Expression of a new type human interleukin-2 gene in Pichia pastoris

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.