High-level Expression, Purification And Study Of Bioactivity Of Fusion Protein Melittin-IL-2(⁸⁸Arg,¹²⁵Ala) In Pichia Pastoris

Objective To construct expression system of fusion protein melittin-IL-2(88Arg,125Ala) in pichia pastoris, and obtain the high-level expression of interest protein. Then purify interest protein, and study the bioactivity of it. Method The purpose gene was obtained through PCR technique from the recombinant vector of pET-15b/M-IL-2(88Arg,125Ala), and cloned into the vector pPICZa A. The sac I-linearized plasmid pPICZaA/M-IL-2(88Arg,125Ala) was transformed into P. pastoris GS115by electroporation. ZeocinTM screened the positive yeast strains. The expression was induced with0.5%methanol at28℃, pH6.0. The fusion protein M-IL-2(88Arg,125Ala) was purified by ammonium sulfate fractionation, dialysis and Ni2+affinity chromatography. The fusion protein was assayed with SDS-PAGE and Western-blot. Antibacterial activity of the purified interest protein was assayed by liquid turbidity analysis. CCK-8assay was used to detect the inhibitory effects of the purified interest protein on the SKOV3cells. Result It was proved that the fragment amplified was inserted into the P. Pastoris expression vector pPICZaA correctly by PCR and gene sequencing. After electroporation of pichia, the rencombinant plasmid was integrated into the regions of homology within the yeast genome. RT-PCR detecting indicated that we have obtained two multicopy recombinant strans growing on YPD with400μg/mL ZeocinTM. SDS-PAGE indicated that the fusion protein molecular weight is about26KD, conforming the theoretical value. And M-IL-2(88Arg,125Ala) possesses strong antigen-specificity by western blot detection. Antibacterial assay indicated that and Bioassay results indicated that the fusion protein has antibacterial activity and could directly inhibit the growth of human ovarian cancer SKOV3cells and Hela cells in vitro. Conclusion This study provides an alternative strategy for large-scale production of bioactive M-IL-2(88Arg,125Ala) using P. pastoris as an expression host and paves the way to clinical practice.