Kim K Y, Lim H K, Lee K J, Park D H, Kang K W, Chung S I, Jung K H
Mogam Biotechnology Research Institute, 341 Pojung-Ri, Koosung-Myun, Yongin, Kyonggi-Do, 449-910, Korea.
Protein Expr Purif. 2000 Oct;20(1):1-9. doi: 10.1006/prep.2000.1300.
The elastase-specific inhibitor, guamerin, was expressed and secreted into a culture medium using the methylotrophic yeast Pichia pastoris, and the resulting recombinant guamerin was purified from the culture media using a two-step procedure composed of a hydrophobic interaction and reverse-phase chromatography. Up to 90 g/L of dry cell weight, the guamerin-producing recombinant P. pastoris was cultivated and guamerin was secreted into the culture medium at a level of 0.69 g/L. The recombinant guamerin was highly purified (>98%) with a recovery yield of 68%. Analyses of the purified guamerin revealed the same N-terminal amino acid sequence, amino acid composition, and molecular mass as found in the native leech protein. The recombinant guamerin exhibited the tight binding to porcine pancreatic elastase. Furthermore, the recombinant guamerin did not produce a humoral immune response in mice.
利用甲基营养型酵母毕赤酵母表达弹性蛋白酶特异性抑制剂胍蛋白并分泌到培养基中，然后通过由疏水相互作用和反相色谱组成的两步法从培养基中纯化得到重组胍蛋白。以高达90 g/L的干细胞重量培养产生胍蛋白的重组毕赤酵母，胍蛋白分泌到培养基中的水平为0.69 g/L。重组胍蛋白高度纯化（>98%），回收率为68%。对纯化的胍蛋白的分析显示，其N端氨基酸序列、氨基酸组成和分子量与天然水蛭蛋白相同。重组胍蛋白与猪胰弹性蛋白酶紧密结合。此外，重组胍蛋白在小鼠中未产生体液免疫反应。
