Expression of a New Type Human Interleukin-2 Gene in Pichia pastoris
LIU Yan 1),3)* , HU Ying he 2) , OUYANG Ke qing 3) , CAI Shao xi 3)
( 1) College of Life Sciences, Southwest Normal University, Chongqing 400715, China; 2) Hugene Corporation, San Diego, CA 92130, USA; 3) College of Bioengineering, Chongqing University, Chongqing 400044, China
Abstract：In order to improve the stability and bioactivity of interleukin 2 and decrease its toxic side effects, it is necessary to reconstruct rhIL 2 for obtaining a high level expression of human IL 2 in Pichia pastoris. Mature human Interleukin 2 gene was amplified from IL 2 cDNA by PCR, using the technique of site specific mutagenesis with a synthetic oligonucleotide primer, where the codon for cysteine 125 of human IL 2 was replaced with alanine, leucine 18 with methionine and leucine 19 with serine. The gene was ligated with yeast expression vector pPIC9K. The constructed plasmid, pPIC9K MvhIL 2, was linearized by Sal Ⅰ and transformed into Pichia pastoris KM71 by Pichia EasyCom TM Transformation Kit. Through selection of G418 resistant transformation and expressing clones, a high expression transformant was obtained. Pichia pastoris yeast cells integrating the plasmid pPIC9K MvhIL 2 produced a high level of human IL 2 about 45 7% of the total yeast body protein with immunocompetence by Western blotting. The specific activity of the purified MvhIL 2 was 4 0×10 7 IU/mg protein, which was 4～5 times of the NhIL 2.
刘堰,胡应和,欧阳克清,蔡绍皙. 突变型人白细胞介素-2基因在巴斯德毕赤酵母中的表达[J]. 中国生物化学与分子生物学报, 2003, 19(05): 618-624.
LIU Yan 1),3)* , HU Ying he 2) , OUYANG Ke qing 3) , CAI Shao xi 3). Expression of a New Type Human Interleukin-2 Gene in Pichia pastoris. Chinese Journal of Biochemistry and Molecular Biol, 2003, 19(05): 618-624.