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CHARACTERIZATION OF THE ZPS1P CELL WALL PROTEIN FROM
STEPHANIE L. DROBIAK
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
Stephanie L. Drobiak
This document is dedicated to my fiance and my family for all their help and support
during the last few years.
I would like to thank my future husband and my family for their moral support,
my lab mates for their continuous help and friendship, and my mentor, Dr. Thomas
Lyons, for his endless guidance and patience.
TABLE OF CONTENTS
ACKNOW LEDGM ENTS ........................................ iv
LIST OF FIGURES ...................................... vi
ABSTRACT.................. .................. vii
1 INTRODUCTION ................................................. ..............
Zpslp-like Proteins from Candida albicans and Aspergillus spp. ...................... 2
Zpsip from Saccharomyces cerevisiae............... ................... 10
Zinc-dependent Metalloproteases of the M35 Clan....................... ........ 12
Comparison of the Zpslp-like Proteins and the M35 Metalloproteases............ 14
2 RE SU LTS AN D D ISCU SSION ................................................................. ......16
Regulation of ZPSI Gene Expression........................... .............. 16
Partial Purification of Zpslp from Inclusion Bodies .......................................... 17
4 M ATERIALS AND M ETHODS................................ .................. 27
Growth Media................. ... .. ..... ................27
Solutions and Buffers for Yeast Transformations and P-Galactosidase Assays... 28
Bacterial and Yeast Strains ................................. ......................... .. .... 29
Y east T ransform nations ....................................................................... 30
P-G alactosidase A ssays................... ... .................................... 3 1
Cloning of ZPS1 and Construction of an E. coli Expression Plasmid.................. 32
Expression of Zpslp in E. coli..................... ............ .............. 33
Estimation of Protein Purity by SDS-PAGE ............... ..... ................. 34
LIST OF REFERENCES ....................... ......... ........35
BIOGRAPHICAL SKETCH .................................................. ............... 39
LIST OF FIGURES
1-1. Multiple sequence alignment of fungal cell wall proteins with related
1-2. Active-site residues of deuterolysin.43........... ............................ ............ 13
1-3. Basic structural features of the Zpslp-like proteins and the metalloproteases in the
M 35 clan....................................... ................................ ......... 14
1-4. Active site structures. On the right is the known active site of the aspzincins,
deduced from the crystal structures of deuterolysin43 and G/MEP.49 On the left is a
possible structure of an active site within the Zpslp-like proteins. .......................15
2-1. Zinc and iron responsiveness of the ZPS]-lacZ reporter. P-Galactosidase activity in
wild-type cells and zap] mutant cells grown in CSD. ..........................................16
2-2. Zinc and iron responsiveness of the ZPS]-lacZ reporter. 3-Galactosidase activity in
wild-type cells and riml01 mutant cells grown in CSD. .......................................18
2-3. SDS-PAGE analysis of E. coli transformants containing the pET-22b(+)-ZPS]
expression vector ............. ................ ........ .........19
2-4. SDS-PAGE analysis of soluble and insoluble components of the cell lysate obtained
from breakage of E. coli expressing Zpslp.................. .......................20
2-5. SDS-PAGE analysis of the soluble and insoluble products obtained after
solubilization and refolding of the inclusion body pellet...................................20
2-6. SDS-PAGE analysis of the major protein peak collected after SEC (combined
fractions 9 12)..................................... ......... 21
2-7. SDS-PAGE analysis of the washed inclusion body pellet, solubilized in Buffer A
containing 8 M Urea. .......... ... .. ........................23
2-8. SDS-PAGE analysis of the soluble and insoluble products obtained after
solubilization and refolding of the inclusion body pellet................ .................24
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
CHARACTERIZATION OF THE ZPS1P CELL WALL PROTEIN FROM
Stephanie L. Drobiak
Chair: Thomas Lyons
Major Department: Chemistry
Fungal cell wall proteins are involved in establishing infection through interaction
with host ligands and by mediating morphological changes that enhance pathogenicity.
In recent years, research has focused on a family of fungal cell wall proteins that are
structurally related to zinc-dependent metalloproteases of the M35 clan. Members of this
protein family include Zpslp from Saccharomyces cerevisiae, Pral from Candida
albicans, CpAspf2 from Coccidioides posadassii, Aspndl from Aspergillus nidulans, and
Aspf2 from Aspergillusfumigatus. The proteins from C. albicans and Aspergillus spp.
are known cell-surface antigens during fungal infections, and both Pral and Aspf2 bind
specific ligands within mammalian hosts. Although expression of these proteins during
fungal infection is well documented, their biological function remains unknown. In this
thesis, we report preliminary work toward characterization of Zpslp from S. cerevisiae.
Results indicate expression of ZPSI to be regulated in response to zinc- and iron-
limitation, as well as extracellular pH. In addition, we present the partial purification of
recombinant Zpslp from bacterial inclusion bodies. Analysis of Zpslp is intended to
provide the framework for future expression, purification, and characterization of the
Zpslp-like proteins from the medically important fungi C. albicans and Aspergillus spp.
Many fungi are responsible for both superficial and systemic infections in man.
Immunocompromised individuals are susceptible to fungal infections caused by a variety
of pathogens, including Candida albicans and several Aspergillus species. Relevant
diseases caused by these species include candidiasis, aspergilloma, invasive aspergillosis,
and allergic bronchopulmonary aspergillosis (ABPA). Although these mycoses are well
documented, many factors contributing to fungal pathogenesis are still not well
understood. Efforts to better understand virulence factors often focus on components of
the fungal cell wall.
The fungal cell wall is a complex mixture of carbohydrates (80 to 90%), proteins
(6 to 25%), and minor amounts of lipid (1 to 7%).1 As the outermost part of the cell, the
wall initiates physical interaction between the microorganism and the environment,
including the host. The host-parasite interaction, resulting in adhesion, is the first critical
step in establishing infection and modulation of the host immune response. In addition,
the cell wall mediates fungal cell-cell adhesion flocculationn), a first step in the
morphological change from a unicellular yeast to growth as multicellular filaments
(mycelia or hypha). Formation of mycelia enhances pathogenicity, allowing the invasion
of host tissues, and is influenced by environmental variables including extracellular pH2'3
and nutritional status.4 For these reasons, fungal cell wall proteins (CWPs) have been of
heightened interest. Not only are CWPs involved in intercellular binding, many possess
enzymatic activity involved in cell wall biosynthesis and maintenance, and acquisition of
extracellular nutrients.1 When the actions of CWPs negatively impact the viability of the
host, the proteins are considered virulence factors that advance the establishment of
infection. Due to their accessibility at the cell surface, and their critical role in
intercellular interactions, CWPs are ideal targets for the development of antifungal drugs.
A family of cell wall proteins from various fungi has been the focus of much
research in recent years. Members of this family include Zpslp from Saccharomyces
cerevisiae, Pral from Candida albicans, CpAspf2 from Coccidioides posadasii, and
Aspndl and Aspf2 from Aspergillus nidulans and Aspergillusfumigatus, respectively.
These CWPs share a number of key structural features, have high sequence homology,
and exhibit significant similarity to a family of zinc-dependent metalloproteases of the
M35 clan, known as the aspzincins5 (Figure 1-1). At present, the biochemical function of
these proteins remains unknown.
The focus of this research is to characterize the Zpslp cell wall protein from the
yeast Saccharomyces cerevisiae. This document entails the preliminary work toward
characterization of Zpslp through study of its structure, function, and gene regulation in
S. cerevisiae. Analysis of Zpslp is intended to provide the framework for future
expression, purification, and characterization of the Zpslp-like proteins from the
medically important fungi C. albicans (Pral) and Aspergillus spp. (Aspndl and Aspf2).
The relevance of the Zpslp-like proteins is discussed below.
Zpslp-like Proteins from Candida albicans and Aspergillus spp.
The homologues from A. fumigatus and A. nidulans are known as Aspf26 and
Aspndl,7 respectively. In C. albicans, the homologue is known by many names: Pral
(pH regulated antigen),8 FBF (fibrinogen binding factor),9 FBP1 (fibrinogen binding
protein),10 and mp58 (58-kDa fibrinogen-binding mannoprotein).11
NPII Secondary Structure NX(
ellp MKFSSGKS l FATIASLALSAPVTY-. T Y .- t. FI T -1
SPutetu eC teavame SIcu t- F5
T T -
SNxT = putting Nzlyryslation rite PVY Y rD Y N P I HG DL -
Zn=Zinligand i n -
XMiCUM important active siresidue VVNG E
XC TX Boxing/Shading = 45% identity MVNG i
Boldboxing=invarriantritkilreidue -M S ;AJ L A
p disulfidebond AT FVG SA ROTLNA SO NIA
afinep Secondary Structure -
-a- -p- --- -a-
1p r NYGVDDVYYIKR F 3ANGS IFTVMG VFE QLMEASKGA LMR D I01 A A N GHHROSAA P TV D Y 5E TTS K
1 'EET I
-a- "B l -[ -
RIC 33 So Y F I T T t. .AL
ITa a UK IR KVY V R E
r= r1e TTT IA GDG TA I
3 r. A I
--c --a- -P- -P- -- t -a
XAnt arrd etght c e ei ir
-L 1LA3 NCLO1
te i O- g t o i a io .lo o i A LK [ N Q
Apnd1 33lacks [J F a Thr- Nich -- egiIon.. H v t C-t regio o .Ap:
T.a S E N T i I
bchemal ca r funcT iF or all thets excep Zprthe m rep hant d
Asergillus ai o n ag ng-------aie- OT
AA rre N
function.TTG- T 1 - - - r . . .
A 1s D S- a- -How r th C-tri ri o Asp
ioc h mica l Sf o orrQ Rih Ceil Wall Bindingt Domain Metap Bindinf Site?
As. rI ll -- I.m. .d m .. i fTn i
N Multiple sequence alignment of funga I antigens with related metalloproteases.
WXAEB i7e N For all proteins except Zps Ip the mature peptide is shown.
gr1p i7 s Secondary structure based on crystal structures for NPII (top) and gfmep (bottom).
Figure 1-1. Multiple sequence alignment of fungal cell wall proteins with related
Aspf2, Aspndl, and Pral are all secreted proteins with four N-glycosylation sites (Asn-
X-Ser/Thr) and eight cysteine residues perfectly conserved, suggesting a similar
function.6711 Both Pral and Aspf2 contain a serine- and threonine-rich region of
potential 0-glycosylation close to the C-terminus, a purported cell wall binding domain
(CWBD).12 Studies have shown that Pral does indeed contain O-linked sugar moieties."
Aspndl lacks this Ser- and Thr- rich region. However, the C-terminal region of Aspndl
is glutamate- and glutamine-rich, the purpose of which is unclear. Although their
biochemical function remains unknown, these three proteins from Candida and
Aspergillus are immunodominant antigens in fungal infections.
Purification and characterization of various Aspergillus spp. cell wall proteins for
use in immunodiagnosis of ABPA and other diseases led to the identification of Aspf2
and Aspndl as proteins that elicit a strong immune response.6'7'13'14 These discoveries
were made by immunoblotting water-soluble extracts of Aspergillus spp. with sera from
patients with ABPA. Sera from those infected with different forms of aspergillosis
contain elevated levels of immunoglobulin G (IgG) and immunoglobulin E (IgE)
antibodies specific for Aspergillus antigens, including Aspndl and Aspf2. These
antigens are consistently recognized by serum samples from aspergilloma patients, but
not with sera from control or healthy individuals.7'14 Deglycosylated forms of the
purified proteins remain reactive to the antibodies, suggesting the N-glycosidic groups
are not required for recognition by the aspergillosis serum samples tested. These data
indicate that the epitopes recognized are located mainly in the polypeptide region.7;13
This hypothesis is further supported by antibody reactivity with the recombinant forms of
Aspndl and Aspf2 which, when over-expressed in the prokaryote E. coli, lack the
glycosidic moieties. 1516 Furthermore, all reactivity was abolished following protease
During characterization of the Aspndl antigen from A. nidulans, Calera et al.
observed sera that reacted with Aspndl also consistently reacted with antigens from A.
fumigatus. Therefore, they tested the reactivity of purified anti-Aspndl specific IgG with
several different A. fimigatus antigens including Aspf2 (also known as gp55). The
various antigens reacted with the anti-Aspndl, suggesting a close relationship between
the immunodominat antigens from the two Aspergillus species, possibly through the
existence of common peptide epitopes.7 Likewise, Banerjee et al. detected binding of
anti-Aspf2 antibodies by Aspndl. The relatedness of Aspndl and Aspf2 is supported
through analysis of their primary structure. The similarity between the two proteins
suggests they share several epitopes and should therefore elicit the formation of IgG and
IgE antibodies able to recognize both antigens. To answer this question, Banerjee et al.
compared the reactivity of purified Aspndl and Aspf2 toward IgE antibodies from ABPA
serum. The mean IgE binding of purified Aspf2 was almost three fold higher than
binding of Aspndl. Differences in Aspnd and Aspf2 binding to IgE may result from
differences in posttranslational modifications or the tertiary structures of these proteins.6
Comparison of IgE binding by both native and recombinant forms of these antigens
indicates that the recombinant forms most likely have structures functionally comparable
to the native proteins, since IgE binding is dependent on proper three-dimensional
Similarly, Pral from C. albicans elicits a strong immune response with sera from
patients infected with candidasis. Antibodies against Pral are present in sera from
patients with systemic candidasis,17;18 and Pral itself has been detected in the cell wall of
clinical isolates of C. albicans.17 Deglycoslyation of Pral did not affect reactivity with
anti-Pral antibodies, again suggesting the epitopes recognized are located mainly in the
polypeptide region. An extensive epitope-scanning study, employing a complete set of
overlapping dodecapeptides deduced from the Pral sequence, identified several
immunoreactive continuous B-cell epitopes within the protein sequence. Six regions of
elevated reactivity were identified, including four internal regions and both the amino and
carboxy termini of the mature polypeptide. Of these regions, the C-terminal domain was
highly reactive towards the anti-Pral antibodies and therefore subjected to further epitope
Analysis of the epitopic region at the C-terminal domain of the Pral polypeptide
identified the nonapeptide 290HTHADGEVH298 as the minimal region required to retain
antibody-binding activity. Researchers further probed the significance of this epitopic
region by synthesizing a synthetic peptide corresponding to the last ten amino acid
residues at the C-terminus. The synthetic peptide, coupled to keyhole limpet hemocyanin
(KLH), was used to immunize two mice. The serum samples obtained from the two
immunized mice were able to recognize Pral from cell wall extracts of C. albicans with
high specificity. Interestingly, this C-terminal sequence (CHTHxxGxxHC) is conserved
in both Aspndl and Aspf2, as are the four internal epitope regions (Table 1-1). The
conservation of linear epitopes within this family of cell wall proteins from various
fungal pathogens provides additional support suggesting their role in the host-parasite
Table 1-1. Sequence homology of five of the identified IgG epitopes. 18
Pral Antigen Sequence
epitope sequence Aspnd1 Aspf2
LRFGSK LRWGNE LRWGNE
RKYF RKYF RKYF
NDGWAGYW LEGWGGHW LEGWGGHW
DVYA EVYA EAYA
HTHADGEVH HTHEGGELH HTHEGGQLH
Not only do these fungal antigens interact with antibodies within the host, but
Aspf2 and Pral also bind specific host ligands. Fungal adhesion to host cells and tissues
initiates establishment of infection and is considered a potential virulence factor. Aspf2
binds the extracellular matrix protein laminin,6;19 and Pral binds the serum protein
Proteins in the extracellular matrix (ECM) are known to bind to A. fumigatus
conidia (an infectious airborne form of the fungus). Binding of conidia to ECM ligands
is believed to be a crucial first step initiating aspergillosis, and specific recognition of
these ligands may greatly influence pathogenicity.2021 One component of the ECM is
laminin, a multidomain glycoprotein and major component of the basement membrane.
Interaction of laminin with cell surface ligands facilitates cell-cell adhesion, cell
migration and cell differentiation.1 As reported by Banerjee et al., laminin shows a dose-
dependent interaction with Aspf2. Both native and recombinant Aspf2 demonstrate high
binding affinity to laminin, with greater affinity observed by the native protein. With
specific binding to laminin, and significant homology to Pral (which binds fibrinogen),
the involvement of Aspf2 in fungal adherence to the ECM may play an important role in
Blood serum proteins (e.g., serum albumin, transferring, fibrinogen, complement
fragments C3d and iC3b) are additional targets for fungal binding. Interactions of C.
albicans with fibrinogen have been well characterized.1 In 1987, Bouali et al. identified
a fibrinogen binding factor (FBF) on the surface of C. albicans germ-tubes and
mycelium, the fungal forms most often found in infected tissues.9 Five years later, in
1992, Casanova et al. identified this FBF as a 58-kDa fibrinogen-binding mannoprotein
(mp58), which is now known to be Pral. Binding of Pral to fibrinogen is apparently
specific, since binding to other mammalian proteins tested (laminin, fibronectin, C3d,
type IV collagen) was not observed. O-deglycosylated Pral was unable to interact with
fibrinogen, implying this carbohydrate domain may play a role in binding. The in vivo
production of Pral during candidasis and its ability to bind fibrinogen suggest a role in
Another factor supporting an active role of Pral in candidasis is its differential
expression in response to pH.8 The ability of C. albicans to grow and differentiate over a
broad pH range is critical for its survival in a variety of environments and host tissues
(e.g., blood, pH ~ 7.2; vaginal tract, pH ~ 4.5).3 Extracellular pH is an environmental
signal that regulates the yeast-to-mycelia transition in vivo, a morphological change that
greatly enhances invasion of host tissues.2 In C. albicans, gene expression is regulated
by the RimlOlp transcription factor in response to alkaline pH.22 Studies have shown the
C. albicans RimlOlp pH response pathway to be required for several host-pathogen
interactions, and therefore essential for pathogenesis.23 Pral is a RimlOlp target gene
maximally expressed at neutral pH, with no detectable expression below pH 6.0.
However, ambient pH is not the sole factor influencing expression. When cultured in
rich medium (YPD) buffered at pH 7.0, no Pral production was detected. This result
implies partial regulation by nutritional status, a hypothesis that remains to be tested.8
Although the effect of nutritional status on Pral expression has not been assessed,
the nutrient regulation of the Apsergillus spp. antigens has been investigated.
Researchers recognized that production of Aspndl and Aspf2 only occurred when the
fungi are grown in certain conditions, especially in Czapek-Dox (CD) medium (3g
NaNO3, 0.5g MgS04'7H20, 0.5g KC1, 55mg FeS04, Ig KH2PO4, and 30g sucrose per
liter). Therefore, the various components of CD medium were tested to determine which
is influencing antigen production. Elucidation of the regulatory elements responsible for
Aspndl and Aspf2 expression may provide clues to their function and potential roles in
Variations of CD medium were quantitatively and qualitatively tested against a
control medium, AMM (1% glucose, 0.6% NaNO3, 0.052% MgS04, 0.052% KC1, 0.15%
KH2PO4, and traces ofFeS04 and ZnS04), known not to stimulate Aspndl or Aspf2
production under normal conditions. The type and amount of carbon or nitrogen source
did not affect antigen production, nor did addition of iron to the CD medium. However,
addition of [[-molar concentrations of zinc eliminated antigen synthesis in CD, while
removal of zinc from AMM medium induced antigen production. Addition of other
divalent metals (Co2+, Ni2+, CU2+, Ca2+) had no inhibitory effects, with the exception of
Cd2+ and Mn2+ (only slight inhibition). Currently, researchers are attempting to identify
and characterize potential zinc response elements (ZREs) in the promoter regions of
ASPND1 and ASPF2.24 Detected in the promoter region ofASPND] are at least five
potential PacC binding sites.7 PacC is a pH responsive transcription factor in Aspergillus
spp. and is homologous to C. albicans RimlOlp.25 The presence of putative PacC sites
suggests possible regulation of Aspndl expression in response to ambient pH.
Regulation of Aspndl and Aspf2 expression by zinc deficiency may play an
important role in pathogenesis. During infection, the host environment is one of
nutritional limitation. In efforts to starve invading pathogens, part of the acute-phase
response of the human immune system is to redistribute micronutrients like iron and zinc
to the liver.26 Therefore, this regulation may illustrate the role of zinc's nutritional status
as a signal for fungal pathogens of a host environment, initiating transcription of genes
involved in zinc acquisition and transport or commencement of pathogenesis. Bacterial
hemolysins offer precedent for the metalloregulation of virulence factors.27 The partial
regulation of Pral expression by nutritional status may also have important implications
in pathogenesis. The effect of zinc limitation on Pral expression may be worth
investigation, since zinc deficiency has been shown to induce mycelium formation in
several dimorphic yeasts.4:28
Zpsip from Saccharomyces cerevisiae
Within the family of cell wall proteins, the homologue from Saccharomyces
cerevisiae is Zpslp (Yoll54w). Although S. cerevisiae is typically nonpathogenic, it is
able to infect immunocompromised individuals29 and colonize complement factor five-
deficient mice.30 Zpslp is a secreted cell wall protein,31 with two putative N-
glycosylation sites and six cysteine residues conserved with respect to Pral, Aspndl, and
Aspf2. Unlike the homologues from Candida and Aspergillus spp., Zpslp has a
truncated C-terminal region lacking the potential cell wall binding domain and the
conserved antigenicity determinant. Like its related fungal antigens, the function of
Zpslp is also unknown. Disruption of the ZPS1 gene failed to reveal any strong
phenotype and resulted in a viable strain, indicating that Zpslp is non-essential.32
Although the function of Zpslp is unknown, many research groups have provided
information about its regulation. Interestingly, Zpslp expression is regulated by some of
the same factors as the other fungal antigens, including zinc limitation and extracellular
In S. cerevisiae, the transcription factor Zaplp is activated by zinc deficiency.33
DNA microarray data has shown Zaplp regulates expression of 46 genes in S. cerevisiae
under zinc deficient conditions. Of these genes, one of the most heavily induced is ZPS1.
This result was confirmed by measuring zinc-regulation of a ZPS]-lacZ reporter
construct, resulting from fusion of the ZPS1 promoter region (-1000 bp to ATG) to the
lacZ reporter gene. Zaplp activates gene transcription during zinc deficiency by binding
to a zinc response element (ZRE) upstream of the target gene's start codon. The
consensus ZRE recognized by Zaplp is ACCTTNAAGGT. Within the ZPS1 promoter
region are two putative ZREs between -300 and -340 bp upstream of the start codon:
ACCTTCAGGGT (-328 to -318) and ACCCTGAAGGT (-313 to -303). DNA
microarray data indicated that ZPS1 was induced 14 fold by zinc deficiency, while ZPS1-
lacZ fusion constructs were 10 times more inducible.34
Expression of Zpslp is also affected by alkaline pH. This regulation is dependant
on the S. cerevisiae RimlOlp transcription factor, which is homologous to the RimlOlp
and PacC transcription factors of Candida and Aspergillus, respectively. Using the
ZPS]-lacZ construct, researchers observed a 100-fold increase in expression at pH 8
compared to pH 4, while alkaline induction did not occur in a rim]01A strain. In
addition, ZPS1 is more highly expressed in yeast strains harboring a hyperactive allele of
RimlOlp at pH 4.35 This direct regulation of ZPS1 expression by RimlOlp is intriguing,
for not only is RimlOlp structurally similar to Zaplp, but these two proteins also interact
in vivo,36 suggesting they may co-regulate ZPS1 expression.
Potential regulation of ZPS1 by environmental iron status has also been implied in
work studying iron-regulatory systems in yeast.37 In S. cerevisiae, iron homeostasis is
regulated by the Aftlp transcription factor in response to low-iron conditions.38 In
addition, S. cerevisiae contains a homologue of Aftlp, known as Aft2p, which regulates
transcription of many of the same genes as Aftlp during iron deficiency.37 In strains
harboring a hyperactive allele of Aft2p, activation of ZPS1 was increased over 8 fold
when compared to wild type strains. This effect is dependent on Zaplp, suggesting Aft2p
activity affects zinc metabolism.37 The data obtained through study of ZPS1 regulation
further support its similarity to the fungal antigens from C. albicans and Aspergillus spp.
Zinc-dependent Metalloproteases of the M35 Clan
Based on sequence comparison and structural predictions, the Zpslp-like proteins
show similarity to zinc-dependent metalloproteases of the M35 clan5 (known as the
aspzincins). Included in this subfamily of secreted metalloendopeptidases (MEPs) are
deuterolysin (neutral proteinase, NPII, aspzincin) from Aspergillus oryzae,39
penicilloysin (PlnC) from Penicillium citrinum,40 mep20 from both Aspergillus fumigatus
and Aspergillus flavus,41 and the AVR Pi-ta avirulence determinant from Magnaporthe
grisea.42 Many of these species are known pathogens. This protein family is
characterized by a leader sequence directing the protein into the secretary pathway, a
long pro-peptide that is cleaved during secretion, a mature polypeptide that contains three
disulfide bonds, and two highly conserved motifs: HExxH and GTxDDxxYG.43 A
crystal structure of deuterolysin,43 supported by site directed mutagenesis studies,39
indicated the two histidine residues of the HExxH motif, the second aspartate residue of
the GTxDDxxYG motif, and two water molecules were the zinc binding ligands. The
conserved glutamate is a catalytic residue, promoting the nucleophilic attack of a water
molecule on the carbonyl moiety of the substrate. The conserved tyrosine residue
interacts with the second zinc bound water molecule, possibly stabilizing the transition
state by hydrogen bonding interactions.43 Figure 1-2 shows the crystal structure of the
active site residues.
More distantly related members of the M35 clan include GfMEP from Grifola
frondosa, PoMEP from Pleurotus ostreatus,44 AmMEP from Armillariella mella,45 eprAl
from Aeromonas hydrophila,46 asaPi from Aeromonas salmonicida,47 XAC2763 from
Xanthomonas axonopodis, and XCC2062 from Xanthomonas campestris.48 Many of
these species are also pathogenic. Furthermore, AmMEP from the edible mushroom A.
mella is known to hydrolyze fibrinogen.45 The crystal structure of GJMEP has been
solved. Despite one less disulfide bond, GJMEP possesses a near identical fold and
active site as deuterolysin, suggesting a conserved mechanism.49
The substrate specificities of deuterolysin, PlnC, and mep20 are toward basic
polypeptides. Both deuterolysin and PlnC show high activities on the basic nuclear
proteins histone, protamine, and salmine, but very low activities on milk casein,
hemoglobin, albumin, and gelatin.39 Further analysis of deuterolysin's substrate
specificity indicates high proteolytic activity toward the peptide bonds next to pairs of
basic residues.50 GJMEP and PoMEP have strict specificity toward acyl-lysine bonds,
also basic in nature.44 Analysis of the GJMEP structure reveals and electrostatically
negative region that attracts a positively charged lysine side chain of a substrate.49
Comparison of the Zpslp-like Proteins and the M35 Metalloproteases
Although Zpslp and the related fungal antigens possess similarities to the M35
clan (i.e., secretary signal, conserved cysteine residues), they differ in their most highly
conserved motifs. Figure 1-3 illustrates the basic structural features of the Zpslp-like
proteins and the M35 proteases.
Zpsip x Ci M HH ExxE C
Pral,Aspf2 I IZC HRMxH DKOED C CS.T-rigCHi
in the M35 clan.
The Zpslp-like proteins lack the HExxH and GTxDDxxYG motifs found in the
metallo-proteases. However, they contain highly conserved CTRxxH and D/ExxD/E
the metalloproteases in the M35 clan with a possible structure in the Zpslp-like proteins.
e.,AlI Cgta? pr~WiMUp< CLC C HBExH GTxDDlfVGI
XCC2I02 I = leader peptide (scretory signal) ZL2+
Figure 1-3. Basic structural features of the Zpslp-like proteins and the metalloproteases
in the M3 5 clan.
The Zpslp-like proteins lack the HExxH and GTxDDxxYG motifs found in the
metallo-proteases. However, they contain highly conserved HRxxH and D/ExxD/E
motifs, which may serve as functional replacements enabling metal binding and
potentially proteolytic activity. Figure 1-4 compares the known active site structure of
the metalloproteases in the M35 clan with a possible structure in the Zpslp-like proteins.
IH H H 1
V- I Y146 Ito I
4128 --D HI0 EZa
D 0143 < E205
H132 H 190
Aspzincins Zpslp-like proteins
Figure 1-4. Active site structures. On the right is the known active site of the aspzincins,
deduced from the crystal structures of deuterolysin43 and GJMEP.49 On the
left is a possible structure of an active site within the Zpslp-like proteins.
Despite their similarity to the M35 metalloproteases, it is quite possible that the
Zpslp-like proteins do not act as metal-binding proteins or possess proteolytic activity.
However, the HRxxH and D/ExxD/E motifs highly conserved within the Zpslp family of
cell wall proteins may act as peptide binding ligands, enhancing potential virulence
within a host. The significance of these motifs can be thoroughly probed through study
of purified Zpslp structure and function.
RESULTS AND DISCUSSION
Regulation of ZPS1 Gene Expression
ZPS1 regulation in S. cerevisiae was studied using the ZPS]-lacZ reporter
construct. P-Galactosidase activity, reported in Miller units, was measured as a function
of growth condition. To confirm the dependence of Zaplp on ZPS1 regulation, we
monitored the responsiveness of the ZPS]-lacZ reporter to zinc deficiency.
Simultaneously, we further probed the apparent regulation by iron status by measuring
activity of the ZPS]-lacZ reporter in response to growth under iron deficient conditions
and combined zinc- and iron-limitation (Figure 2-1).
E 2500 H WT
2 2000 *zapl
U 1500 -
9 1000 -
Fe-/Zn- Fe+/Zn- Fe-/Zn+ Fe+/Zn+
Figure 2-1. Zinc and iron responsiveness of the ZPS]-lacZ reporter. P-Galactosidase
activity in wild-type cells and zap] mutant cells grown in CSD with or
without 10[M iron and/or zinc added.
The ZPS]-lacZ reporter construct was indeed regulated by zinc in a Zaplp
dependent manner, with no detectable expression in a zap] knockout strain. Induction
was only observed when the yeast were grown under zinc-deficient conditions, with no
measurable increase in ZPS]-lacZ activity when the yeast were grown under solely iron
deficient conditions. When the yeast were grown under both zinc- and iron-limitation, a
significant increase in ZPS]-lacZ activity was observed. As the literature previously
suggests, the increased ZPS1 expression by iron-deficiency may be due to Aft2p.37 To
further investigate this hypothesis, future work may involve monitoring ZPS1 expression
in strains lacking Aft2p, Aftlp, or both.
Previously, reports have described ZPS1 regulation by RimlOlp in response to
alkaline pH.35 Therefore, we attempted to study ZPS]-lacZ activity in response to iron
and/or zinc deficiency at both acidic and alkaline pH. Under standard growth conditions,
the Chelex-treated synthetic defined medium (CSD) used to limit zinc and iron
availability is at pH 4 (optimal for yeast growth). When the CSD medium was buffered
to pH 8.0, the metals in the medium became insoluble and precipitated out of solution.
Therefore we were limited to monitoring the effects of RimlOlp on ZPS]-lacZ
expression at acidic pH (Figure 2-2). When compared to wild type yeast, strains lacking
RimlOlp exhibit a significant decrease in ZPS]-lacZ activity, which remained a function
of zinc-deficiency. This result suggests that RimlOlp affects ZPS1 expression even at
acidic pH, possibly by enhancing Zaplp regulation of this gene. These observations
further support the hypothesis that Zaplp and RimlOlp co-regulate ZPS1.
Partial Purification of Zps1p from Inclusion Bodies
We are currently attempting to purify recombinant Zps1p from Escherichia coli
for use in characterizing Zpslp structure and function. Although Zpslp is not native in
E. coli, expression of yeast proteins in bacterial systems has several advantages,
including high yield and lack of glycosylation moieties, which may complicate protein
C 2500- m WT
'V 2000- riml01
Fe-/Zn- Fe+/Zn- Fe-/Zn+ Fe+/Zn+
Figure 2-2. Zinc and iron responsiveness of the ZPS]-lacZ reporter. 3-Galactosidase
activity in wild-type cells and riml01 mutant cells grown in CSD (pH 4.0)
with or without 10[M iron and/or zinc added.
As described in the Materials and Methods section, ZPS1 (lacking the leader
peptide sequence) has been cloned by the polymerase chain reaction (PCR) and inserted
into the pET-22b(+) expression vector for isopropyl-P-D-thioglactopyranoside (IPTG)
inducible expression by bacteriophage T7 RNA polymerase in BL21(DE3) E. coli. When
expressed, the mature form of Zpslp should have an apparent molecular weight of ~ 25.5
kDa. Approximately eight hours of induction by IPTG is required for optimum Zpslp
yield (Figure 2-3).
Following large scale induction (as described in Methods section), the resulting
pellet was thawed and resuspended in 20 mL of cold 50 mM Tris(hydroxymethyl)
aminomethane (Tris), buffered at pH 7.4, containing 1 mM phenylmethanesulfonly
fluoride (PMSF), a protease inhibitor used to prevent degradation of Zpslp. The cells
were lysed using several cycles of French press at 4oC. Multiple rounds of French press
were required to adequately break the large cell pellet resulting from 8 h of growth. The
resulting lysate was centrifuged at 19,000 rpm for 20 min at 4oC and the supernatant was
decanted and saved.
M C1 2h 4h 6h 8h C2
Figure 2-3. SDS-PAGE analysis of E. coli transformants containing the pET-22b(+)-
ZPS1 expression vector not induced (lane C) or induced with IPTG for the
number of hours indicated (lanes 2 8 h). For comparison, products from
non-induced cells after 8 h growth are also shown (lane C2). In lane M is a
molecular weight marker.
At this time, the soluble supernatantt) and insoluble (pellet) components of the lysate
were analyzed by SDS-PAGE to determine the location of Zpslp (Figure 2-4). Zpslp
was present in the insoluble fraction, indicating the protein accumulates as inclusion
bodies (dense aggregates of misfolded polypeptide). Formation of recombinant Zpslp
inclusion bodies is not unexpected, given that expression of recombinant Aspndlp in E.
coli also results in inclusion body formation.15
To solubilize the inclusion bodies, 5 mL of 50 mM Tris (pH 7.4) containing 8 M
Urea was used to denature the Zpslp aggregates by gentle mixing overnight. Next, we
attempted to refold the denatured protein by single-step dilution. This entailed slowly (>
24 h) dripping 50 mL of buffer into the sample so to gradually decrease the concentration
of Urea to ~ 0.7 M. Dilution was followed by dialysis to remove all traces of the
denaturant and the sample was centrifuged at 8,000 rpm for 20 min at 40C to collect any
insoluble material. The soluble and insoluble components were analyzed for Zpslp
content by SDS-PAGE (Figure 2-5). The results indicated Zpslp was successfully
solubilized, with only trace amounts in the insoluble fraction.
SDS-PAGE analysis of soluble and insoluble components of the cell lysate
obtained from breakage of E. coli expressing Zpslp. Lane M, molecular
weight marker; Lane C, non-induced E. coli (8 h growth); Lane I, IPTG
induced E. coli (8 h growth); Lane S, soluble fraction; Lane P, insoluble
fraction. In attempts to load the maximum sample volumes to each lane,
runoff into neighboring lanes occurred (Lanes X).
Figure 2-5. SDS-PAGE analysis of the soluble and insoluble products obtained after
solubilization and refolding of the inclusion body pellet. Lane M, molecular
Next, we attempted to purify the soluble Zpslp by size-exclusion chromatography
(SEC). The protein sample was concentrated, applied to a column containing Sephadex
G-75 (Sigma) size-exclusion resin (molecular weight cutoff ca. 80 kDa), with a bed
volume of approximately 310 mL. The protein was eluted using 50 mM Tris (pH 7.4).
After elution of the void volume, 2 mL fractions were collected and analyzed for protein
using the method of Bradford.51 The Bradford protein assay indicated that the protein
eluted as one major peak (fractions 9 12) shortly after collection of the void volume.
Fractions 9 12 were combined and the content was analyzed by SDS-PAGE (Figure
M C I E
Figure 2-6. SDS-PAGE analysis of the major protein peak collected after SEC
(combined fractions 9 12). Lane C, non-induced E. coli (8 h growth); Lane
I, IPTG induced E. coli (8 h growth); Lane E, protein eluate from SEC
The results indicated poor separation of Zpslp from contaminating proteins. The
lack of separation may result from aggregation of Zpslp with other peptides, possibly due
to unfavorable disulfide bridging involving one ofZpslp's six cysteine residues.
Therefore, use of a reducing agent such as dithiothreitol (DTT) during the refolding,
concentrating, and chromatographic steps may prove effective in decreasing unfavorable
disulfide bond formation.
To reduce the concentration of contaminating proteins that could unfavorably
interact with Zpslp forming aggregates, a purification strategy was adapted from a
published method.52 This method involves washing the inclusion body pellet with the
detergent sodium deoxycholate (DOC) to remove impurities. First, the frozen cell pellet
obtained after IPTG induction was thawed and resuspended in 20 mL of cold Buffer A
(5% Glycerol, 50 mM NaCl, 0.5 mM EDTA, 50 mM Tris-HCl) containing 1 mM PMSF
and 0.1 mM DTT. The cells were lysed using several cycles of French press at 40C.
Next, DOC was added to the lysate to give a concentration of 0.2% (approximately 240
[IL of a 20% DOC stock), which is used to help liberate slightly insoluble proteins. The
solution was mixed well, allowed to stand for 10 min at room temperature, and
centrifuged at 13,000 rpm for 10 min at 40C. The supernatant was decanted and saved for
future analysis (Supernatant 1).
Following collection by centrifugation, the inclusion body pellet appears as a
white bull's-eye, which is the inclusion body protein, surrounded by a brownish layer.
The brownish layer consists of contaminating cellular debris that can be effectively
solubilized by washing the pellet with 2% DOC. Therefore, the inclusion body protein
was washed by resuspending the pellet in 18 mL of Buffer A (containing 1 mM PMSF
and 0.1 mM DTT) and 2 mL of 20% DOC. The solution was allowed to stand for at least
10 min at room temperature before being centrifuged at 13,000 rpm for 10 min at 40C.
The supernatant was decanted and saved for future analysis (Supernatant 2). The
remaining pellet was washed one additional time and, after centrifugation, the
supernatant was decanted and saved for future analysis (Supernatant 3). At this time, the
washed inclusion body pellet was solubilized by resuspending in 5 mL Buffer A
containing 8 M Urea and gently agitated overnight at 40C. Prior to refolding by single-
step dilution, the protein purity was assessed by SDS-PAGE to determine the
effectiveness of the DOC wash (Figure 2-7). The gel showed few major bands, one being
Zpslp, thus demonstrating the value of the DOC wash in purifying the inclusion body
M C I P
22 kDa -
Figure 2-7. SDS-PAGE analysis of the washed inclusion body pellet, solubilized in
Buffer A containing 8 M Urea (Lanes P). Lane M, molecular weight
marker; Lane C, non-induced E. coli (8 h growth); Lane I, IPTG induced E.
coli (8 h growth).
Next, we attempted to refold the solubilized inclusion body protein by single-step
dilution using Buffer A. As before, this procedure involved slowly decreasing the
concentration of Urea by dilution followed by dialysis. In an attempt to discourage
unfavorable disulfide bridging, 0.1 mM DTT was added to the buffer during the refolding
process. After dialysis, the sample was centrifuged at 8,000 rpm for 20 min at 40C to
pellet insoluble materials, and the supernatant was decanted and concentrated. The
supernatant and pellet collected after refolding were analyzed by SDS-PAGE for protein
content, as were the soluble fractions (Supernatant 1 3) collected after each treatment
with DOC (Figure 2-8). Unfortunately, refolding was unsuccessful and Zpslp was
present in the insoluble fraction.
M C 1 2 3 S P
29 kDa -
22 kDa -
Figure 2-8. SDS-PAGE analysis of the soluble and insoluble products obtained after
solubilization and refolding of the inclusion body pellet. Also shown are the
supernatant fractions collected after each purification step. Lane M,
molecular weight marker; Lane C, non-induced E. coli (8 h growth); Lane I,
IPTG induced E. coli (8 h growth); Lane 1, Supernatant 1 (post-lysis); Lane
2, Supernatant 2 (after first DOC wash); Lane 3, Supernatant 3 (after second
DOC wash); Lane S, soluble fraction (after refolding); Lane P, insoluble
fraction (after refolding).
It is unclear why the solubilized inclusion body protein failed to refold despite its
improved purity and the addition of DTT (to prevent unfavorable disulfides). One
possible explanation is that the rate of dilution was accelerated due to poor control of the
flow rate. It is critical that the rate of dilution is slow. At high denaturant concentrations,
the unfolded protein is well solvated and flexible. Rapidly altering solvent dynamics
toward an aqueous environment forces the protein to collapse into a compact and rigid
structure. Unfortunately, the resulting structure is often misfolded or aggregated and
therefore insoluble. Gradual dilution allows for refolding at intermediate concentrations
of urea, where the denaturant concentration is low enough to force protein molecules to
collapse, yet allowing flexible motion enabling proteins to reorganize their structures and
stay in solution. Therefore, it may be beneficial to alter the refolding strategy so to
provide a slower and more controlled rate of denaturant dilution. Alternative refolding
strategies include, but are not limited to: one-step dialysis, step-wise dialysis, and buffer-
exchange by gel filtration.53 Although expressing recombinant Zpslp from E. coli is
advantageous due to high protein yield, it is possible the protein will not properly refold
after solubilization from inclusion bodies. If future efforts to refold and purify Zpslp
from E. coli inclusion bodies are unsuccessful, it may be necessary to purify Zpslp
directly from S. cerevisiae.
The work presented represents initial steps toward characterization of Zpslp from
S. cerevisiae. ZPS1 expression is regulated by extracellular pH and zinc-deficiency,
environmental signals known to elicit Zpslp-like antigen production in Candida and
Aspergillus spp., respectively. These results suggest that the regulation, and
consequently function, of the cell wall proteins is conserved among these fungi. This
hypothesis is supported by their high sequence homology. Because of their localization
within the fungal cell wall and the observed binding of Pral and Aspndl to host
molecules, the Zpslp-like proteins in C. albicans and Aspergillus spp. are believed play a
role in establishing infection. The Zpslp-like proteins may function as virulence factors
by mediating critical host-parasite interactions or through involvement in morphological
processes. Therefore, future characterization of Zpslp may include investigating the
protein's potential role in fungal cell-cell adhesion flocculationn) or adherence to host
ligands (e.g.., ECM or serum proteins).
The partial purification of recombinant Zpslp from bacterial inclusion bodies is
an important first step toward characterization ofZpslp. Once purified, a wealth of
information can be obtained by studying both Zpslp structure and function. Due to
similarities between the Zpslp-like proteins and zinc-dependent metalloproteases, future
work using purified Zpslp ought to include metal binding studies and testing for
proteolytic activity towards a variety of substrates.
MATERIALS AND METHODS
For standard growth of E. coli, LB medium was used. The recipe per liter is 10 g
NaC1, 10 g Bactotryptone, and 5 g Yeast Extract. When required, ampicillin was added
to a final concentration of 200 [tg/mL. In preparation of plates, 15 g of agar was added
YPD medium was used for routine, non-selective yeast growth. The recipe per
liter is 10 g Yeast Extract, 20 g Bactopeptone, and 20 g Dextrose. In preparation of
plates, 15 g of agar was added per liter.
For maintenance of recombinant yeast strains, selective (SD) medium was used.
The base recipe per liter is 5 g (NH4)2SO4, 20 g Dextrose, and 1.7 g Yeast Nitrogen Base
without amino acids or (NH4)2SO4 (Difco; Sparks, MD). To satisfy the auxotrophic
strains used in this study, the medium was supplemented with 0.1 g L-Histidine, 0.1 g L-
Leucine, and 0.1 g L-Lysine per liter. Although the strains required Uracil, this was
omitted from the medium for selective growth. This medium will be referred to as SD-
Ura. For plates, 15 g of agar was added per liter.
To limit zinc and iron availability, Chelex-treated synthetic defined medium
(CSD) was used. The recipe per liter, using H20 at 18 MQ purity, is 20 g Dextrose, 5.1 g
Yeast Nitrogen Base without amino acids or divalent cations or potassium phosphate
(BiolOl; Vista, CA), and 0.1 g each of L-Histidine, L-Leucine, and L-Lysine. Again, for
selective purposes, Uracil was omitted from the medium. To remove metals from the
media, 25 g of Chelex-100 ion exchange resin (Sigma) was added, and the mixture was
stirred for a minimum of 2 h. After removal of the resin, 10 mL of potassium phosphate
monobasic (100 g/L) was added and the pH was adjusted to 4.0 using HC1. Next,
divalent metal ions were added to the medium to the following concentrations (as
recommended by BiolOl): 0.4 mg/L MnS04, 0.04 mg/L CuS04, 100 mg/L CaCl2, and
500 mg/L MgS04. The resulting solution was filter sterilized into a polycarbonate flask
washed with Acationox detergent (Baxter Scientific Products; McGraw Park, IL). The
resulting solution contains residual zinc and iron at concentrations less than 100 nM
(approximate value), and is referred to as CSD-Ura(-Zn/-Fe). For zinc or iron replete
medium, the desired metal is added back to the medium to a final concentration of 10
Solutions and Buffers for Yeast Transformations and P-Galactosidase Assays
10x TE (250 mL):
100 mM Tris and 10 mM EDTA pH 7.5. Sterilize.
LiTE solution (50 mL):
5 mL sterile 10x TE, 5 mL sterile 1 M Lithium Acetate 40 mL sterile
PEG-LiTE solution (50 mL):
5 mL sterile 10x TE, 5 mL sterile 1 M Lithium Acetate, 40 mL sterile
44% (w/v) PEG-3350
Carrier DNA (10 mL):
100 mg salmon testes DNA and 10 mL ultrapure H20. Shear DNA by
drawing the mixture up into a 10 mL syringe with an 18g needle 15 times,
boil, and restore volume to 10 mL. Store as 1 mL aliquots at -200C.
Z-buffer, pH 7.0 (1 L):
0.06 M Na2HPO4, 0.04 M NaH2PO4-H20, 0.01 M KC1, 0.001 M
Bacterial and Yeast Strains
Listed below are the bacterial and yeast strains used in this work.
Table 4-1. Yeast strains used for the work described.
Strain Mutation Source Genotype
MAT u; his3; leu2; ura 3;
BY4742 Wild type lys2
MAT a; his3; leu2; ura 3;
Y11367 zapl EUROSCARF lys2
MAT u; his3; leu2; ura 3;
Y10936 riml01 EUROSCARF lys2
Table 4-2: Bacterial strains used for the work described.
TOP 10 F mcrA A(mrr-hsdRMS-mcrBC) 4801acZAM15 AlacX74
deoR recAl araD139 A(ara-leu)7697 galU galK rpsL (StrR)
BL21(DE3) F- ompT hsdSB(rBm,-) gal dcm (DE3)
Using the lithium acetate method, yeast strains of interest were transformed with a
plasmid containing the ZPS]-lacZ fusion (with Ura+ selection), which was previously
constructed34 in YEp35354 by gap repair.55 This was accomplished by growing the yeast
in 5 mL YPD at 30'C at 250 rpm overnight. The following day, 300 |pL of the overnight
culture was transferred to a new tube containing 5 mL YPD and incubated for 2 hr. at
30'C at 250 rpm. Next, the cells were harvested by centrifugation at 3500 rpm for 3 min
and the supernatant was decanted. The remaining cell pellet was washed by adding 5 mL
LiTE solution and vortexing. Again, the cells were harvested by centrifugation (as
described above). The cells were resuspended in residual LiTE solution by vortexing and
50 |pL of the cell suspension was transferred into a sterile 1.5 mL centrifuge tube. Added
to the centrifuge tube containing cells were 2 |pL (~ 400 [tg) of plasmid DNA containing
the ZPS]-lacZ fusion and 10 |pL (~ 10 [tg) of salmon sperm carrier DNA (boiled for 5
min and flash cooled on ice prior to use). Next, 500 |pL of PEG-LiTE solution was
added. The mixture was vortexed briefly and incubated at 300C at 250 rpm for 30 45
min. After incubation, the sample was heat shocked for 10 15 min at 420C. The cells
were pelleted at 4000 rpm for 1 min in a microcentrifuge. The supernatant was aspirated
and 500 |pL LiTE solution was added to the pellet and subsequently vortexed. Finally, 50
- 200 |pL of transformant was plated on SD-Ura plates to (select for the YEp353 plasmid)
and incubated at 300C for 3 5 days. Plates which grew colonies were stored at 40C for
A single colony of yeast transformed with the ZPS]-lacZ fusion plasmid was
transferred to 5 mL of SD-Ura and incubated at 300C at 250 rpm overnight. This liquid
culture was used to inoculate metal-free 14 mL polystyrene tubes containing 5 mL of
CSD-Ura with the appropriate combinations of zinc and iron as follows: -Zn/-Fe, 45 |pL
cell culture; -Zn/+Fe, 30 |pL cell culture; +Zn/-Fe, 30 |pL cell culture; +Zn/+Fe, 20 |pL
cell culture. The cultures were grown for 12 h and then stored on ice for approximately
20 min. Next, the cells were harvested by centrifugation for 3 min at 3500 rpm at 40C.
The supernatant was discarded and the resulting pellet was washed by adding 5 mL cold
Z-buffer and vortexing. The cells were harvested by centrifugation (as above) and the
supernatant was discarded.
The cells were resuspended in 2 mL cold Z-buffer and 1 mL of the cell
suspension was transferred to a 5 mL glass assay tube containing 50 |pL CHCl3 and 50 |pL
0.1% SDS. The contents of the tube were vortexed to permeablize the cells and then
incubated at 300C for 10 min to equilibrate. After incubation, the tube was vortexed
vigorously for 3 sec and its contents (principally CHCl3) were allowed to settle for
approximately 10 se. before transferring 100 |pL of the suspension to a 96-well plate (in
triplicate). The assay reaction was initiated by adding 20 |pL of 4 mg/mL o-nitrophenyl-
P-D-galactopyranoside (ONPG). The sample was mixed and the reaction was allowed to
proceed until the darkest samples were an intense yellow color. The reaction was
stopped by adding 50 |pL of 1 M Na2CO3 and the reaction time (min) was noted. The
absorbance at 420 nm was measured (reference wavelength, 600 nm) using a SAFIRE
microplate reader (Tecan) and XFLUOR software. The absorbance at 600 nm (OD600) Of
the remaining cell suspension (from above) was also measured using a BIO-RAD
SmartSpecTM 3000 bench-top spectrophotometer. 3-galactosidase activity was measured
in Miller Units using the method of Guarente,56 and activity units were calculated as
follows: (AA420 x 1000)/(min x mL of culture used x OD600).
Cloning of ZPS1 and Construction of an E. coli Expression Plasmid
The gene that encodes the mature form of Zpslp (lacking the signal peptide) was
PCR cloned from S. cerevisiae strain BY4724 genomic DNA using forward and reverse
primers containing Nde I and EcoR I restriction sites, respectively. The primers were
obtained from Integrated DNA Technologies, Inc. (Coralville, IA) and were designed as
ZPS1 for: 5'- AAC TTT AAG AAG GAG ATA TAC ATA
TGC CTG TCA CTT ACG ACA CCA A -3'
ZPS1 rev: 5'- CAA GCT TGT CGA CGG AGC TCG AAT
TCT TAC AAG TTA CCT AGA CAG C -3'
The PCR reaction was catalyzed using Taq DNA polymerase and the
thermocycling conditions employed were as follows: one cycle at 95oC for 3 min; and
25 cycles at 95oC for 30 sec, 50oC for 30 sec, 72oC for 1.5 min; and a final extension at
72oC for 8 min.
The PCR product was digested for 4 h at 37oC using the restriction enzymes Nde
I and EcoR I (New England Biolabs; Beverly, MA). The pET-22b(+) vector was
obtained from Novagen (La Jolla, CA). The pET-22b(+) plasmid was also digested using
Nde I and EcoR I (as described above). Following restriction digestion, the cut PCR and
pET-22b(+) samples were subjected to agarose gel electrophoresis (0.8% agarose) and
purified using the QIAquick Gel Extraction Kit, following the manufacture's protocol
(QIAGEN Inc.; Valencia, CA). These purified samples were subsequently used to ligate
the cloned ZPS1 gene into the pET-22b(+) vector between the Nde I and EcoR I
restriction sites using T4 DNA ligase (New England Biolabs), incubated overnight at
16oC. The ligation product was used to transform electrocompetent E. coli TOP10 cells
by electroporation following standard procedures.57 The E. coli transformant (10 150
1L) was plated on LB agar plates containing ampicillin (for plasmid selection) and
incubated at 37oC overnight. Plates that grew colonies were stored at 4oC for future use.
To obtain large quantities of the pET-22b(+)-ZPS1 construct, a single colony from the
transformation product was used to inoculate 5 mL of LB medium containing ampicillin.
The cells were grown at 37oC at 250 rpm overnight. Using the Promega (Madison, WI)
Wizard Plus Miniprep DNA purification system, the pET-22b(+)-ZPS1 plasmid was
purified from the overnight culture according to the manufacture's directions. Using the
purified plasmid, the sequence of the cloned ZPS 1 gene was confirmed by the ICBR
DNA sequencing core laboratory at the University of Florida.
Expression of Zpslp in E. coli
To obtain Zpslp using the T7 expression system, the pET-22b(+)-ZPS] plasmid
was transformed into BL21(DE3) E. coli by electroporation using standard methods. The
E. coli transformant (10 150 [LL) was plated on LB agar plates containing ampicillin
and incubated at 370C overnight. Plates that grew colonies were stored at 40C for future
use. A single colony from the transformation product was used to inoculate 15 mL of LB
medium containing ampicillin. The cells were grown at 370C at 250 rpm overnight and
10 mL of culture was used to inoculate 1 L of LB medium containing ampicillin. The 1
L culture was incubated at 370C at 250 rpm for approximately 2 h until reaching an OD600
of 0.4 1.0. At this time, Zpslp expression was induced by addition of IPTG (Isopropyl-
P-D-thioglactoside) to a final concentration of 1 mM. The culture was then incubated at
30'C at 250 rpm for 8 h. Finally, the cells were harvested by centrifugation (3000 rpm
for 15 min at 40C) and washed two times using 50 mM Tris (pH 7.4). The resulting
pellet was stored frozen at -200C overnight.
Estimation of Protein Purity by SDS-PAGE
SDS-PAGE gels containing 14% (w/v) polyacrylamide were prepared and
analyzed by standard methods. Samples were prepared by adding equal volumes of 2x
Laemmli sample buffer, boiling for 10 min, followed by centrifugation at 14,000 rpm in a
microfuge for 1 min to pellet any insoluble debris. The gels were run at 70 V, using a
Tris-glycine electrode buffer. All gels were stained with Coomassie blue.
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Stephanie L. Drobiak is from Brooklyn, Connecticut. She graduated from
Wheaton College in Norton, Massachusetts, in 2001 with a Bachelor of Arts in
biochemistry. In the fall of 2001, she entered the graduate program in the Department of
Chemistry at the University of Florida. Upon completion of her master's, she will
continue working towards her PhD at the University of Florida.