Expression and purification of a mutant of human interleukin-2 in Pichia pastoris.
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.
Liu
Yan
Y
College of Life Science, Southwest University, Chongqing 400715, P.R. China. liuyan922@hotmail.com
Xiao
Xun-Yan
XY
Sun
Min
M
Hu
Ying-He
YH
Ou-Yang
Ke-Qing
KQ
Cai
Shao-Xi
SX
Hua
Zi-Chun
ZC
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
C508594:IL2 protein, human; D007376:Interleukin-2; D011994:Recombinant Proteins