Authors: Liu, Yan¹; Xiao, Xun-Yan²; Sun, Min²; Hu, Ying-He³; Ou-Yang, Ke-Qing³; Cai, Shao-Xi⁴; Hua, Zi-Chun⁵

Source: Applied Biochemistry and Biotechnology, Volume 133, Number 1, April 2006 , pp. 77-86(10)

Publisher: Humana Press

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

Document Type: Research article

DOI: http://dx.doi.org/10.1385/ABAB:133:1:77

Affiliations: 1: College of Life Science, Southwest University, 400715, Chongqing, P.R. China, Email: liuyan992@hotmail.com 2: College of Life Science, Southwest University, 400715, Chongqing, P.R. China, 3: College of Bioengineering, Chongqing University, 400044, Chongqing, P.R. China, 4: College of Bioengineering, Chongqing University, 400044, Chongqing, P.R. China, Email: sxcai@cqu.edu.cn 5: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093, Nanjing, P.R. China, Email: zchua@nju.edu.cn

Publication date: 2006-04-01

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• In this Subject: Anatomy & Physiology ,  Biotechnology ,  Organic Chemistry ,  Biochemistry
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