Production and characterization of clinical grade Escherichia coli derived Plasmodium falciparum 42 kDa merozoite surface protein 1 (MSP142) in the absence of an affinity tag

The 42 kDa cleavage product from the carboxyl end of the Plasmodium falciparum merozoite surface protein 1 (MSP142) is an important blood-stage malaria vaccine target. Several recombinant protein expression systems have been used for production of MSP142 including yeast (Saccharomyces cerevisiae and Pichia pastoris), Escherichia coli, baculovirus and transgenic animals. To date, all of the reported recombinant proteins include a 6× His affinity tag to facilitate purification, including three MSP142 clinical grade proteins currently in human trials. Under some circumstances, the presence of the 6× His tag may not be desirable. Therefore, we were interested to produce clinical grade MSP142 without a 6× His affinity tag from E. coli inclusion bodies. We produced a recombinant MSP142 with a P. falciparum FUP (Uganda-Palo Alto) phenotype which accounts for a substantial proportion of the MSP142 protein observed in African isolates. EcMSP142-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 8 M guanidine–HCl and the EcMSP142-FUP protein refolded by rapid dilution. Refolded EcMSP142-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 EcMSP142-FUP clinical antigen is available for preclinical evaluation and human studies.