Production and purification of human lipocortin-I secreted by recombinant Saccharomyces cerevisiae = 재조합 Saccharomyces cerevisiae로부터 인체 리포코틴-I의 분비 생산 및 정제

Abstract

Human lip)cortin-I (LCD is a calcium ion-dependent and phospholipid -binding protein which exhib its an anti기 iflammatory activity by inhibiting phospholipase A2 activity. In this study, the LCI gene containing it ; own terminator region was joined to GA上 10 promoter-ppL (prepro-leader sequence of mating facte" a). An ATG start codon of LCI gene was placed at downstream with KR endoprotease recognition . ite (Lys-Arg) of ppL. Recombinant S. cerevi.stae harboring the LCI expression/secretion vector, pYG J)T5, was aerobically grown on a liquid YPDG medium at 30"C for 72h「s. The whole cell and culture ;upernatant were separated after centrifugation, and the expressed LCI was analyzed by SDS-PAGE and western blotting methods. A majority fraction of the expressed LCI was found to be accumulatec in the intracellular fraction, resulting in very low secretion efficiency of about 7.4%. About 500rr i/k of LCI was extracellularly produced by the fed-batch culture employing the controlled -feeding of ；lucose and galactose. The secreted LCI was purified by ultrafiltration and hydroxylapatite column chrc natography, and a purity of more than 99% was obtained.