Office for Research and Graduate Studies (ORAGS)

Protein Core (Center for Vaccine Development)

Background:  The Protein Core Facility has been in the Center for Vaccine Development in one form or another for well over a decade.  Known within the CVD as the Antigen Purification Unit, the lab has been producing protein and carbohydrate antigens, largely for studying sera from animal models that have been challenged with trial vaccines or vaccine components.  Recently, Dr. Keith Inman was asked to re-establish the former Antigen Purification Unit with a broader mission, to include an effort in protein structural biology.  Dr. Inman’s appointment as Assistant Professor of Pediatrics began in July of 2004, simultaneous to his moving into lab space adequate for the task.  An excellent technician joined the lab in May 2005, and the lab should be operating at full speed within the next few months.

Scientific Objectives:  It is the mission of the Protein Core Facility (PCF) at the Center for Vaccine Development (CVD) to support and advance research capabilities at the CVD and elsewhere by providing high quality protein and antigen purification support, and to facilitate access to structural biology resources.  Current and planned services of the Protein Core Facility include construction and optimization of expression systems, protein purification, protein folding, and a number of analytical techniques.  The latter include protein quantitation and purity analysis (e.g., endotoxin detection), bioinformatics analysis, and aid in beginning structural biology projects.

A major goal of the CVD Protein Core is to provide CVD researchers with purified antigen for use in vaccine research.  The chief concern for our products is immunogenic purity.  Emphasis is placed on careful monitoring and removal of bacterial endotoxin and other immunogens from recombinant protein preparations, while minimizing epitope degradation (for example, deamidation or improper folding).  Another key component of our mission will be to bridge the gap between protein discovery and structural biology.  We will offer fee-for-service optimization of protein expression, protein refolding, and sample preparations necessary for NMR and X-ray crystallography projects.  Our approach to expression optimization will be through high-throughput screening of vectors, host strains, and culture conditions.  Host organisms will initially be standard Escherichia coli expression strains, but will ultimately include other systems, such as yeast, insect, and mammalian cells, and cell-free systems.  After expression conditions have been worked out, purification protocols can then be developed and optimized by the PCF, the lab originating the work, or elsewhere.  PCF purification strategies include screening conditions for refolding, where required.  Finally, when sufficient quantities of purified, properly folded protein can be obtained for structural studies, we will offer to screen conditions for NMR and/or crystallization.

Personnel:

Keith G. Inman, Ph.D., Director.  Dr. Inman’s training in Protein NMR and methods of protein purification and analysis were completed in the laboratory of Dr. David Weber, in the Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore.  Following completion of his Ph.D. in 2003, he trained briefly as a post-doctoral fellow under Jim Nataro in the Department of Pediatrics, also at the School of Medicine and Center for Vaccine Development, working on various proteins of Gram-negative pathogens, while beginning to set up the PCF.  As Director of the lab, Dr. Inman will be responsible for designing purification and refolding schemes and overseeing their implementation, as well as scheduling the work and price schedules for the PCF with guidance from the Faculty Advisory Committee as required.

Jason E. Savage, M.S., Research Assistant.  Mr. Savage received his Master’s degree in August 2004 and has been with the PCF since May 9, 2005.  His background is in Molecular Biology, having worked on an RNA virus of Drosophila melanogaster.  Protein purification is not something he has extensive experience with; however, he is familiar with the principles involved in most of the techniques involved and is quickly becoming adept at running our FPLC and lab operations in general.  Mr. Savage’s duties will include performing most of the routine protein purifications for the Core, as well as maintain laboratory inventories and equipment under the close supervision and guidance of the Director.

Tatiana C. da Silva, M.S., Research Fellow (temporary).  Ms. Silva has been working for the Protein Core Facility since August of 2004, dividing her time between Core projects and those of Dr. James Nataro.  Her background is in organic synthesis and pharmaceutical sciences, and was hired originally to bridge a gap between technicians.  Her tenure with us is probably nearly complete, when she finishes her current project for a contract with SIGA Technologies.

Equipment:

1.         ÄKTAprime FPLC.  Amersham Biosciences (now GE Healthcare)
2.         DU640 Spectrophotometer.  Beckmann Instruments, Inc.
3.         Tangential Flow Apparatus.  “Flexstand” from Amersham Biosciences.
4.         Tangential Flow Apparatus.  “Quixstand” from Amersham Biosciences. 
5.         Analytical balance, .01 mg.  Scientech SM128D.
6.         Sorvall RC-5B.  GS-3 and SS-34 rotors.
7.         Criterion electrophoresis and electroblotting system.
8.         Hybridization oven.
9.         Multitron shaker/incubator, two-unit.
10.       Microtiter plate shaker, 4-plate, for high-throughput screening of conditions for protein  expression or refolding.  (On-order.)

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