Production and characterization of clinical grade Escherichia coli derived Plasmodium falciparum 42 kDa merozoite surface protein 1 (MSP1(42)) in the absence of an affinity tag.
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.
Shimp
Richard L
RL
Malaria Vaccine Development Branch (MVDB), NIAID/NIH/DHHS, Rockville, MD, USA.
Martin
Laura B
LB
Zhang
Yanling
Y
Henderson
Brian S
BS
Duggan
Peter
P
MacDonald
Nicholas J
NJ
Lebowitz
Jacob
J
Saul
Allan
A
Narum
David L
DL
D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D057927:Hydrophobic and Hydrophilic Interactions; D020066:Merozoite Surface Protein 1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D008970:Molecular Weight; D010963:Plasmodium falciparum / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012995:Solubility; D013997:Time Factors
D020066:Merozoite Surface Protein 1; D011994:Recombinant Proteins