III型细胞毒素铜绿假单胞菌外毒素S两种重组形式的表达与纯化

Expression and purification of two recombinant forms of the type-III cytotoxin, Pseudomonas aeruginosa ExoS.

作者信息

Maresso Anthony W, Barbieri Joseph T

机构信息

Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

出版信息

Protein Expr Purif. 2002 Dec;26(3):432-7. doi: 10.1016/s1046-5928(02)00544-2.

DOI:10.1016/s1046-5928(02)00544-2

PMID:12460767

Abstract

Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N-terminus (residues 1-232) possesses Rho GTPase-activating (GAP) activity, while the C-terminus (residues 233-453) comprises an ADP-ribosyltransferase domain. Amino acid residues 51-72 of ExoS are involved in membrane binding and aggregation, which has complicated purification schemes. Here, it is reported on the expression, purification, and characterization of two recombinant forms of ExoS that lack this membrane-binding domain, designated rExoS78-453 and rExoSdelta51-72. Purification of these forms was achieved using sequential NTA/Ni(2+)-affinity, gel filtration, and anion-exchange chromatography. Both forms of ExoS possessed Rho GAP activity and ADP-ribosyltransferase activity comparable to wild-type ExoS. Mass spectrometry showed that rExoS78-453 and rExoSdelta51-72 had molecular masses similar to their predicted molecular masses.

摘要

铜绿假单胞菌外毒素S（ExoS）是一种双功能III型细胞毒素。其N端（第1 - 232位氨基酸残基）具有Rho GTP酶激活（GAP）活性，而C端（第233 - 453位氨基酸残基）包含一个ADP核糖基转移酶结构域。ExoS的第51 - 72位氨基酸残基参与膜结合和聚集，这使得纯化方案变得复杂。本文报道了两种缺失该膜结合结构域的ExoS重组形式的表达、纯化及特性鉴定，分别命名为rExoS78 - 453和rExoSdelta51 - 72。通过依次使用NTA/Ni(2+)亲和、凝胶过滤和阴离子交换色谱法实现了这些形式的纯化。两种形式的ExoS均具有与野生型ExoS相当的Rho GAP活性和ADP核糖基转移酶活性。质谱分析表明，rExoS78 - 453和rExoSdelta51 - 72的分子量与其预测分子量相似。