Recombinant human mast-cell chymase: an improved procedure for expression in Pichia pastoris and purification of the highly active enzyme.
Recombinant human mast-cell chymase: an improved procedure for expression in Pichia pastoris and purification of the highly active enzyme.
复制标题
重组人肥大细胞糜酶：一种在毕赤酵母中表达和纯化高活性酶的改进程序。
DOI：
10.1042/ba20040074
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发表时间：
2005
影响因子：
2.8
通讯作者：
Johnson,DavidA
中科院分区：
文献类型：
--
作者：
Lockhart,BrentE;Vencill,JessicaR;Felix,CheriseM;Johnson,DavidA
人肥大细胞糜蛋白酶（EC 3.4.21.39）是一种胰凝乳蛋白酶样丝氨酸蛋白酶，储存在肥大细胞颗粒中并从肥大细胞颗粒释放。该酶已在毕赤酵母中通过编码成熟活性糜酶的cDNA同源重组到毕赤酵母基因组中而表达。通过活性筛选选择产生最高水平的重组人糜酶的细胞，并将它们在发酵罐中生长。甲醇诱导导致活性糜酶分泌到毕赤酵母生长培养基中，并且在培养基中检测到增加的酶水平持续5天。通过简单的两步程序从培养基中纯化活性酶，活性产率为22%，该程序包括疏水相互作用色谱法，然后在固定化肝素上进行亲和色谱法。肝素柱的主峰在SDS/PAGE上含有30.6 kDa的单一条带。纯化的重组人糜酶活性为96%，产量为2.2 mg/l生长培养基。
Human mast‐cell chymase (EC 3.4.21.39) is a chymotrypsin‐like serine protease that is stored in and released from mast‐cell granules. This enzyme has been expressed inPichia pastorisby homologous recombination of the cDNA coding for the mature active chymase into thePichiagenome. Cells producing the highest levels of recombinant human chymase were selected by activity screening and they were grown in a fermentor. Methanol induction resulted in the secretion of active chymase into thePichiagrowth media and increasing levels of enzyme were detected in the media for 5 days. Active enzyme was purified from the culture media with a 22% yield of activity by a simple two‐step procedure involving hydrophobic‐interaction chromatography followed by affinity chromatography on immobilized heparin. The major peak from the heparin column contained a single band of 30.6 kDa on SDS/PAGE. The purified recombinant human chymase was 96% active and the yield was 2.2 mg/l of growth media.