High-level expression, purification and study of bioactivity of fusion protein M-IL-2(88Arg, 125Ala) in Pichia pastoris

Abstract M-IL-2( 88 Arg, 125 Ala) is a fusion protein comprising melittin genetically linked to a mutant human interleukin 2( 88 Arg, 125 Ala). In this study, we constructed an expression system of M-IL-2( 88 Arg, 125 Ala) in Pichia pastoris : GS115/pPICZα A /M-IL-2 ( 88 Arg, 125 Ala), and achieved the high-level expression of the fusion protein. The maximum yield of the fusion protein M-IL-2( 88 Arg, 125 Ala) reached up to 814.5 mg/L, higher than the system in E scherichia coli. The fusion protein was purified by means of ammonium sulfate fractionation, dialysis and nickel ion affinity chromatography. The molecular weight of the fusion protein is about 26 kDa, conforming the theoretical value. And M-IL-2( 88 Arg, 125 Ala) possesses strong antigen-specificity by Western blot detection. Bioassay results indicated that the fusion protein could directly inhibit the growth of human ovarian cancer SKOV3 cells and Hela cells in vitro . This study provides an alternative strategy for large-scale production of bioactive M-IL-2( 88 Arg, 125 Ala) using P. pastoris as an expression host and paves the way to clinical practice.