Effects of antonymous mutation of 17th condon of salivary HRP5 on its expression in Pichia pastoris / 解放军医学杂志

ABSTRACT

Objective To induce the the 17th condon antonymous mutagenesis of salivary histatin 5(HRP5) cDNA,express the mutant and hrp5 in Pichia pastoris,and to study the effects of mutation on expression.Methods According to the Pichia pastoris' codon bias,two pairs of primers(H1 and H2,H3 and H4) were designed.H1 and H2,H3 and H4 have complementary 3' end,and the EcoR I site was added to the 5' end of H1 and H3,Sal I site to H2,H4.The cDNA of hrp5 and hrp5' was generated with PCR by H1 and H2,H3 and H4,respectively.The secrete vector pPICZ?-A,hrp5 and hrp5' were digested with EcoR I +Sal I,linkede by T4 DNA ligase and transformed to E.coli TOP10 comptetent cell,positive colonies were screened on LB plates with Zeocin.The recombinant plasmids pPICZ?-A-hrp5 and pPICZ?-A-hrp5' identified by digestion and DNA sequencing were amplified largely,linearized by Sac I and transformed to GS115 comptetent cell by electroporation,positive colonies were screened on YEPD plates with Zeocin,the recombinant GS115 were confirmed by PCR,cultured and induced expression by methanol.The amount and anticandidal activity of the expressed products was compared with synthetic HRP5.Results Both hrp5 and hrp5' were integrated into the genome of GS115 and expressed successfully,the anticandidal activity of the recombinant HRP5 and HRP5' was identical with synthetic HRP5,the amount of expressed HRP5 and HRP5' was 4?mol/L and 5?mol/L,respectively.Conclusion Both the recombinant HRP5 and HRP5' showed better anticandidal activity.The amount of expressed prodcts increased 25% by substituting Asn for Lys17 without changing anticandidal activity.