Citation
Structure-Based Drug Design

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Title:
Structure-Based Drug Design
Creator:
McDowell, Megan
Publication Date:
Language:
English

Subjects

Subjects / Keywords:
Bottles ( jstor )
Broths ( jstor )
Diseases ( jstor )
Flasks ( jstor )
Gels ( jstor )
Genes ( jstor )
Inclusion bodies ( jstor )
Kidney dialysis ( jstor )
Molecular weight ( jstor )
Pipettes ( jstor )
Major histocompatibility complex
Proteins--Analysis
Genre:
Undergraduate Honors Thesis

Notes

Abstract:
Immunohistochemical and structural analysis of encoded proteins from the Major Histocompatibility Complex (MHC) were performed with the objective of revealing novel insights into their pathological roles. The MHC is the region located on the short arm of chromosome 6. Studies have shown that this region is implicated in over 100 human diseases, including autoimmune diseases. Gene STG, located on chromosome 6 order reading frame 15, is specifically linked with follicular lymphoma and is present in the taste buds, skin, and tonsils. STG was expressed in an E. coli expression system, and then inclusion body preparations were performed. A BCA reaction was completed to determine the protein concentration before dialysis was performed. An SDS-PAGE was also completed to ensure the protein of interest was collected. The BCA reaction showed the protein concentration to be 1806.3 µg/mL. The SDS-PAGE, however, showed that the protein of interest (33.6 kD) was not present. Further testing on gene STG was halted. For future research, gene STG could be tested again to ensure a similar result. Also, genes G8 and G18 showed promising results after an inclusion body prep and SDS-PAGE were performed. Thus, further testing on these genes, along with others on chromosome 6, should be analyzed. ( en )
General Note:
Awarded Bachelor of Science in Agricultural and Biological Engineering; Graduated May 4, 2010 summa cum laude. Major: Agricultural and Biological Engineering
General Note:
Advisor: Dr. James Leary
General Note:
College/School: College of Engineering
General Note:
Legacy honors title: Only abstract available from former Honors Program sponsored database.

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University of Florida
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University of Florida
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Copyright Megan McDowell. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Structure Based Drug Design Megan McDowell Spring 2010 Summa Cum Laude Bachelor of Science in Biological Engineering

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2 Table of Contents Abstract ................................ ................................ ................................ ................................ ........... 3 Introduction ................................ ................................ ................................ ................................ .... 4 Materials and Methods ................................ ................................ ................................ ................... 4 Overview ................................ ................................ ................................ ................................ ..... 4 Small Inclusion Body Prep ................................ ................................ ................................ ........... 5 BCA Assay Protocol ................................ ................................ ................................ ..................... 6 SDS PAGE ................................ ................................ ................................ ................................ ..... 7 Dialysis ................................ ................................ ................................ ................................ ......... 7 Results ................................ ................................ ................................ ................................ ......... 8 Conclusion ................................ ................................ ................................ ................................ ....... 9 Works Cited ................................ ................................ ................................ ................................ ... 10

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3 Ab stract Immunohistochemical and structural analysis of encoded proteins from the Major Histocompatibility Complex (MHC) were performed with the objective of revealing novel insights into their pathological roles. The MHC is the region located o n the short arm of chromosome 6. Studies have shown that this region is implicated in over 100 human diseases including autoimmune diseases Gene STG, located on chromosome 6 order reading frame 15, is specifically linked with follicular lymphoma and is present in the taste buds, skin, and tonsils. STG was expressed in an E. coli expression system, and then inclusion body preparations were performed. A BCA reaction was completed to determine the protein concentration before dialysis was pe rformed. An SDS PAGE was also completed to ensure the protein of interest was collected. The BCA reaction showed the protein concentration to be 1806.3 g/mL. The SDS PAGE, however, showed that the protein of interest (33.6 kD) was not present. Further testing on gene STG was halted. For future research, gene STG could be tested again to ensure a similar result. Also, genes G8 and G18 showed promising results after an inclusion body prep and SDS PAGE were performed. Thus, further testing on these genes, along with others on c hromosome 6, should be analyzed.

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4 Introduction The Major Histocompatibility Complex (MHC) is the region located on the short arm of chromosome 6 that is strongly implicated in over 100 human diseases, including most autoimmune diseases. One hundred and twenty four genes located in this region are thought to be expressed, some of which have known functions in the immune response. Genes in this region are highly polymorphic and there are strong associations between MHC alleles and diseases such as Type I D iabetes ( e.g. HLA DQ8) and Rheumatoid Arthritis (e.g., HLA DR4) (National Center for Biotechnology Information) However, due to a phenomenon called linkage disequilibrium (non random association of alleles at two or more loci) it is not feasible to disse ct the role of each of the 124 MHC genes in human diseases solely using genetic methods. Therefore, it is necessary to utilize additional methods to characterize the role of each MHC encoded protein in normal and pathological conditions. There are a number of uncharacterized MHC encoded proteins that are implicated in human diseases, but have been studied due to limitations in the genetic approach. Over the course of my research, the genes P5, G8, G18, RD, and STG were amplified and purified but only the re sults for STG are d iscuss ed in this paper. STG was investigated in human tissues and found to be located in the taste buds, skin, and tonsils (Sanchez, 2004) A recent study has shown that gene STG is strongly associated with follicular lymphoma (Skibola CF, 2009) For this study, the hypothesis is that the Immunohistochemical and structural analysis of MHC encoded proteins will reveal novel insights into their pathological roles. Materials and Methods Overview E a ch human MHC gene, such as STG was ex pressed in an E. coli expres sion system. For example, STG was expressed in bacterial cell cultures and inclusion body preparations were carried out. Ins oluble inclusion body proteins we re solub i lized using 8M urea. BCA reactions wer e used to determine the protein concentration prior to refolding by dialysis against T ris buffered saline, pH 7.4. SDS PAGE wa s used to separate proteins according to their molecular weight. The results of the BCA reaction and SDS PAGE help ed determine if further testing should be performed. The purpose f or dialysis wa s to allow the protein to refold into the correct conformation. After refolding, the protein wa s concentrated and analyzed by injection on a Superdex 200 column attached to a Fast Protein Liquid C hromatogr aphy (FPL C) machine. This step wa s used to analyze and purify the protein following refolding. SDS PAGE was run on fractions collected after separati on on the Superdex 200 column. One mg of purified protein

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5 was used for the generation of monoclonal antibodies by th e ICBR core laboratory for immunohistochemical studies. Ten mg of purified protein were used in 1,536 crystallization trials. Crystallization conditions were optimized to yield large crystals (greater than 1 mm in the longest dimension) for analysis by X r ay diffraction studies. The following sections detail each method used in the analysis of gene STG. Small Inclusion Body Prep The overnight prep was begun between late afternoon and night, allowing no more than 12 hours to pass before the next procedure was completed. After putting the glycerol stock (STG) on ice, a 100 mL sterile flask was labeled with the date and initialed. Then, 55 mL of LB broth was placed in flask, ensuring a hot flame was near for sterility. Fifty five micro liters of Carbenicil lin were then added to the flask (100 g/mL of final concentration). Using a 1000 L pipette tip, the glycerol stock was scraped and mixed into the LB Broth and Carbenicillin solution. The top of the flask was then flamed and covered with sterile foil. The flask was then placed in an automatic shaker at 37C overnight. The following morning, a 2000 mL flask was labeled and initialed. Five hundred mL of LB broth were added to the flask, along with 500L of Carbenicillin. The top of the flask was flame d and covered with sterile foil, then placed in the shaker at 37C until warmed. The 50 mL of overnight prep was then added to the 2000 mL flask containing LB broth and Carbenicillin. The flask was then placed in the shaker for three hours until the Opti cal Density (OD) at 600 nm reached between 0.6 0.8. Using a spectrophotometer, the baseline was first set to 0 using only LB broth. The OD measurement was then taken with 1 mL of sample in a cuvette. When the OD reached the appropriate range, Isopropyl D 1 thiogalactopyranoside (IPTG) was added at 1mM of final concentration. The solution was then put back into the shaker at 37C for three hours. The flask was then removed and the solution was split evenly into two 1 L centrifuge bottles. The bottles of solution were balanced precisely on a weighing scale. The centrifuge bottles were then spun at 4000 RPM for 20 minutes at 4C. The supernatant was then removed and 5 mL of BugBuster were added to each pellet, vortexed, and mixed with a pipette. Then the solutions were pipetted into a 50 mL oakridge tube. The process was repeated by pipetting 5 mL of BugBuster into each 1 L bottle mixture, then pipetting the remaining solution into the same 50 mL oakridge tube. The oakridge tube was then stored at 80C overnight. The next day, the 50 mL oakridge tube was retrieved from the freezer and thawed at room temperature. Benzonase was retrieved from the 20C freezer and placed in an ice bucket. Once the prep solution thawed out completely, 20 L of Benzo nase was added to the oakridge tube. The oakridge tube was then placed on a rocker at room temperature for one hour. The solution was then divided between two 50 mL oakridge tubes. The two tubes were spun at 4C

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6 at 13,000 RPM for 15 minutes. The supern atant was then transferred from the tubes into a 50 The soluble fraction was then stored in 20C for future testing. The pellets were resuspended with 15 mL of 1:10 BugBuster diluted in sterile filtered dd H 2 O. Another 15 mL of 1:10 BugBuster was added to the tubes, totaling 30 mL of BugBuster in each tube. The tube was then spun at 10,0 0 0 RPM for 15 minutes at 4C. The supernatant was then removed with a pipette and discarded. The pellet was resuspended with 30 mL of 1:10 BugBuster and vortexed. Again, the tube was spun at 13,000 RPM for 15 minutes at 4C. The supernatant was again removed and disposed. This process continued until the pellet was relatively white. Once the pellet appeared clean, the pellet was resuspended in 2 mL of 1:10 BugBuster and vortexed. Two 1.5 mL tubes were then labeled, and 1 mL of solution was aliquoted into each tube. In some cases, more than two tubes will be needed. The tubes were then spun at top speed at 4C for 15 minutes in a cold room. The supernatant was then removed from each tube with a pipette and discarded The pellet was then resuspended in 1 mL of ddH 2 O and placed in 20C freezer. BCA Assay Protocol A total volume of 5500 L of working reagent 5392 L of reagent A mixed with 108 L of reagent B, was prepared in a 10 mL tube. Nine standards were then prepared using 2000 g/mL of BSA stock (see Table 1 below) Four unknowns were prepared of 1/4x, 1/8x, 1/16x, and 1/32x concentration. Each standard and unknown of 25 L was plated into two wells each. Then, 200 L of working reagent was pipetted into each well and mixed thoroughly. The plate was then incubated for 15 minutes at 37C. The absorbance was then read at 562 nm. The background absorbance wa s subtracted, and a standard curve was generated by plotting the standard absorbance vs. concentration. The standard curve was then used to evaluate the concentration of the protein sample. Vial V D iluent ( L) V BSA stock (L) Final [BSA] (g/mL) A 0 60 2000 B 25 75 1500 C 65 65 1000 D 35 35 of B 750 E 65 65 of C 500 F 65 65 of E 250 G 65 65 of F 125 H 80 20 of G 25 I 80 0 0 Table 1. Prepared standards for BCA Reaction.

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7 SDS PAGE One tube of inclusion bodies was unthawed and spun for 10 minut es at 13.2 RPM at room temperature. Then, the supernatant (ddH 2 O) was removed from the tube and disposed. Then 1 mL of 8M urea was added to the tube and left overnight without mixing. The next day, the pellet and solution was pipetted up and down to mix thoroughly. The tube was then spun for 10 minutes at 13.2 RPM at room temperature. The supernatant was then removed and saved. Then 0.5 mL of 8M urea was added to the pellet, mixed thoroughly with a pipette, and spun for 10 minutes at 13.2 RPM. The sup ernatant was removed and saved and the pellet was discarded. The gel electrophoresis box was then set up by first retrieving the protein gel from the refrigerator, removing the comb, and rinsing the plate with deionized water. The gasket was wet with 1X Tris glycine SDS buffer to ensure a tight seal, while the inside of the electrophoresis box was filled to the top with SDS buffer. The protein sample was then mercaptoethanol) with 10L of collected s upernatant. The cuvettes were then loaded into the GeneAmp PCR machine for 15 minutes (94C to 4C setting). Ten microliters of a molecular marker were pipetted into the first well and then 10 L of protein sample were pipetted into the other wells. Th e gel electrophoresis was run at 150 volts for approximately one hour. The gel plate was then removed and placed in a container of Coomassie Brilliant Blue R 250 for 30 minutes. The gel was then rinsed with deionized water and then placed in a container of destaining liquid (mixture of methanol, bromphenol blue, acetic acid, and water). The gel was then compared to Dialysis First, 6.49 g of urea were mixed w ith 360 L of Tris buffer in a small beaker This mixture was poured into a graduated cylinder, and ddH 2 O was added up to the 18 mL line. The graduated cylin der was then placed in a small beaker with a stir bar. Both of these containers were then placed into an ice bucket. STG was then added drop wise (approximately one drop per minute) into the buffer solution. Once all of the STG had been mixed into the buffer solution, the graduated cylinder was covered, labeled, and placed in a cold room overnight, while stirring constantly. T he next day, the dialysis bags were prepared by soaking in dialysis buffer for 30 minutes. The solution from the cold room was pipetted into the dialysis bag and clipped tightly. Then, 2 L of buffer sol ution were poured into a small beaker, and the dialy sis bag was carefully placed in the solution. The beaker was placed in the cold room at 4C overnight, stirring the solution constantly using a stir bar and magnetic stir plate. The next day the buffer solution was replaced with 2 L of fresh solution and left in the cold room overnight, stirring

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8 continuously. The next day, the buffer solution was again replaced with 2 L of fresh solution and left in the cold room overnight, stirring continuously. Results The results of the BCA reaction are shown below i n Figure 1. The concentration of the protein sample was determi ned to be 1806.3 g/mL, which was sufficient to continue testing. However, the results of the SDS PAGE (see Figure 2) showed a substance at molecular weight 20 kD STG has a molecular weight of 33.6 kD. Figure 3 shows the molecular weight marker used in the experiment and helps demonstrate where the correct location should have been. Since no growth was shown at 33.6 kD, the substance amplified in the experiment was not STG. Although dialys is had already been performed with the STG, further testing on STG was halted after analyzing the SDS PAGE results. Figure 1. BCA Reaction Results.

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9 Figure 2. Section of SDS PAGE gel results. Figure 3. Molecular weight protein marker example. Co nclusion The SDS PAGE showed that the gene of interest, STG, was not expressed after the inclusion body preparation. Another inclusion body prep and SDS PAGE analysis could be performed on gene STG to ensure accurate results were obtained in the first ex periment. In addition, the beginning analysis on genes G8 and G18 have shown promising results and should be tested further, along with other genes on the MHC. Overall, th is research project was valuabl e in gaining experience in the field of research and medicine. As a future physician with a background in biological engineering, this experience has given me a unique perspective from which to make interdisciplinary contributions to the field of medicine.

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10 Works Cited National Center for Biotechnology Information. (n.d.). NCBI. Retrieved April 2010, from Entrez Gene http://www.ncbi.nlm.nih.gov/gene/29113#geneGenomic%20regions,%20transcripts,%20and% : 20products Sanchez, F. (2004). STG does not associate with psoriasis in the Swedish population. Experimental Dermatology 413 8. Skibola CF, B. P. (2009). Genetic variants at 6p21.33 are associated with susceptibility to follicular lymphoma. Nature Genetics 873 875.