATG)
ATG)
ATA)
ATA)
ATG)
ATA)













In Vitro Binding Assay ELISA

Three representative clones were chosen for ELISA tests, P7.28g (P7) for the

HPYTFLN aptamer, N4.28g (N4) for the YPASYMQ aptamer and P10.28g (P10) for the

HPHTFLN aptamer. Each clone was amplified until the titer reached 1013 pfu/ml. The

unselected phage from the original phage display library was also amplified until the titer

reached 1013 pfu/ml for use as a control. Serial dilutions of each phage were prepared

with titers ranging from 1012 pfu/ml to 107 pfu/ml. The target control was Bovine Serum

Albumin (BSA). Figure 17 shows a 96-well ELISA plate loaded with 150 p1 of reaction

mixture, which was pipetted out of 0.5 ml microcentrifuge tubes and read at 492nm using

a Microplate Autoreader EL309. There was no reaction between phage (unselected

original M13 phage(WT), HPYTFLN aptamer (P7), YPASYMQ aptamer (N4) or

HPHTFLN aptamer (P10)) and BSA, the "pseudo-target". There also was no reaction

between unselected original M13 phage (WT) and PthA full-length protein (pthA) or the

COOH-terminal 200 aa truncated PthA GST fusion protein (NLS). HPYTFLN aptamer

(P7), YPASYMQ aptamer (N4) and HPHTFLN aptamer (P10) all reacted to PthA full-

length protein (pthA) and to the COOH-terminal 200 aa truncated PthA GST fusion

protein (NLS). The reaction of the aptamers to the full length PthA protein looked

stronger than to the truncated protein. Figure 18 is the summary of ELISA data read at

492nm. The result indicated that HPYTFLN (P7), YPASYMQ (N4) and HPHTFLN

(P10) aptamer phage all had strong binding affinity to PthA full-length protein and the










































Figure 17. In vitro Binding Assay of Aptamers.

ELISA Test for different concentrations of WT (no selective random phage), P7
(HPYTFLN phage), N4 (YPASYMQ phage) and P10 (HPHTFLN phage) against
different targets including BSA (Bovine Serum Albumin), pthA (PthA full length protein)
and NLS (PthA COOH-terminal 200 aa truncated protein).









44





2
0

S--IN4




c| 1
o P10

o i





























Figure 18. The in vitro binding afnity of aptamers: P7 (HPYTFLN),
N4 (YPASYMQ) and P10 (HPHTFLN).


















COOH-terminal 200 aa truncated PthA protein. The binding affinity diminished with


dilution. There was not much difference in binding affinity among the three different


aptamers.
m N4
C C



CC 1









1012 1011 100 1109 108 107

Serial Dilution of Phage


Figure 18. The in vitro binding afnity of aptamers: P7 (TPYTFLN),
N4 (YPASYMQ) and PI 0 (HPHTFLN).






COOH-terminal 200 aa truncated PthA protein. The binding affinity diminished with


dilution. There was not much difference in binding affinity among the three different


aptamers.













PCR-UDG Cloning of Aptamers

Two aptamer sequences, encoding YPASYMQ and HPYTFLN, were synthesized

and amplified by PCR for UDG-cloning into pBI221 using the pYD12.9F/BamHI

fragment as the template (Figure 19, band C). PCR products were tested by 0.7%

agarose gel electrophoresis. Figure 19 band E shows the GZ1 and GZ2 primer amplified

PCR products with the sequence of YPASYMQ and Figure 19 band G shows the GZ3

and GZ4 primer amplified PCR products with the sequence of HPYTFLN. Figure 19

band D and band F are negative controls without template. The PCR products were at the

expected size of 5.8 kb. After Uracil DNA glycosylase treatment, the linear PCR

products were reclosed (Figure 20). Further testing by double restriction enzyme

digestion yielded the expected aptamer size of 45 base pairs between the Xbal site and

the BamHI sites (Figure 10). Figure 21 indicates that both pGZ7.5 and pGZ7.6 have 45

bp insertions (Figure 21, band D and band F). The 90 bp band is likely an aptamer

dimer. The insertion sequence was confirmed by DNA sequencing. Two clones were

saved for further testing. The pGZ7.5 for the YPASYMQ apatmer (Figure 20 band F)

and pGZ7.6 for the HPYTFLN aptamer (Figure 20 band M). Maps of pGZ7.5 and

pGZ7.6 are shown in Figure 22.













A B C D E F G H
































Figure 19. PCR amplification of aptamers P7 and N4.

A: kDNA/HindIII fragments; B: pYD12.9F plasmid DNA, C: pYD12.9F/BamHI
template, D: pGZ1 primer + pGZ2 primer (no template), E: pGZ1 primer + pGZ2 primer
+ pYD12.9F/BamHI template, F: pGZ3 primer + pGZ4 primer (no template),
G: pGZ3 primer + pGZ4 primer + pYD12.9F/BamHI template, H: 1 Kb DNA Ladder.











ABCDEFGHI J KLMN

















Figure 20. Uracil DNA glycosylase treatment of PCR products.

A: kDNA/HindIII fragments, B to G: pGZ1 primer and pGZ2 primer amplified
"YPASYMQ" clones (F clone saved as pGZ7.5), H to M: pGZ3 primer and pGZ4 primer
amplified "HPYTFLN" clones (M clone saved as pGZ7.6), N: 1 kb DNA Ladder.

A B C D E FG H
















Figure 21. 15.0% polyacrylamide gel electrophoresis of apatmers.

A to C: Other PCR-UDG clones digested by Xbal+BamHI,
D: pGZ7.5+Xbal+BamHI, E: pGZ7.5+XbaI+SmaI, F: pGZ7.6+Xbal+BamHI,
G: pGZ7.6+XbaI+SmaI, H: 100 bp DNA Ladder.
















CAuwaMAnrFI


Hhbl M PMI
\SfrBl /p


An o00


Figure 22. Plasmid maps of pGZ7.5 and pGZ7.6.
















Agrobacterium tumefaciens Mediated Transient Expression of Aptamers in Bean, Sweet
Orange and Tomato Leaves


Constructs of pGZ7.5 and pGZ7.6 cloned into Agrobacterium binary vector

(pYD40.1 and pYD40.2) are shown in Figure 23. EcoRI digestion confirmed the size of

insertion and SstI digestion indicated the orientation of inserted fragments. Four clones

were saved: pGZ8.1 carries the coding sequence for the YPASYMQ aptamer andpthA

(Figure 23, band 2), pGZ8.2 carries the coding sequence for the YPASYMQ aptamer

(Figure 23, band 17), pGZ8.3 carries the coding sequence for the HPYTFLN aptamer and

pthA (Figure 23, band 28) and pGZ8.4 carries the coding sequence for the HPYTFLN

aptamer (Figure 23, band 31). Maps of these four clones shown in Figure 24.

Figure 25 Panel A shows Agrobacterium tumefaciens GV2260 constructs after

infiltration into young California light red kidney bean leaves. Compared to the dark

brown Hypersensitive Reaction (HR) lesion of the positive control from pYD40.1

(expressing pthA), pGZ8.1 (expressing the YPASYMQ aptamer andpthA) and pGZ8.3

(expressing HPYTFLN aptamer andpthA) showed reduced HR symptoms. pGZ8.1

showed significantly reduced HR, indicating that the YPASYMQ aptamer may work

better in plant than the HPYTFLN aptamer.

Figure 25 Panel B shows Agrobacterium tumefaciens GV2260 constructs

inoculated into young sweet orange leaves. Compared to the strong canker symptom

expressed by pYD40.1 (pthA), pGZ8.1 (expressing the YPASYMQ aptamer andpthA)











1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20



































21 22 23 24 25 26 27 28 29 30 313233 343536 37 38 39 40


Figure 23. Aptamers P7 and N4 cloned in Agrobacterium binary vector pYD40.1 and
pYD40.2.

1, 21: XDNA/HindIII fragments; 20, 40: 1 kb DNA Ladder; 2 to 10: pYD40.1+pGZ7.5;
11 to 19: pYD40.2+pGZ7.5; 22 to 30: pYD40.1+pGZ7.6; 31 to 39: pYD40.2+pGZ7.6.
Each group has three different clones. Each clone has three different treatments,
such as undigested plasmid DNA, digested by EcoRI and digested by SstI.
(Save No.2 clone as pGZ8.1, No.17 clone as pGZ8.2, No.28 clone as pGZ8.3,
and No.31 clone as pGZ8.4, respectively).







































Figure 24. The plasmid maps of pGZ8.1, pGZ8.2, pGZ8.3 and pGZ8.4.











A. Bean










B. Citrus











C. Tomato


pGZ8.4
Figure 25. Inoculation of Agrobacterium tumefaciens strain GV2260 constructs to
the leaves of bean (panel A), citrus (panel B) and tomato (panel C).

pYD40.1 ispthA gene only in binary vector, pYD40.2 is the binary vector, pGZ8.1 is
YPASYMQ aptamer sequence withpthA gene in binary vector, pGZ8.2 is YPASYMQ
aptamer sequence only in binary vector, pGZ8.3 is HPYTFLN aptamer sequence with
pthA gene in binary vector and pGZ8.4 is HPYTFLN aptamer sequence only in vector.










and pGZ8.3 (expressing the HPYTFLN aptamer andpthA) showed reduced canker

symptoms. Also, pGZ8.1 reduced canker symptoms more than pGZ8.3, which confirmed

the observations in bean. pYD40.2 (binary vector only), pGZ8.2 (expressing the

YPASYMQ aptamer only) and pGZ8.4 (expressing the HPYTFLN aptamer only) showed

no effects on any plants tested.

Figure 25 Panel C shows inoculation of tomato young leaves using the same

Agrobacterium constructs. There was no difference in HR symptoms between pYD40.1

(expressing pthA), pGZ8.1 (expressing the YPASYMQ aptamer andpthA) and pGZ8.3

(expressing the HPYTFLN aptamer and pthA). This result indicated that the two

aptamers had no effect on HR expression in tomato, as expected. Published reports and

our own lab results (Gabriel et al., 1996) showed that the COOH-terminal region of

members of the pthA gene family are not important for elicitation of the HR in tomato.

The most plausible explanation for this results is that both the YPASYMQ aptamer and

the HPYTFLN aptamer bind to the COOH-terminal region of PthA protein, thus, it didn't

show the effect of block PthA function.

















CHAPTER 4
DISCUSSION



A Phage Display Peptide Library was used to screen for 7-peptide aptamers that

specifically bind to PthA full-length protein and a PthA C-terminal 200aa truncated

protein. Two recombinant protein were constructed, pthA full length gene in pET-19b

vector for His.Tag fusion and pthA COOH terminal 600bp gene fragment in pGEX-4T-3

vector for GST fusion.

The His.Tag fusion protein was best expressed by vigorously shaking at 370C

followed by 0. mM IPTG induction at OD600=0.5, changing the temperature to 280C for

3 hr. The best condition for GST fusion protein expression was vigorously shaking at

37C in 2X YT medium till OD600=0.5, followed by adding of 0. mM IPTG and shaking

at 300C for 3 hr. The low concentration of IPTG and lower temperature induction

appeared to make the fusion proteins more soluble and more easily purified by resin

affinity purification. Use ofE. coli BL21(DE3)pLysS as a host strain significantly

enhanced the expression rate of the fusion protein compared to non-host E. coli strain

DH5a. Also, since Ampicillin and Chloramphenicol had a synergistic effect,

Carbenicillin was used to replace Ampicillin in combination with chloramphenicol for











antibiotic selection. Proteinase inhibitors such as PMSF appeared to inhibit proteinase

activity during sonication and protected the fusion protein from degradation. Nonionic

detergents such like NP-40 increased the solubility of fusion proteins. All reactions were

kept on ice to obtain the maximum yield of fusion proteins. Quick sonication on ice

helped keep a high protein yield and high-speed centrifugation was used to collect

soluble proteins. For His.Tag protein purification, the His.Bind resin needed to be

regenerated before use and well washed, charged and equilibrated. Such treatment

significantly enhanced the efficiency of the affinity column. A flow rate of about 10

column volumes per hour was required and if necessary, the pass-through was collected

and reloaded on top of the column and run again to maximize the binding of His.Tag

protein to the column. The His.Tag protein always come out in the first 5 ml of eluate.

Later eluate contained other proteins with some affinity to the His.bind resin. Protein

was dialyzed and redissovled in as small a volume as possible to increase the

concentration.

For GST fusion protein, low speed (500xg) centrifugation was necessary to

sediment the Glutathione Sepharose 4B without compacting the pellet. Gentle agitation

during elution was required. The Glutathione Elution Buffer was freshly made to keep it

in a reduced state. Dialysis against 50mM NH4HCO3 is one of the modifications used for

easy purification of lyophilized proteins. After freeze drying, the NH4HCO3 evaporated

and the protein sample was dissolved in any desired salt solutions without changing of

ion strength. After eight hours of electro elution at 10mA per tube, the Protein Elution

Buffer without SDS was continued another 30 min.











There were two target proteins in Phage display biopanning, PthA full-length

His.Tag protein and PthA COOH-terminal 200aa truncated GST fusion protein. Because

the pET-19b vector only had 10 Histidine repeats, compared to the size of PthA full-

length protein (130 kDa), the His.Tag region was very small. Therefore, PthA full length

His.Tag protein could be directly used as the target for phage display biopanning. But for

the GST fusion protein, the Glutathione S-transferase itself has a molecular weight of 26

kDa, and the size of PthA COOH-terminal 200 aa peptide was 29 kDa (Figure 3, bold

letters). In this case, Glutathione S-transferase could readily interact with the truncated

PthA peptide and have some side effect. Therefore, the Glutathione S-transferase could

be separated using a cleavage enzyme and the PthA COOH-terminal peptide could be

recovered by gel purification. In this experiment, during phage display biopanning, the

amplified phage was first incubated with pure Glutathione S-transferase. Any phage

bound to GST was eliminated during this pre-absorbing treatment, then, unbounded

phage was applied to against GST fusion protein in biopanning. After three rounds of

separate selection, a fourth round of cross-selection using full length PthA was carried

out. For PthA full length protein biopanning, PthA full-length protein was used as the

target in all selections. Three different aptamer encoding sequences were found:

YPASYMQ, HPYTFLN and HPHTFLN. YPASYMQ was found 4 times out of 11

(36.4%), HPYTFLN was found 5 times out of 11 (45.5%) and HPHTFLN was found 2

times out of 11 (18.2%). In the phage population from selection against the PthA

COOH-terminal 200 aa truncated GST fusion protein, YPASYMQ was found 7 times out

of 10 (70.0%) and HPYTFLN was found 2 times out of 10 (20.0%). No HPHTFLN











sequence was found in this case. Based on previous reports (Hoess et al., 1994), some

phage containing sequences with repeats of Phenylalanine or Histidine especially could

bind to polystyrene well. One such sequence, encoding the sequence ofHYGFPPP, wass

considered to be a pseudo-binding sequence. Based on the in vitro binding affinity assay,

ELISA readings indicated HPYTFLN and HPHTFLN had very similar reactivity both to

PthA full length protein and PthA COOH-terminal 200 aa truncated GST fusion protein,

so HPHTFLN could be considered as the subgroup of HPYTFLN with only one base pair

change in the DNA sequence. It is interesting that there were only two aptamer

sequences found in two independent selective pathways, and that they were equally

distributed within two selection phage populations.

In vitro binding affinity assay was carried out by ELISA reading at 492 nm. The

optimal reading value should be in the range of 0.5 to 2.0. M13 phage itself may bind to

ELISA plates. M13 phage always showed high background readings and it was very hard

to tell the difference between the phage binding to plate or the phage binding to target.

After a couple of trials, I found that 0.5 ml microcentrifuge tubes (usually used for PCR

reaction) were very good for reducing the background of M13 in ELISA reactions. To

reduce background, one set of tubes were blocked by Blocking Buffer. The phage was

first put into only blocked empty tubes to incubate for 60 min at room temperature, then,

pre-absorbed phage was transferred to the tubes with blocked target. This step helped to

eliminate non-specific phage only binding to the tube surface. The unbound phage was

used in binding to the target. As a target control, BSA was used. YPASYMQ and

HPYTFLN didn't bind to BSA. Also, as a phage control, original unselected phage was











used. The ELISA result showed that unselected phage didn't bind to PthA full length

protein or PthA COOH-terminal 200 aa truncated GST fusion protein. Since the original

library complexity is 2 x 1013 pfu/ml, the frequency of two aptamer sequences in the

unselected original phage population is almost zero, the reaction of unselected original

phage could be considered as the background. In the ELISA results, aptamers bound to

PthA full length protein with higher affinity than bound to PthA COOH-terminal 200 aa

truncated GST fusion protein. This indicated that the aptamers interacted more with the

full-length PthA protein.

PCR amplification was carried out by a program called "touch-down". Based on

the Tm of primers (GZ1 at 580C, GZ2 at 54C, GZ3 at 580C and GZ4 at 54C), a

program was designed with annealing temperatures ranging from 56C to 500C,

dropping one degree of centigrade once in a total of seven respective cycles, followed by

a regular 30 cycles of PCR carried out at 94C for 1 min, 65 C for 1 min and 720C for 5

min. Elongation times were determined by the length of the expected PCR amplified

molecules. For an expected PCR product of 5.8 Kb, elongation time was set up at 5 min.

Uracil DNA glycosylase was used to remove Uracil. The intervening bases was then

melted off at 65 C, and the 3' overhangs reannealed (See Figure 10). The aptamers in

pBI221 were engineered to form the peptides of sequence M-YPASYMQ-GGGS-

PGGQSLM-GUS and M-HPYTFLN-GGGS-PGGQSLM-GUS. The starting methionine

seemed to have no effect on aptamer function. The Glu-Glu-Glu-Ser forms a flexible

arm to ensure that the aptamer peptide protrudes out of the protein fusion complex. The

GUS protein provides both stability to the aptamer and is useful for detection of plant











transformation. The aptamer fusion sequence was given a CaMV35S promoter and

terminated by a NOS terminator, so it could be used in plant expression (Figure 22).

The two aptamer constructs were cloned into the Agrobacterium binary vector

pYD40.1 (with pthA) and pYD 40.2 (vector only), and mated into Agrobacterium

tumefaciens GV2260 strains, then vacuum infiltrated into California light-red kidney

bean young leaves and syringe inoculated into sweet orange and tomato leaves.

pYD40.1 (pthA alone) showed very strong Hypersensitive Reaction (HR) brown lesions

on California light red kidney bean leaves, while pGZ8.1 (YPASYMQ aptamer with

pthA) and pGZ8.3 (HPYTFLN aptamer with pthA) showed significantly reduced HR

symptoms. The YPASYMQ aptamer appeared to work better than the HPYTFLN

aptamer. A similar result also showed in sweet orange inoculations. Compared to the

strong canker symptom of pYD40.1 (pthA alone), the YPASYMQ aptamer with pthA and

the HPYTFLN aptamer with pthA showed reduced canker symptoms. Also, the

YPASYMQ aptamer reduced canker symptoms more than the HPYTFLN aptamer, which

confirmed the observations in bean. The aptamers alone, when expressed in plant cells,

showed no effect. Inoculating tomato showed there is no difference between pthA alone

orpthA with aptamer which indicated the aptamer may interact the COOH terminal of

pthA gene family.

Figure 26 shows the predicted antigen index of PthA full-length protein. Figure

27 shows the predicted KD hydrophilicity and KD hydrophobicity of PthA full-length

protein. Figure 28 shows the predicted surface probability of PthA full-length protein.





























Figure 26. The predicted Antigenicity Index of PthA.


Figure 27. The predicted KD Hydrophilicity and KD Hydrophobicity of PthA.


































Figure 28. The predicted Surface Probability of PthA.


From all three figures, the COOH-terminal has highest chance to be exposed to the

surface of the PthA protein in the natural folded state, and the COOH-terminal also has

the strongest antigenic and hydrophilic characteristics. It is predicted that the COOH-

terminal end of PthA full length protein plays a very important functional role in protein-

protein interactions and antigenic recognition. Since the aptamers appear to interact with

the COOH-terminal of PthA and block pathogenicity, the COOH terminal end is likely to

be critical in the canker and HR signal transduction pathways.

In the future, the aptamer sequence could be used for permanent transformation of

citrus, in attempt to engineer "resistant" or "immune" trees.


















CHAPTER 5
SUMMARY AND CONCLUSIONS



This work was to determine if peptide aptamers could be selected that bind to

PthA. Two different proteins were expressed, PthA full-length protein (constructed in

pET-19b His.Tag fusion expression vector) and PthA COOH-terminal 200 amino acids of

PthA fused to GST (constructed in pGEX-4T-3 expression vector). Phage display

library biopanning was performed to screen 7-peptide aptamers specifically binding to

full-length PthA protein and a truncated PthA consisting of the C-terminal 200 amino

acids. From 11 random clones chosen from the phage population after four rounds of

selection against full length PthA, the sequence encoding HPYTFLN appeared 5 times

(45.5%), the sequence encoding HPHTFLN appeared twice (18.2%) and the sequence

encoding YPASYMQ appeared 4 times (36.4%). From 10 random clones chosen from

the phage population after three rounds of selection against the COOH-terminal 200 aa of

PthA and a 4th round of selection against full-length PthA, the sequence encoding

HPYTFLN appeared twice (20.0%) and the sequence encoding YPASYMQ appeared 7

times (70.0%). ELISA tests indicated that all three aptamers, YPASYMQ, HPYTFLN

or HPHTFLN, can bind to full-length PthA as well as the COOH-terminal 200 amino











acids of PthA. There were no significant differences in binding affinities among these

three aptamers. Two of the aptamer sequences were cloned into pBI221. The two

aptamer constructs were YPASYMQ (pGZ7.5) and HPYTFLN (pGZ7.6). Both were

separately cloned into Agrobacterium binary vectors with and without pthA. They were

transferred into Agrobacterium tumefaciens strain GV2260, and inoculated into

California light-red kidney bean, sweet orange and tomato leaves. The results showed

that the YPASYMQ aptamer had a strong effect to block the Hypersensitive Response

(HR) normally elicited by pthA expressed in beans and canker elicited by pthA expressed

in citrus. The HPYTFLN aptamer had a reduced, but significant effect. In tomato,

neither aptamer reduced the HR symptom elicited by expression ofpthA in cells. This

result is in consistent with other published reports that the COOH-terminal region of

other members of the pthA gene family are not important for elicitation of the HR in

tomato. These results confirmed that the aptamers exerted their effect by binding to the

COOH-terminal end of pthA, and indicated that aptamers might be used to control citrus

canker disease.


















APPENDIX A
MEDIA AND STRAINS USED



Media


LB:


Bacto tryptone

Bacto yeast extract

NaCI


10 g


5g

5g


pH was adjusted to 7.5 by adding IN NaOH, distilled water was added up

to the final volume of 1 liter, the medium was autoclaved before use.


SOBG:


Tryptone

Yeast extract

NaCl (5 M)

KC1 (1 M)


MgC12 (2 M)

MgSO4 (1 M)


20 g

5g

2 ml

2.5 ml

5 ml

10 ml











Distilled water was added up to the volume of 990 ml, pH was adjusted to

7.0 by adding IN NaOH, the medium was autoclaved and equilibrated to

room temperature. At this point, 10 ml of 1M glucose (18%) was added.

The final volume was 1 liter.


2X YT:


Tryptone

Yeast extract

NaCl


16 g

10 g

5g


pH was adjusted to 7.0 by adding IN NaOH, distilled water was added up

to final volume of 1 liter, the medium was autoclaved.


YEB:


Beef extract

Yeast extract


Peptone

Sucrose

MgSO4


2 mM


pH was adjusted to 5.6 by adding IN HC1, distilled water was added up to

the final volume of 1 liter, the medium was autoclaved and equilibrated to

room temperature. At this point, the following items was added:











MES 10 mM

Acetosyringone 20 [pM

MMA:

MS salt stock (10X) 100 ml

MES 10 mM

Sucrose 20 g

Acetosyringone 200 [pM

pH was adjusted to 5.6 by adding appropriate IN HC1 or IN NaOH,

distilled water was added to the final volume of 1 liter. The medium was

filter sterilized.




Strains


E. coli DH5ca (competent cells):


supE44, AlacU169 (480 lacZAM15), hsdR17, recAl, endAl, gyrA96,

thi-1, relAl

E. coli BL21(DE3)pLysS (fusion protein expression host):

F-, ompT, hsdSB (rB-, mB-), gal, dcm (DE3)pLysS, Cmr 34










E. coli ER2537 (M13 phage host):

F, lacIq, A(lacZ)M15, proA+B+/JhuA2, supE, thiA(lac-proAB), A(hsdMS-

mcrB) 5(rk-mk-McrBC-)

E.coli HB101/pRK2073 (mating helper):

supE44, hsdS20(rB, mB-), recA13, ara-14, proA2, lacY1, galK2, rpsL20,

xyl-5, mtl-1, Spr 50

Agrobacterium tumefaciens GV2260:

C58C1, Rif 75

















APPENDIX B
PURIFICATION OF HIS.TAG PROTEIN (FULL-LENGTH PTHA)



1. pYY50.13 Plasmid Preparation:

E. coli DH5cu/pYY50.13 (pthA full-length gene in pET-19b vector) was streaked

out on LB plates with ampicillin at a final concentration of 100[pg/ml. A single colony

was used to inoculate 2 ml of LB medium with ampicillin at a final concentration of 100

[pg/ml and the inoculated culture was incubated overnight at 37C in a rotary shaker at a

speed of 220 rpm. The bacterial culture was transferred into 1.5 ml microcentrifuge

tubes and centrifuged at 5,000 rpm for 5 min. The pellets were re-suspended in 200p1 of

Buffer P1, 200 p1 of Buffer P2 was added and mixed gently, and the mixture was

incubated at room temperature for 5 min. 200 p1 of chilled Buffer P3 was then added

and mixed immediately; the mixture was incubated on ice for 15 min. The mixture was

then centrifuged at 10,000 rpm for 15 min at 40C and the clean supernatant was

transferred into a fresh 1.5 ml microcentrifuge tube. DNA was precipitated at room

temperature with 0.7 volume of isopropanol for 5 min. The mixture was centrifuged at

10,000 rpm for 15 min at 40C and the supernatant was poured off. The DNA pellet was

washed with 200 pAl of 70% ethanol and the pellet was air-dried for 10 min. pYY50.13











DNA was re-dissolved into 30 p1 of TE (pH 8.0). The quality of plasmid DNA was

tested by agarose gel electrophoresis. The concentration of pYY50.13 DNA was adjusted

to 1 [pg/pl1.

Buffer P1:

Tris-HCl (pH 8.0) 50 mM

EDTA 10 mM

Buffer P2:

NaOH 200 mM

SDS 1%

Buffer P3:

Potassium acetate (pH 5.5) 3.0M

2. Transformation toE. coliBL21(DE3)pLysS:

E. coli BL21(DE3)pLysS competent cells were thawed on ice and of 50 p11

transferred to a 1.5 ml microcentrifuge tube and mixed with 1 p11 of pYY50.13 DNA on

ice for 10 min without shaking. The mixture was heat shocked for 1 min at 420C. The

cells were put back on ice for 5 min. One ml of SOBG medium was added, and the

mixture was incubated at 370C in a rotary shaker at a speed of 220 rpm for 45 min. 150

1il of transformed culture was spread on LB agar plates with carbenicillin at a final

concentration of 50[pg/ml and chloramphenicol at a final concentration of 34 [pg/ml, and

the plates were inverted and incubated at 370C overnight.











3. Expression ofE. coli pYY50.13/BL21(DE3)pLysS (PthA full-length protein):

A single colony from the above transformants was inoculated in 2 ml of LB broth

with carbenicillin at a final concentration of 50 [pg/ml and chloramphenicol at a final

concentration of 34 [pg/ml as a starter culture and incubated in a rotary shaker at a speed

of 220 rpm at 370C overnight. 500 pil of the overnight culture was diluted 1:100 into 50

ml of LB with 50 [pg/ml carbenicillin and 34 [pg/ml chloramphenicol in a 250 ml flask,

and continuously incubated in a rotary shaker at a speed of 220 rpm for 3 to 5 hours at

370C till OD600=0.5. For incubation, 50pl of 100mM IPTG was added to 50 ml of early-

log-phase bacterial culture (the final concentration of IPTG was 0. 1mM) and the culture

was incubated in a rotary shaker at a speed of 220 rpm at 280C for 3 hr. The bacterial

culture was centrifuged at 7,000 rpm at 40C for 10 min. The pellet was washed once with

IX Binding Buffer. The cells were re-suspended in 5 ml of IX Binding Buffer. The

suspensions were kept on ice.

IX Binding Buffer:

Imidazole 5 mM

NaCl 0.5 M

Tris-HCl (pH 7.9) 20 mM

4. His.Bind Resin Preparation:

His.Bind Resin (Novagen) was gently mixed by inversion until the slurry was

completely suspended. The slurry was transferred into a 10 ml column (1cm in

diameter). The storage buffer (with 20% ethanol) was completely removed, and the











remaining resin occupied about 1 ml of settled bed volume. The column was washed,

charged and equilibrated with 3 volumes of sterile deionized water, 5 volumes of 1X

Charge Buffer and 3 volumes of IX Binding Buffer, respectively.

IX Charge Buffer:

NiSO4 50 mM

5. PthA Cell Extract Preparation:

0.1% NP-40 and ImM PMSF was added to the chilled cell suspension in 1X

Binding Buffer. The mixture was kept on ice for 10 min. The cell mixture was sonicated

with SONICATOR (Model W-225) (% duty cycle=50, out put control=6, pulsed cycle)

for 60 seconds in a ice bath until the sonicated solution was no longer viscous. The

sonicated mixture was kept on ice. The lysate was centrifuged at 12,000 rpm for 20 min

at 40C. The supernatant was transferred to a fresh tube and filtered through a 0.45 micron

membrane. About 5 ml of prepared crude extract was collected and kept on ice until the

next step.

6. His.Tag Column Chromatography to Purify PthA:

The IX Binding Buffer was allowed to drain to the top of the column bed. 5 ml

of prepared crude extract was loaded on the top of the column and the column was

adjusted to a flow rate of about 10 column volumes per hour. When all cell extract had

passed through, the column was washed with 10 volumes of IX Binding Buffer and 6

volumes of IX Wash Buffer. The bound PthA was finally eluted with 6 volumes of 1X

Elute Buffer. The PthA eluate (about 5ml) was collected in a fresh tube which was kept











on ice. The column was cleaned with 5 volumes of 1X Strip Buffer. Finally, the column

was washed with deionized water until equilibrium was achieved.

IX Wash Buffer:


Imidazole


NaCl


Tris-HCl (pH 7.9)


60mM

0.5M

20mM


IX Elute Buffer:


Imidazole


NaCl


Tris-HCl (pH 7.9)


1X Strip Buffer:

EDTA

NaCl

Tris-HCl (pH 7.9)


7. Dialysis of PthA:


The PthA column eluate (about 5 ml) was transferred into SPECTRAPOR

membrane tubing which was dialyzed against 50 mM NH4HCO3 at 40C with gentle

agitation overnight. The dialysis solution was changed in every 2 hrs.

8. Lyophilization of PthA:


0.5M


20mM


100mM

0.5M

20mM











The dialyzed PthA was pipetted out of the dialysis bag and put into Falcon 14 ml

centrifuge tubes. The protein sample was frozen at -70C overnight. The dialyzed PthA

was freeze-dried in a SENTRY Freeze Dryer overnight.

9. SDS-Polyacrylamide Gel Electrophoresis of PthA:

The freeze dried PthA was re-suspended in 200 ptl of IX Treatment Buffer, The

mixture was boiled for 10 min and then centrifuged at 10,000 rpm for 10 min. Only the

upper 80% supernatant of treated samples was pipetted out for electrophoresis loading.

IX Treatment Buffer:

Tris-HCl (pH 6.8) 0.125 M

SDS 4%

Glycerol 20%

DTT 0.2 M

Bromophenol Blue (pH 6.8) 0.02%

Protein samples were separated by SDS-PAGE, using an 8% separating gel and

4% stacking gel.

8% Separating gel:

distilled water 16.37 ml

1.5 M Tris-HCl (pH 8.8) 8.75 ml

30% Acrylamide/Bis 9.3 ml


10% SDS


350 ptl











10% Ammonium persulfate


TEMED

4% Stacking gel:

distilled water

0.5 M Tris-HCl (pH 6.8)

30% Acrylamide/Bis

10% SDS


10% Ammonium persulfate

TEMED


210 1l


21 Itl


8.54 ml

3.5 ml

1.82 ml

140 1l


75 p 1


20 I1


30% Acyrlamide/Bis (30%T, 2.67%C) stock:


Acrylamide

N, N'-bis-methylene-acrylamide


29.2 g

0.8 g


(Distilled water was added up to 100 ml. The solution was stored at 40C

in dark.)

SDS-PAGE was run at 100 V for 5 hr. After gel electrophoresis, the gel slice was

soaked into 200 ml of ice-cold 0.2 M KC1. The gel slice was kept on ice for 2 min.

Proteins appeared as white pale colored bands on the gel slice. A razor blade was used

to cut the 130 kD protein band out and the recovered gel slice was chopped into small

pieces.











10. Gel Elution of PthA:

The recovered gel slices were loaded into Model 422 Electro-Eluter (Bio-Rad)

assembly. The gel slices were washed with distilled water for 5 min to eliminate excess

KC1. PthA was electroeluted at 10 mA per tube for 8 hours. The Protein Elution Buffer

without SDS was changed at this point and electrophoresis was continued for an

additional 30 min to remove SDS in the protein sample.

Protein Elution Buffer:

Tris base 25 mM

Glycine 192 mM

SDS 0.1%

The PthA eluate was collected in a volume of 700 pl, which was transferred into

SPECTRAPOR membrane tubing and dialyzed against 50mM NH4HCO3 at 4C

overnight with gentle agitation. The dialyzed PthA electro gel eluate was freeze-dried

overnight.

11. PthA Quantification by UV Spectrophotometry:

PthA was re-dissolved in 100 pAl of 0.1M NaHCO3 (pH 8.6), and 10 pAl was

diluted 1:30 in 300 ptl of 0.1M NaHCO3 (pH 8.6). The optical density was measured in a

Gilford spectrophotometer. The OD280 was 0.3363, the OD260 was 0.3286, the A280/A260

ratio was 1.023. The concentration of electro gel purified PthA was caculated by A280 X

Correction Factor = 0.3363 X 0.81 = 0.27 mg/ml. The original concentration of PthA in

0.1M NaHCO3 (pH 8.6) was 8.1 mg/ml. PthA were stored at -200C.

















APPENDIX C
PURIFICATION OF GST FUSION PROTEIN (PTHA C-TERMINAL 200AA
TRUNCATED PROTEIN)



1. pGNLS-3-2 Plasmid Preparation:

E. coli DH5cL/pGNLS-3-2 (pthA C-terminal 600bp truncated gene fused with GST

in pGEX-4T-3 vector) plasmid DNA was prepared as described in Appendix B1.

2. Transformation toE. coli BL21(DE3)pLysS:

E. coli BL21(DE3)pLysS competent cells were prepared as described in

Appendix B2.

3. Expression of E. coli pGNLS-3-2/BL21(DE3)pLysS (PthA C-terminal 200aa

Truncated Protein):

E. coli pGNLS-3-2/BL21(DE3)pLysS cells were induced as described in

Appendix B3, except that the culture medium was 2X YT medium, the cells were washed

and resuspended in 1X PBS.

iX PBS (pH 7.3):

NaCl 140 mM

KC1 2.7 mM











Na2HPO4 10 mM

KH2PO4 1.8 mM

4. Preparation of Glutathione Sepharose 4B:

The bottle of Glutathione Sepharose 4B (Pharmacia Biotech) was gently shaken

to resuspend the matrix. The matrix was washed with IX PBS. A 50% Glutathione

Sepharose 4B slurry was prepared in a bed volume of 50 pl. A micropipette was used to

remove 100 p1 of slurry and the slurry was transferred to a 1.5 ml microcentrifuge tube.

The matrix was sedimented by centrifugation at 500 x g for 5 min. The supernatant was

carefully removed and decanted. The matrix was washed again with 700 p|l of cold 1X

PBS, inverted to mix, and centrifuged at 500 x g for 5 min. Again, the supernatant was

discarded. The matrix was kept at 40C ready for use.

5. GST Fusion Cell Extract Preparation:

The truncated PthA::GST fusion protein was prepared as described in Appendix

B5.

6. Glutathione Sepharose 4B Batch Purification of GST Fusion:

One ml of the prepared cell extract was added to the Glutathione Sepharose 4B

affinity matrix in a 1.5 ml microcentrifuge tube; the tube was slowly inverted to mix with

gentle agitation at 40C for 30 min. The slurry was centrifuged at 500 x g for 5 min and

the supernatant was carefully removed. One ml of cold IX PBS was added and inverted

to mix with gentle agitation at 40C for 10 min. The mixture was centrifuged at 500 x g

for 5 min and the supernatant was carefully removed. The washing steps was repeated











twice. One ml of Glutathione Elution Buffer was added, the tube was inverted to mix

with gentle agitation at 40C for 30 min. The slurry was centrifuged at 500 x g for 10 min.

The supernatant was kept on ice as "glutathione protein elute". The rest of Glutathione

Sepharose 4B matrix should be washed by 1X PBS at least three times to clean up.

Glutathione Elution Buffer:

Reduced glutathione 10 mM

Tris-HCl (pH 8.0) 50 mM

7. Dialysis of GST Fusion Protein:

The total Glutathione eluate (about 5 ml) was transferred into SPECTRAPOR

membrane tubing and dialyzed against 50mM NH4HCO3 at 4C overnight. Fresh

solution was changed every two hours. The solution was agitated at very low speed.

8. Lyophilization of GST Fusion:

The dialyzed Glutathione eluate was freeze-dried as described in Appendix B8.

9. SDS-PAGE of GST Fusion Protein:

The freeze-dried Glutathione protein sample was electrophoresed as described in

Appendix B9. The 55 kD protein band was recovered.

10. Gel Elution of GST Fusion Protein:

The GST fusion protein was electro-eluted as described in Appendix B10.

11. Protein Quantification by UV Spectrophotometry:

The concentration of PthA C-terminal 200aa truncated protein in 0.1M NaHCO3








79


(pH 8.6) was 10.0 mg/ml, determined as described in Appendix Bl1. The GST fusion

protein sample was stored at -20C.






















APPENDIX D
PHAGE DISPLAY PEPTIDE LIBRARY BIOPANNING SCREENING OF
APTAMERS



Phage Display Biopanning

1. A solution of 100 [lg/ml of the target in 0.1M NaHCO3 (pH 8.6) was prepared. There

were two targets: PthA full-length protein (His.Tag) and the PthA C-terminal 200aa

truncated protein (GST fusion protein). Also, a solution of 100 [lg/ml of Glutathione S-

transferase (GST) was prepared as a pre-absorb target in 0.1 M NaHCO3 (pH 8.6).

2. 150 |pl of each target was added to two separate microtiter wells. In a separate well,

150 |pl of GST was added. The microtiter plate was swirled repeatedly until the surface

was completely wet. The plate was incubated at 40C overnight with gentle agitation

within a humidified container.

3. E. coli ER2537 stock culture was inoculated into 10 ml of LB medium, and incubated

with vigorous shaking at a speed of 220 rpm at 370C overnight. The overnight culture

was 1:100 diluted into 100 ml LB, and continuously incubated at 370C in a rotary shaker

at a speed of 220 rpm for 3 hr until the OD600 reached 0.5.

80











4. The coating solution from each well was poured off and the plate was firmly slapped

face-down onto a clean paper towel to remove residual solution. Each plate or well was

completely filled with Blocking Buffer and incubated at least 1 hour at 40C with gentle

agitation.

Blocking Buffer:

NaHCO3 (pH 8.6) 0.1 M

BSA 5 mg/ml

NaN3 0.02%

5. The Blocking Buffer was discarded by shaking. The well was washed 6 times with

TBST at room temperature.

TBST:

Tris-HCl (pH 7.5) 50 mM

NaCl 150 mM

Tween-20 0.1% (v/v)

6. 2 x 1011 M13 phage from the original library (New England BioLabs, Ph.D.-7T

Phage Display Heptapeptide Library, 2 x 1013 pfu/ml) was diluted by 1 ml of TBST. The

diluted phage were pipetted onto coated plates and rocked gently for 60 min at room

temperature. For PthA full-length protein (His.Tag) screening, the diluted phage was

directly added. For the GST fusion protein screening, the diluted phage was first added

to the GST coating well and incubated for 60 min, then the pre-absorbed phage was











transferred to the GST fusion protein coating well and incubated for another 60 min at

room temperature. Such a pre-absorbing step was proved to be very efficient to eliminate

GST non-specific binding background.

7. The non-binding phage was discarded and the plate was washed 10 times with 1X

TBST.

8. The bound phage were eluted with 100 Ipl of free target solutions, i.e., 100 [lg/ml of

PthA full-length protein (His.Tag) in TBS and 100 [lg/ml of GST fusion protein in TBS,

respectively. The plates were rocked gently for 60 min at room temperature. The eluate

was transferred to a 1.5 ml microcentrifuge tube.

TBS:

Tris-HCl (pH 7.5) 50 mM

NaCl 150 mM

9. One Ipl of elute phage was titered based on the following steps. The remaining phage

elute was stored at 40C.

10. The elute phage was amplified till the titer up to 2 x 1013 pfu/ml.

11. The biopanning steps were repeated twice. The only difference was changing the

Tween-20 to 0.5% in washing steps after second round phage display biopanning. For

GST fusion protein, each round of selection was pre-absorbed by GST.











Phage Amplification

1. 10 ml of LB medium was inoculated with a single colony of E. coli ER2537 and was

shaken vigorously in a rotary shaker at a speed of 220 rpm at 370C overnight. The

overnight starting culture was diluted 1:100 into 100 ml of LB medium. The dilute

bacterial culture was incubated at 370C for 3 hr until the OD600 reached 0.5.

2. One microliter of phage eluate was added into 20 ml of early-log-phase E. coli

ER2537 culture, transferred to a 250 ml flask, and incubated at 370C in a rotary shaker at

a speed of 220 rpm for 4.5 hr.

3. The culture was centrifuged at 10,000 rpm for 15 min at 40C. The upper 80%

supernatant was transferred to a fresh tube and re-centrifuged briefly.

4. The upper 80% of the supernatant was pipetted out to a fresh tube and 1/6 volume of

PEG/NaCl was added. The M13 phage was precipitated at 40C overnight.

PEG/NaCl:

Polyethylene glycol-8000 20% (w/v)

NaCl 2.5 M

5. The precipitation mixture was centrifuged at 10,000 rpm for 20 min at 40C. The

supernatant was discarded. The residual supernatant was carefully removed by a pipette.

6. The pellet was re-suspended in 1 ml of TBS. The suspension was transferred to a 1.5

ml microcentrifuge tube and centrifuged at 10,000 rpm for 5 min at 40C to remove the

residual cells.











7. The supernatant was transferred to a fresh tube and re-precipitated by adding 1/6

volume of PEG/NaC1. The precipitation mixture was incubated on ice for 60 min then

centrifuged at 10,000 rpm for 10 min at 40C. The supernatant was discarded and the

residual supernatant was carefully removed by a micropipette.

8. The pellet was resuspended in 200 Ipl of TBS with 0.02% NaN3. The suspension was

centrifuged at 10,000 rpm for 5 min to pellet any remaining insoluble matter. The

supernatant was transferred to a fresh tube. This was the amplified phage.

9. The amplified phage was titered according to the next step. All the eluate were stored

at 40C.




Phage Titer

1. Ten ml of LB medium was inoculated with a single colony of E. coli ER2537 and

incubated in a rotary shaker at a speed of 220 rpm at 370C overnight. The overnight

culture was 1:100 diluted into 100 ml LB medium and continuously incubated at 370C

for 3 hr to reach OD600=0.5.

2. Agarose Top Agar was melted in a microwave oven and 3 ml of melted top agar was

dispensed into a 15ml Falcon centrifuge tubes (one tube for one phage dilution). All the

tubes were equilibrated at 450C water bath ready for use.

Agarose Top Agar:


Bacto-tryptone


10 g











Yeast extract 5 g

NaC 5 g

MgC12.6H20 1 g

Agarose 7 g

pH was adjusted to 7.5, H20 was added up to volume of 1 liter, the

medium was autoclaved and stored in room temperature.

3. LB agar plates were pre-warmed at 370C (one plate per phage dilution).

4. Ten-fold serial dilutions of M13 phage were prepared in LB medium. The dilution

range was from 101 to 1012

5. 200 p1 of mid-log E. coli ER2537 culture was transferred into a 1.5 ml

microcentrifuge tube (one tube for one phage dilution).

6. 10 ptl of each phage dilution was pipetted to each microcentrifuge tube with bacteria,

mixed quickly and incubated at room temperature for 1 min (The infection time was very

critical).

7. The infected cells were transferred to the 450C pre-warmed Agarose Top agar tube,

mixed well, and were immediately poured onto a 370C pre-warmed LB agar plate. Top

agar was spread evenly by tilting plate for several times.

8. The plate was cooled at room temperature for 5 min, inverted and incubated at 370C

no longer than 18 hours. Longer incubation may cause deletion of the phage sequence.

9. The plates were inspected and the phage plaques was counted. A good range of











dilution was the plate having about 100 plaques. The dilution factor was multiplied to get

phage titer in plaque forming units (pfu) per ml.




Characterization of Binding Clones

1. 10 ml of LB medium was inoculated with a single colony of E. coli ER2537 and the

inoculated culture was incubated in a rotary shaker at a speed of 220 rpm at 370C

overnight. The overnight starting culture was diluted 1:100 into 100 ml of fresh LB

medium, and continuously incubated at 370C for 3 hr to reach OD600=0.5.

2. One milliliter of early-log-phase bacterial culture was transferred into 15 ml test tubes

(one for each clone to be characterized).

3. The phage titer plate with about 100 plaques was chosen. A sterile wooden stick was

used to stab each plaque and transfer to the bacterial culture tube.

4. The test tube with bacteria and phage mixture was incubated at 370C in a rotary shaker

at a speed of 220 rpm for 4.5 hr.

5. The culture was transferred to a 1.5 ml microcentrifuge tube and centrifuged at a

speed of 10,000 rpm for 1 min. The upper 80% of the supernatant was transferred to a

fresh tube and re-centrifuged for an additional 1 min. The upper 80% of the supernatant

was transferred to another fresh tube.

6. 300 ptl of the upper 80% of the supernatant was diluted 1:1 with 100% glycerol and

stored at -200C as the phage stock. It could be re-amplified as necessary.











7. The rest of 500 p1l of upper 80% supernatant was transferred to a fresh 1.5 ml tube.

8. 200 p.1 of PEG/NaCl was added, the tube was inverted several times to mix well, and

incubated in room temperature for 10 min.

9. The mixture was centrifuged at 10,000 rpm for 10 min and the supernatant was

discarded.

10. The pellet was re-suspended in 100 p1l of Iodide Buffer and 250 p1l of 95% ethanol

was added later. The mixture was incubated at room temperature for 10 min.

Iodide Buffer:

Tris-HCl (pH 8.0) 10 mM

EDTA 1 mM

Nal 4M

11. The mixture was centrifuged at 10,000 rpm for 10 min at 40C. The supernatant was

discanted. The pellet was carefully washed by 70% ethanol and dried by a vacuum.

12. The pellet was re-suspended in 30 p1l of TE (pH 8.0).

13. The single-stranded M13 phage DNA was tested by 0.7% agarose gel

electrophoresis.

14. 10 p1 of each sample was enough for DNA sequencing. The DNA sequencing

primer was -28gIII (5'-GTATGGGATTTTGCTAAACAAC-3') which was synthesized

by University of Florida ICBR DNA Synthesis Core Facility.


















APPENDIX E
IN VITRO BINDING AFFINITY OF APTAMERS ELISA



1. Host strain E coli ER2537 was streaked out in a LB agar plate.

2. A single colony was inoculated to 2 ml of LB medium as starting culture. The culture

tube was incubated in a rotary shaker at a speed of 220 rpm at 370C overnight.

3. The overnight starting culture was diluted 1:100 into 20 ml of LB medium in a 250 ml

flask, incubated in a rotary shaker with a shaking speed of 220 rpm at 370C for about 3

hr, until the OD600 reached 0.5 (early-log-phase).

4. 1 1l of M13 amplified phage stock (phage upper 80% supernatant 1:1 mixed with

100% glycerol, stored at -200C) was transferred to the 20ml of early-log-stage E. coli

ER2537 culture, and the inoculated culture was incubated in a rotary shaker with a

shaking speed of 220 rpm at 370C for 4.5 hr.

5. The culture was transferred to a Falcon centrifuge tube and centrifuged at 10,000 rpm

at 40C for 10 min. The upper 80% of the supernatant was transferred to a fresh tube and

re-centrifuged at 10,000 rpm at 40C for 5 min.

6. The upper 80% supernatant was pipetted out to a fresh tube and 1/6 volume of

PEG/NaCl was added to the mixture. The M13 phage was precipitated overnight at 40C.




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