Stabilization of a chimeric malaria antigen in separation and purification through efficient inhibition of protease activity by imidazole

Abstract

Abstract A chimeric antigen M.RCAg-1 of Plasmodium falciparum expressed in Escherichia coli was previously demonstrated inhibiting the growth of malaria parasites in vitro, but its further development has been retarded by the antigen’s instability during the downstream process. In this study, it was definitely demonstrated the instability was caused by the susceptibility of M.RCAg-1 to metalloprotease(s) released from the disintegrated host cells. Interestingly, imidazole showed better inhibition effects on the degradation than EDTA. Hence, a purification procedure was successfully developed to produce M.RCAg-1 with a purity of up to 95% and an overall recovery of nearly 600 mg/L culture. When performed this protocol following the Good Manufacturing Practice regulations, the endotoxin level, the host protein content and residual DNA level, all met the FDA standards. MALDI-TOF MS demonstrated a consistent molecular weight with the theoretical value and CD revealed a mainly disordered random coil secondary structure. Immunizing mice with M.RCAg-1 with Freund’s adjuvant elicited high levels of specific antibodies. Moreover, M.RCAg-1 itself could be stable at 4 °C for up to 6 months. Our results would provide an efficient and robust protocol for the large-scale production of M.RCAg-1 which would warrant the further development of this promising malaria vaccine candidate.