[Interleukin-2 gene cloning and expression in S. cerevisiae].
Mature human Interleukin-2 gene was amplified from IL-2 cDNA by PCR methods. The PCR product was cloned into pUC12 plasmid at Sma I site, then precisely engineered into an intermidiate vector pSK43SB which were digested with Hind III, mung bean nuclease, and Sal I. Added BamH I linker to Cla I site of pSK43SB-IL2 to produce one more BamH I site, then digested pSK43DB-IL2 with restriction enzyme BamH I, the BamH I fragment with an alpha-factor leading sequence, IL2 gene and cycl terminater were produced. It was then cloned into a typical high efficiency episomal expression vector YEpHc8. Human IL-2 protein was purified from supernatant secreted by yeast transformants of YEpHc8-IL2 leading by alpha-factor promoter with IL-2 right translational reading frame and accurate cleavage site. High level expression of IL-2 with highly activity has been purified. A sugar specific silver staining method and 3H sugar labelling experiment has shown the detection of carbohydrate component.
Zhang
X
X
Institute of Basic Medical Sciences, CAMS, Beijing.
Huang
B
B
Cai
L
L
D003001:Cloning, Molecular; D006031:Glycosylation; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D007376:Interleukin-2; D011993:Recombinant Fusion Proteins