Optimized Method for Purification of Expressed Plasmodium Vivax Duffy Binding Protein II (PvDBP II): Implication for Vivax Malaria Vaccine Development

Background: The purity and correct folding of a recombinant protein is critical for any structural, biochemical and vaccine design studies. Plasmodium vivax Duffy binding protein II is a leading vaccine candidate for vivax malaria. In the present study, the purification process of recombinant DBP IX (a variant form of PvDBP II) was optimized to achieve the highest yield and purity. Moreover, naturally acquired IgG antibodies to the expressed protein have been evaluated. Material and Methods: DBP IX was cloned and expressed as a his tagged protein in E. coli. The recombinant protein was purified using Ni NTA agarose and different purification parameters were optimized to achieve the highest yield and purity. The quality of the purified rDBP IX was assessed by different procedures such as SDS PAGE gel analysis in both reducing and non reducing conditions, followed by indirect immunofluorescence antibody test and ELISA using the sera of P. vivax infected patients (n= 202). R esults: DBP IX was successfully cloned, expressed and optimally purified. Differential mobility of the rDBP IX on the SDS PAGE gel in reducing and non reducing conditions, confirmed the presence of disulphide bonds. In addition, anti rPvDBP IX antibody produced in mice recognized the native PvDBP II, suggesting that epitopes in the recombinant protein were similar to the corresponding native form. Finally by performing ELISA experiments, it was demonstrated that natural P. vivax infection produces IgG against rDBP IX (42.1%) whilecytophilicIgG1 antibody (35.4%) was the predominantly detected IgG subclass. Conclusion: The results indicated that the optimally purified rDBP IX was of a quality that could be used in vaccine development research and immunological studies of vivax malaria.