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KMID : 0380019950100030343
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Korean Journal of Biotechnology and Bioengineering
1995 Volume.10 No. 3 p.343 ~ p.348
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Production and Purification of Human Lipocortin-I secreted by Recombinant Saccharomyces cerevisiae
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김병문/Kim, Byung Moon
정봉현/이상기/박영훈/남수완/Chung, Bong Hyun/Rhee, Sang Ki/Park, Young Hoon/Nam, Soo Wan
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Abstract
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Human lipocortin-I (LCI) is a calcium ion-dependent and phospholipid-binding protein which exhibits an anti-inflammatory activity by inhibiting phospholipase A, activity. In this study, the LCI gene containing its own terminator region was joined to GAL10 promoter-ppL (prepro-leader sequence of mating factor a). An ATG start codon of LCI gene was placed at downstream with KR endoprotease recognition site (Lys-Arg) of ppL. Recombinant S. cerevisiae harboring the LCI expression/secretion vecto;pYGLPT5, was aerobically grown on a liquid YPDG medium at 30 C for 72hrs. The whole cell and culture supernatant 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 accumulated in the intracellular fraction, resulting in very low secretion efficiency of about 7.4%. About 500mg/h of LCI was extracellularly produced by the fed-batch culture employing the controlled -feeding of glucose and galactose. The secreted LCI was purified by ultrafiltration and hydroxylapatite column chromatography, and a purity of more than 99% was obtained.
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