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Expression and purification of a mutant of human interleukin-2 in Pichia pastoris
Authors:Yan Liu  Xun-Yan Xiao  Min Sun  Ying-He Hu  Ke-Qing Ou-Yang  Shao-Xi Cai  Zi-Chun Hua
Affiliation:(1) College of Life Science, Southwest University, 400715 Chongqing, P.R. China;(2) College of Bioengineering, Chongqing University, 400044 Chongqing, P.R. China;(3) State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, P.R. China
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.
Keywords:Human interleukin-2  site-directed mutagenesis   Pichia pastoris   purification  increased activity
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