Production and identification of PfCP-2.9 mutants. (A) Schematic representation of site-directed mutagenesis of PfCP-2.9. The 17 mutants were designated according to the positions of corresponding mutated amino acid resudues on PfCP-2.9. The N-terminal signal peptide (SP) and C terminal 6×His tag of each construct remained intact, and were involved in subsequent expression and purification, respectively. (B) Assembly of each mutant gene by overlap extension PCR method described in Method section. The 176 U was generated by PCR using the forward universal primer and a reverse mutant primer, while the 176 D fragment generated using the reverse universal primer and a forward mutant primer. The two fragments were combined to generate the entire mutant gene (176F) by PCR using the forward and reverse universal primers. (C) Purification of PfCP-2.9 mutants by Ni-NTA chromotography. The supernatant of culture expressing M176 gene in P. pastoris was applied to Ni-NTA agarose columns. Pre- and post-column fraction as well as elution fractions with various concentration of imidazole was analyzed on a 12% SDS-PAGE gel, followed by Coomassie blue staining. (D) SDS-PAGE analysis of purificed PfCP-2.9 mutants. Each lane indicated each purified PfCP-2.9 mutant; (E) Western blot analysis of PfCP-2.9 mutants. Polyclonal rabbit antibodies to the PfCP-2.9 were used as the primary antibody for this detection.