Preliminary purification and expression of Asian malaria vaccine candidate recombinant erythrocyte-binding antigen 140

Abstract

Background: Causing an estimated 219 million cases in 2017, malaria is a life-threatening mosquito-borne disease caused by Plasmodium parasites, with P. falciparum being the most deadly. P. falciparum antigens involved in erythrocyte recognition and invasion during the merozoite stage are likely targets of protective immune responses. Natural antibody responses against the Erythrocyte Binding Antigen 140 (EBA-140) ligand have been found in individuals in malaria-endemic areas. EBA-140, a member of the Pf erythrocyte binding antigen (EBA) family, is one of the prospective candidates for malaria vaccine development. Methods and materials: In the study, the Philippine malaria parasite strain, FCH/4, was identified using its sequences in Genbank. Erythrocyte-binding antigen 140 (EBA-140) was selected by in silico screening of putative vaccine candidates in P. falciparum using the genomic database PlasmoDB and was then synthesized as codon-optimized full-length EBA-140. This 3284 bp gene was cloned into peT30-a(+) vector, transformed and expressed in E. coli BL21(DE3) cells. The EBA-140 gene insert was confirmed via restriction enzyme digestion and its protein 3D conformation was predicted using I-TASSER. Protein expression was optimized at 28 °C induction for 17 h using 0.3 mM IPTG. Recombinant EBA-140 in the native lysate was initially run on a weak anion-exchanger and then on a hydrophobic interaction column for purification. Results: The recombinant EBA-140 was constitutively expressed in the cytosol and highly expressed in the inclusion bodies upon induction at 28 °C for 17 h using 0.3 mM IPTG. The expressed rEBA-140 was then purified by several optimization steps of anion exchange chromatography, producing a 51.9% pure protein. To further purify the protein, hydrophobic interaction chromatography was employed. Conclusion: Recombinant EBA-140 was successfully expressed and purified optimally using anion exchange chromatography. The hydrophobic interaction chromatography method as polishing step is continuously being optimized to purify the 32% hydrophobic rEBA-140. To further confirm the specificity and identity of the purified fractions, Western blot and peptide mass fingerprinting methods are being optimized. Since no vaccine is yet available for malaria, characterization studies of immunoreactive Plasmodium antigens are of significant importance.