Abstract:
:The 42 kDa cleavage product from the carboxyl end of the Plasmodium falciparum merozoite surface protein 1 (MSP1(42)) is an important blood-stage malaria vaccine target. Several recombinant protein expression systems have been used for production of MSP1(42) including yeast (Saccharomyces cerevisiae and Pichia pastoris), Escherichia coli, baculovirus and transgenic animals. To date, all of the reported recombinant proteins include a 6 x His affinity tag to facilitate purification, including three MSP1(42) clinical grade proteins currently in human trials. Under some circumstances, the presence of the 6 x His tag may not be desirable. Therefore, we were interested to produce clinical grade MSP1(42) without a 6 x His affinity tag from E. coli inclusion bodies. We produced a recombinant MSP1(42) with a P. falciparum FUP (Uganda-Palo Alto) phenotype which accounts for a substantial proportion of the MSP1(42) protein observed in African isolates. EcMSP1(42)-FUP was produced in E. coli inclusion bodies by high cell mass induction with IPTG using 5 L and 60 L bioreactors. Isolated inclusion bodies were solubilized in 8M guanidine-HCl and the EcMSP1(42)-FUP protein refolded by rapid dilution. Refolded EcMSP1(42)-FUP was purified using hydrophobic interaction chromatography, anion exchange chromatography, and size exclusion chromatography, and subject to biochemical characterization for integrity, identity, and purity. Endotoxin and host cell protein levels were within acceptable limits for human use. The process was successfully transferred to pilot-scale production in a cGMP environment. A final recovery of 87.8 mg of clinical-grade material per liter of fermentation broth was achieved. The EcMSP1(42)-FUP clinical antigen is available for preclinical evaluation and human studies.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Shimp RL Jr,Martin LB,Zhang Y,Henderson BS,Duggan P,MacDonald NJ,Lebowitz J,Saul A,Narum DLdoi
10.1016/j.pep.2006.06.018subject
Has Abstractpub_date
2006-11-01 00:00:00pages
58-67issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(06)00192-6journal_volume
50pub_type
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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