来自乳酸乳球菌的X-脯氨酰二肽基氨基肽酶PepX的纯化、结晶及初步X射线分析

Purification, crystallization, and preliminary X-ray analysis of PepX, an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

作者信息

Chich J F, Rigolet P, Nardi M, Gripon J C, Ribadeau-Dumas B, Brunie S

机构信息

Unité de Biochimie et de Structure des Protéines, INRA, Centre de Recherches de Jouy-en-Josas, France.

出版信息

Proteins. 1995 Oct;23(2):278-81. doi: 10.1002/prot.340230216.

DOI:10.1002/prot.340230216

PMID:8592708

Abstract

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 A, b = 102.6 A, and c = 101.6 A, space group P2(1)2(1)2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 A and are suitable for high-resolution structural analysis.

摘要

X-脯氨酰二肽基氨基肽酶PepX是一种最初从乳酸乳球菌乳酸亚种NCDO 763中分离出来的丝氨酸肽酶，已在大肠杆菌中克隆并过量表达。该酶通过两步纯化以活性形式分离出来。使用聚乙二醇4000作为沉淀剂，在pH 5.0条件下，通过悬滴气相扩散法培养PepX晶体。这些晶体为正交晶系，晶胞参数a = 92.8 Å，b = 102.6 Å，c = 101.6 Å，空间群为P2(1)2(1)2，不对称单元中可能包含一个87.5 kDa的单体。这些晶体在X射线下非常稳定，衍射分辨率至少达到2.2 Å，适合进行高分辨率结构分析。