High-level expression of human lipocortin I in the fission yeast Schizosaccharomyces pombe using a novel expression vector.
We have developed a novel expression system that allows the fission yeast, Schizosaccharomyces pombe, to be used for the efficient overproduction of heterologous proteins. As an example of the utility of this system, human lipocortin I was expressed to 50 percent of soluble protein, and 150 mg of highly purified material was obtained from 10 grams of wet cell paste. Expression of lipocortin I was driven by the human cytomegalovirus (hCMV) promoter in a vector that also contains a neomycin resistance gene (neo) under the control of the SV40 early promoter, permitting selection for increasing copy-number with increasing concentrations of the antibiotic G418. The purified protein was equivalent to its native counterpart with respect to antigenicity and biochemical properties such as phospholipase A2 inhibition, actin binding and N-terminal acetylation. We have also used this system to produce comparable amounts of other proteins including rat arginase, rat NDP-kinase and human interleukin-6.
Giga-Hama
Y
Y
Research Center, Asahi Glass Co. Ltd, Kanagawa, Japan.
Tohda
H
H
Okada
H
H
Owada
M K
MK
Okayama
H
H
Kumagai
H
H
D000818:Animals; D017305:Annexin A1 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D009355:Neomycin / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D012568:Schizosaccharomyces; D012995:Solubility
D017305:Annexin A1; D009355:Neomycin