Biochemist
Resume:
Walter C. Thompson
Silver Spring, MD 20906
********@*****.***
ANALYTICAL PROTEIN BIOCHEMISTRY/PROTEIN PURIFICATION EXPERIENCE:
Utilized or developed processes to purify over ten enzymes many being membrane-associated enzymes that required use of a wide array of aqueous buffers, detergents, or buffer additives to maintain or optimize their respective biological activity.
Adapted published assay conditions and methods to measure these enzyme’s activity by the following protein analytical biochemistry methods:
(1) enzyme kinetics using radiochemical, spectrophotometric, or
fluorometric assays (including GTP-binding assays),
(2) enzyme inhibition using IC50 screening of drug candidates,
(3) enzyme-substrate binding constants.
Performed denaturation/refolding protocols for multiple 6-10 cysteine-containing proteins or cytokines using or developing processes to preserve protein structural integrity as confirmed by NMR performed by other researchers. Familiar with protein ultrafiltration concentration methods and techniques to avoid protein aggregation.
Utilized reverse-phase HPLC to separate properly from improperly folded multi-cysteine-containing proteins.
Initially trained in quantitative analytical methods; developed extremely accurate/reproducible trace metal determinations at the part-per-billion level foundational analytical experience found in all subsequent work.
CHROMATOGRAPHIC TECHNIQUES FOR PROTEIN PURIFICATION:
ÄKTA FPLC with UNICORN software at lab scale using nickel-affinity, ion-exchange, or size-exclusion chromatography.
Preparative protein reverse-phase HPLC (Agilent/ChemStation software).
Various bulk- and gravity-flow column affinity purification techniques that include the following: nickel-affinity, GST-affinity, FLAG-affinity, protein A/G affinity, dye-affinity or ligand-affinity (e.g., 2 ,5 -ADP-Sepharose 4B).
EXAMPLES OF CREATIVE, SELF-DIRECTED EXPERIMENTAL METHODS DEVELOPED:
Developed a written protocol documenting a more reproducible process to optimize protein yield for a difficult-to-refold, 6-cysteine-containing cytokine; the newly developed method utilized a different solubility-enhancing thioredoxin tag with the protein expressed in a more refolding-compatible strain of E. coli.
Developed a protein-derivatization method for HPLC purified ubiquitin separating purified ubiquitin from ubiquitin bound with non-specific substances thereby enabling protein-derivatization of the HPLC-purified ubiquitin with a spin-label, MTSL, for subsequent NMR structural analysis by other researchers.
Developed a published protocol for protease digestion of glyceraldehyde- 3-phospate dehydrogenase. Utilized reverse-phase HPLC to create a peptide map of digested peptides that clearly identified the specific site of post-translational modification.
Developed a microscale, size-exclusion-based method to separate “bound” from “unbound” platinum anti-cancer drug thereby enabling quantitative kinetic binding measurements to be made at the part-per-billion level.
Developed a fluorimetrically based technique to estimate light output from wild-type firefly luciferase versus light output from point-mutants produced from wild-type firefly luciferase using standard or novel luciferin substrates.
Implemented set up of a growth hormone radioimmunoassay for a newly established lab; worked out conditions to successfully prepare and purify125I-labeled protein and peptide hormone reagents.
Developed an immunoprecipitation/radiochemical assay to determine endothelial nitric oxide synthase’s enzymatic activity in transiently transfected COS-7 cells.
RELEVANT LAB EXPERIENCE:
Over twenty-five years of government, academic or government contractor lab experience with emphasis on analytical protein biochemistry, protein purification and recombinant DNA methods.
Co-author on 16 scientific publications from five different labs most as a second author indicating a primary technical assistance role.
Proficient in laboratory-scale bacterial protein production, including lab scale protein production in mammalian cell lines post-transfection.
Proficient in recombinant DNA methods (expression plasmid/site-directed point-mutant expression plasmid construction, PCR primer design, PCR plasmid/point-mutant plasmid amplification, bacterial transformation, clone plating and selection, DNA purification to express membrane proteins in E. coli, amplify genes, ligate, transform DNA; perform PCR, DNA purification, site-direct mutagenesis, analysis by restriction digests run on agarose gels; evaluate constructs using restriction digests.
Proficient in RNA techniques: (RNA purification, northern blotting, cDNA from RNA, qRT-PCR).
Over ten years performing basic tissue culture (aseptic technique, cell line thawing/freezing, growth of primary and immortalized cell lines, cell transfection, cell plating and counting, cell-based bioassays, confocal cell slide preparation).
One year use of ÄKTA purifier system with UNICORN software at lab scale (up to 50 mg) including instrument maintenance.
Able to grow 1 to 3 L bacterial using minimal, rich or specialized (e.g., isotopically labeled carbon source) growth media.
Extensive experience evaluating protein expression using Western blots, SDS-PAGE, Western blots, densitometry, phosphorimaging, and other quantitative assays.
Experienced in using ultracentrifuges to isolate membranes and cellular fractions.
Experienced solubilizing membrane-associated proteins using aqueous buffers with various detergents, with biological activity preserved.
Proficient in cell disruption using sonication, lysozyme addition, freeze/thaw, or microfluidization at 23,000 psi., with biological activity preserved.
Trained in good laboratory practice, with excellent recordkeeping and troubleshooting skills including assay troubleshooting, instrument issues and method development.
Skilled in data analysis, data interpretation, and data processing as demonstrated in figures, methods and abstracts published in 16 scientific articles (PowerPoint, Excel, GraphPad Prism, SigmaPlot, Microsoft Office, Photoshop, etc.).
Experienced in presenting PowerPoint data summaries at group meetings.
Experienced in assisting MD, PhD, post-doc’s, as they seek to complete their first scientific publication (e.g., by demonstrating lab techniques, by assisting with equipment usage, by helping with data collection/interpretation, etc.).
Enjoys searching scientific literature to develop or troubleshoot challenging protein purification and relevant biochemical assay methods to answer specific research objectives.
PROFESSIONAL EXPERIENCE:
•Chemist (2011–present), Aerotek Scientific Staffing, Rockville, MD
Pre-screen Environmental Protection Agency Inorganic Superfund reports at PRIZIM, Inc., as a first step in PRIZIM’s determination if these reports meet National Functional Guidelines.
•Research Associate (2009-2010) University of Massachusetts Medical School, Worcester, MA.
Purified firefly luciferase and generated firefly luciferase point-mutants for enzymatic characterization of novel substrates.
Prepared publication-quality graphics and experimental protocols as a co-author for a subsequently published scientific journal article on novel luciferase substrates.
•Cut and Wrap Sanitation (2008–2009) Cabot Creamery Cooperative, Cabot, VT.
Performed sanitation standard operating procedures (SOPs) per relevant good manufacturing practices (GMPs).
•Research Technician (2007–2008) Brandeis University, Waltham, MA.
Purified (by dye- or ligand-affinity chromatography) bacterially produced Crytosporidium parvum inosine monophosphate dehydrogenase (IMPDH) and screened potential drug compounds for IMPDH inhibition (IC50).
•Microbiological Technician (2006–2007) Seldon Technologies, LLC, Windsor, VT.
Utilized SOPs to test performance of carbon-nanotube water filters.
Prepared viral or bacterial stock solutions for carbon-nanotube water filters in accordance with SOPs.
Assisted in maintaining and cleaning filtration holding tanks and equipment.
•Senior Research Technician (2005–2006) SAIC-Frederick, Frederick, MD.
Purified (by nickel-affinity, ion-exchange, or size-exclusion ÄKTA FPLC; or by preparative, reverse-phase HPLC) bacterially produced proteins with defined ratios of incorporated isotopic labels (as determined by liquid chromatography-mass spectroscopy) for subsequent NMR protein structural analysis.
Developed denaturation/refolding/ultrafiltration protein preparation methods for difficult-to-refold-, multicysteine-containing proteins/cytokines with written protocols.
•Biologist (1996–2005) National Institutes of Health, Bethesda, MD.
Purified (by size-exclusion or affinity chromatography) bacterially produced, biologically active recombinant enzymes (affinity- or non-affinity-tagged) for radiochemical (GTP-binding) and immunochemical (SDS-PAGE with western-blot or immunoprecipitation) post-translational modification studies.
Developed procedures to enzymatically digest and HPLC-purify protein peptides to determine specific sites of post-translational modification.
Investigated effects of the apoptosis-inducing agent, staurosporine, on nitric oxide production by endothelial nitric oxide synthase (eNOS).
Developed an immunoprecipitation/radiochemical assay to determine eNOS enzymatic activity in transiently transfected COS-7 cells.
Performed standard molecular biology techniques (PCR, plasmid-DNA isolation, restriction-enzyme digestion, vector/insert isolation, ligation and subcloning, PCR-generated point-mutation, DNA-sequencing).
Performed standard aseptic tissue culture techniques using multiple mammalian cell lines.
•Lab Scientist (1995–1996) University of Maryland, College Park, MD.
Prepared and purified 125I-labeled protein and peptide hormone reagents; implemented set up of a growth hormone radioimmunoassay.
•Biologist (1988–1994) Food and Drug Administration (CBER), Bethesda, MD.
Vaccinated live mice and surgically removed livers to measure vaccine-induced alterations in cytochrome P-450 superfamily enzymatic activities via radiochemical, ELISA or northern-blot analysis.
Isolated primary murine bone-marrow derived mast cells; purified mRNA, synthesized cDNA and performed semi-quantitative RT-PCR.
•Physical Science Technician (1981–1988) National Institutes of Health, Bethesda, MD.
oPerformed quantitative trace-metal analysis and method development at the part-per-billion level by atomic absorption spectroscopy.
EDUCATION:
B.S., microbiology, cum laude (1988) University of Maryland, University College.
PUBLICATIONS:
Reddy, G.R., Thompson W.C. and Miller S.C. (2010) “Robust light emission from cyclic alkylaminoluciferin substrates for firefly luciferase,” J. Am. Chem. Soc.,132(39):13586-7.
Hiroi, T., Someya, A., Thompson, W., Moss, J. and Vaughan, M. (2006) “GEP100/BRAG2: activator of ADP-ribosylation factor 6 for regulation of cell adhesion and actin cytoskeleton via E-cadherin and alpha-catenin,” Proc. Natl. Acad. Sci.,103(28):10672-7.
Tesauro, M., Thompson, W.C. and Moss, J. (2006) “Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells,” Cell Death Differ.,13(4):597-606.
Tesauro, M., Thompson, W.C., Rogliani, P., Qi, L., Chaudhary, P.P. and Moss, J. (2000) “Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298,” Proc. Natl. Acad. Sci., 97(6):2832-5.
Weng, B., Thompson, W.C., Kim, H.J., Levine, R.L. and Moss, J. (1999) “Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation,” J. Biol. Chem., 274(45):31797-803.
Rivera-Nieves, J., Thompson, W.C., Levine, R.L. and Moss, J. (1999) “Thiols mediate superoxide-dependent NADH modification of glyceraldehydes-3-phosphate dehydrogenase,” J. Biol. Chem., 274(28):19525-31.
Zolkiewska, A., Thompson, W.C. and Moss, J. (1998) “Interaction of integrin alpha 7 beta 1 in C2C12 myotubes and in solution with laminin,” Exp. Cell Res., 240(1):86-94.
Bloom, E.T., Thompson, W.C., Horvath-Arcidiacono, J.A. and Burd, P.R. (1995) “Differential effects of interleukin-12 treatment on gene expression by allostimulated T cells from young and aged mice,” Mech. Ageing Dev., 85(2-3):109-24.
Burd, P.R., Thompson, W.C., Max, E.E.and Mills, F.C. (1995) “Activated mast cells produce interleukin 13,” J. Exp. Med., 181(4):1373-80.
Ansher, S.S. and Thompson, W. (1994) “Modulation of hepatic mRNA levels after administration of lipopolysaccharide and diphtheria and tetanus toxoids and pertussis vaccine adsorbed (DTP vaccine) to mice,” Hepatology, 20(4 Pt 1):984-91.
Ansher, S., Thompson, W. and Watson, R. (1994) “Alterations of hepatic drug metabolism in mice following infection with the murine retrovirus LP-BM5,” Immunopharmacology, 27(3):215-23.
Ansher, S., Thompson, W., Bridgewater, J. and Snoy, P. (1993) “Pertussis toxin-induced alteration of murine hepatic drug metabolism following administration of diptheria and tetanus toxoids and pertussis vaccine adsorbed,” Infect Immun., 61(10):4240-7.
Ansher, S., Thompson, W., Snoy, P. and Habig, W. (1992) “Role of endotoxin in alterations of hepatic drug metabolism by diptheria and tetanus toxoids and pertussis vaccine adsorbed,” Infect. Immun., 60(9):3790-8.
Ansher, S.S., Puri, R.K., Thompson, W.C. and Habig, W,H. (1992) “ The effects of interleukin 2 and alpha-interferon administration on hepatic drug metabolism in mice,” Cancer Res., 52(2):262-6.
Ansher, S., Thompson, W. and Habig, W. (1991) “Vaccine-induced alteration in hepatic drug metabolism,” Vaccine, 9(4):277-83.
LeRoy, A.F. and Thompson, W.C. (1989) “Binding kinetics of tetrachloro-1,2-diaminocyclohexaneplatinum (IV) (tetraplatin) and cis-diamminechloroplatinum (II) at 37 degrees C with human plasma proteins and with bovine serum albumin. Does aquation precede protein binding?” J. Natl. Cancer Inst., 81(6):427-36.
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