The major goals of the RPEU are to 1) develop a state-of-the-art recombinant protein expression facility to fill the gap between gene isolation and protein function/structure and 2) provide or assist in the production of milligram-to-gram amounts of adequately folded and post-translationally modified near clinical grade material for preclinical studies of promising parasite vaccines. The RPEU consists of three groups: 1) a molecular biology group for designing and engineering recombinant DNA for transfection into bacteria, yeast and baculovirus; 2) a production group which analyzes protein production and optimizes fermentation/growth conditions; and 3) a protein purification and analysis group that works on post-production process development and does preliminary analysis of the resultant recombinant protein for structural and immunological integrity. The latter group has the facility for purifying proteins in controlled environments similar to those required for clinical grade production. In addition to actual process development, the RPEU also provides detailed Good Manufacturing Practice-grade documentation to support the transfer of technology to GMP pilot plants. The RPEU interacts with a number of collaborators including LPD and extramural scientists whose major goals are studying the functional/immunological and structural characteristics of recombinant parasite proteins. The projects undertaken by the RPEU in the past year include: 1) pre-erythrocytic malaria parasite antigens expressed in yeast (CSP and TRAP); 2) recombinant asexual blood stage antigens expressed in bacteria, yeast, baculovirus, mammalian cells and transgenic animals (MSP1, AMA1, SERA, PfEMP1, Sequestrin); 3) preparation of the Master Cell Bank for GMP production of a malaria transmission-blocking target antigen (TBV25-28) and optimization of production of this antigen in yeast; 4) Pgs25 and Pgs28 for the study of P. gallinaceum ookinete invasion of mosquito midguts, and An. gambiae late midgut trypsin; 5) schistosomiasis antigen (CALPAIN); 6) onchocerciasis antigens (OV 11.21 and aldolase); and 7) leishmania antigens (3'-nucleotidase and chitinase). We have started to explore the utility of recombinant Crithidia as an expression system for heterologous protozoan protein expression. This RPEU has capabilities at 1, 5 and 10 Liter scale. A Class 10000 clean room for preparation of ultra-pure, near endotoxin-free recombinant protein for preclinical studies. The RPEU supplied the documentation for production of clinical-grade P30P2-MSP1 and TBV25-28 and was the lead agency filing an FDA-approved IND application for testing P30P2-MSP1 in a Phase I human clinical trial.
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