title: Expression and purification of a mutant of human interleukin-2 in Pichia pastoris.
abstract: Interleukin (IL)-2 is a pharmacologically important cytokine secreted by T-lymphocytes. Recombinant IL-2 (rIL-2) has been modified and produced in many systems. Mass production of rIL-2 is the prerequisite for its wide application. Using a site-directed mutagenesis strategy, we first generated a gene coding for a new type of mutant of human IL-2 (MhIL-2), in which we replaced the cysteine-125 in human IL-2 with alanine, the leucine-18 with methionine, and the leucine-19 with serine. Then we investigated the possibility of its production of MhIL-2 in a Pichia pastoris system. High-level secreted expression of MhIL-2 was achieved by methanol induction. When purified with ultrafiltration, cation-exchange chromatography, and Sephadex G100 gel filtration, about 100 mg of MhIL-2 with high purity was obtained from 1 L of ferment supernatant. Biologic activity assay revealed that the purified recombinant protein displayed increased activity on proliferation of IL-2-dependent CTLL-2 cells. These results suggest that MhIL-2 is an improved IL-2 mutant that might hold great promise for clinical use, and that P. pastoris is an excellent system for the mass production of biologically active hIL-2.
authors.affiliation: College of Life Science, Southwest University, Chongqing 400715, P.R. China. liuyan922@hotmail.com
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Xun-Yan
authors.initials: XY
authors.lastname: Xiao
authors.forename: Min
authors.initials: M
authors.lastname: Sun
authors.forename: Ying-He
authors.initials: YH
authors.lastname: Hu
authors.forename: Ke-Qing
authors.initials: KQ
authors.lastname: Ou-Yang
authors.forename: Shao-Xi
authors.initials: SX
authors.lastname: Cai
authors.forename: Zi-Chun
authors.initials: ZC
authors.lastname: Hua
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: C508594:IL2 protein, human; D007376:Interleukin-2; D011994:Recombinant Proteins