title: High-level expression, purification and study of bioactivity of fusion protein M-IL-2((88)Arg, (125)Ala) 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 814.5mg/L, higher than the system in Escherichiacoli. 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 26kDa, 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.
authors: Department of Microbiology, Qingdao University Medical College, Qingdao 266071, People's Republic of China.
authors: Lin
authors: L
authors: Li
authors: Department of Microbiology, Qingdao University Medical College, Qingdao 266071, People's Republic of China.
authors: Dongmeng
authors: D
authors: Qian
authors: Department of Microbiology, Qingdao University Medical College, Qingdao 266071, People's Republic of China.
authors: Guangcan
authors: G
authors: Shao
authors: Department of Microbiology, Qingdao University Medical College, Qingdao 266071, People's Republic of China.
authors: Zhiyong
authors: Z
authors: Yan
authors: Department of Biology, Qingdao University College of Life Science, Qingdao 266071, People's Republic of China.
authors: Ronggui
authors: R
authors: Li
authors: Department of Microbiology, Qingdao University Medical College, Qingdao 266071, People's Republic of China.
authors: Xiaomin
authors: X
authors: Hua
authors: Department of Microbiology, Qingdao University Medical College, Qingdao 266071, People's Republic of China.
authors: Xuxia
authors: X
authors: Song
authors: Department of Microbiology, Qingdao University Medical College, Qingdao 266071, People's Republic of China. Electronic address: qdmblm@163.com.
authors: Bin
authors: B
authors: Wang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D008555:Melitten / Q000235:genetics*; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000188:drug therapy*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*
chemical_list: C508594:IL2 protein, human; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D008555:Melitten
keywords: Bioactivity; Expression; Fusion protein; Pichia pastoris; Purification