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BPTI融合蛋白的细胞内表达及胰凝乳蛋白酶的单柱切割/亲和纯化

Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin.

作者信息

Altman J D, Henner D, Nilsson B, Anderson S, Kuntz I D

机构信息

Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.

出版信息

Protein Eng. 1991 Jun;4(5):593-600. doi: 10.1093/protein/4.5.593.

Abstract

The novel and efficient expression system described here produces formerly poorly expressed, proteolytically unstable mutants of bovine pancreatic trypsin inhibitor (BPTI). A new, single column method for the cleavage of a recombinant fusion to BPTI and affinity purification of the BPTI moiety by immobilized chymotrypsin is an integral part of the system. Wild-type and mutant BPTI molecules are expressed in Escherichia coli as fusion proteins forming intracellular inclusion bodies. Transcription initiation is under the control of the E. coli trp promoter. The expressed protein is tripartite fusion comprising (i) a portion of the TrpLE leader peptide, (ii) a synthetic IgG binding domain derived from protein A and (iii) the BPTI variant. Solubilization of the inclusion bodies and refolding of the fusion proteins in a thiol-disulfide shuffling system yields correctly folded inhibitor molecules. In the single column purification and cleavage procedure, immobilized chymotrypsin cleaves the refolded fusion protein and releases affinity purified active BPTI mutants with correct N-termini. Mutant BPTI molecules which do not fold into active inhibitors are also stably expressed in inclusion bodies but cannot be purified by this method. Unlike previously described secretion systems for the production of BPTI, expression levels in this system appear to be independent of both the mutation in the BPTI gene and the activity of the expressed protein. Mutants poorly expressed in secretion systems can now be produced in sufficient quantities for protein folding studies and structural analysis using X-ray crystallography and NMR spectroscopy.

摘要

本文所述的新型高效表达系统能够产生以前表达不佳、蛋白水解不稳定的牛胰蛋白酶抑制剂(BPTI)突变体。一种用于切割与BPTI的重组融合体并通过固定化胰凝乳蛋白酶亲和纯化BPTI部分的新型单柱方法是该系统的一个组成部分。野生型和突变型BPTI分子在大肠杆菌中作为融合蛋白表达,形成细胞内包涵体。转录起始受大肠杆菌色氨酸启动子的控制。表达的蛋白质是一种三方融合体,包括(i)TrpLE前导肽的一部分,(ii)源自蛋白A的合成IgG结合结构域,以及(iii)BPTI变体。在硫醇-二硫键重排系统中溶解包涵体并使融合蛋白重折叠,可产生正确折叠的抑制剂分子。在单柱纯化和切割过程中,固定化胰凝乳蛋白酶切割重折叠的融合蛋白,并释放出具有正确N端的亲和纯化活性BPTI突变体。不能折叠成活性抑制剂的突变型BPTI分子也能在包涵体中稳定表达,但不能用这种方法纯化。与先前描述的用于生产BPTI的分泌系统不同,该系统中的表达水平似乎与BPTI基因中的突变以及表达蛋白的活性均无关。现在可以大量生产在分泌系统中表达不佳的突变体,用于蛋白质折叠研究以及使用X射线晶体学和核磁共振光谱进行结构分析。

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