蛋白酶抑制剂依考汀的表达及其与胰蛋白酶的共结晶。

Expression of the protease inhibitor ecotin and its co-crystallization with trypsin.

作者信息

McGrath M E, Erpel T, Browner M F, Fletterick R J

机构信息

Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0110.

出版信息

J Mol Biol. 1991 Nov 20;222(2):139-42. doi: 10.1016/0022-2836(91)90199-g.

DOI:10.1016/0022-2836(91)90199-g

PMID:1960717

Abstract

We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter. After induction, functional, soluble ecotin comprises 15% of total cellular protein. This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin. Ecotin was co-crystallized with rat trypsin mutant D102N. Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21. The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees. Four molecules each of trypsin and ecotin are found in the asymmetric unit.

摘要

我们利用内源性信号肽和异源tac启动子，在其天然环境——大肠杆菌周质中，将丝氨酸蛋白酶抑制剂依考汀表达至高水平（大于400毫克/升细胞培养物）。诱导后，具有功能的可溶性依考汀占总细胞蛋白的15%。该表达系统有助于启动晶体学研究，以确定依考汀抑制胰腺丝氨酸蛋白酶的结构基础。依考汀与大鼠胰蛋白酶突变体D102N共结晶。共晶体的初步晶体学分析表明，它们的衍射分辨率至少为2.7埃，并表明它们属于单斜空间群P21。晶胞常数为a = 52.0埃，b = 93.3埃，c = 160.7埃，β = 96°。在不对称单元中发现每个都有四个胰蛋白酶和依考汀分子。