Kulich S M, Yahr T L, Mende-Mueller L M, Barbieri J T, Frank D W
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
J Biol Chem. 1994 Apr 8;269(14):10431-7.
We report the purification and proteolytic characterization of the 49-kDa form of exoenzyme S and the cloning of the structural gene for the 49-kDa form of exoenzyme S (exoS). The 49-kDa form of exoenzyme S was purified from SDS-polyacrylamide gels. Conditions were established that allowed efficient trypsin digestion of the 49-kDa form of exoenzyme S. Amino acid sequence determination of the amino terminus and tryptic peptides of the 49-kDa form of exoenzyme S allowed the generation of degenerate oligonucleotides, which were used to amplify DNA encoding an amino-terminal sequence and an internal sequence of the 49-kDa form of exoenzyme S. These DNA fragments were used to clone the entire structural gene for the 49-kDa form of exoenzyme S (exoS) from a cosmid library of Pseudomonas aeruginosa strain 388. The 49-kDa form of exoenzyme S (ExoS) is predicted to be a 453 amino acid protein. The predicted amino acid sequence indicates that ExoS is secreted from Pseudomonas without cleavage of an amino-terminal sequence. BESTFIT analysis identified three regions of alignment between ExoS and the active site of Escherichia coli heat-labile enterotoxin. One region of homology appears to be shared among several members of the family of bacterial ADP-ribosyltransferases.
我们报告了外毒素S 49 kDa形式的纯化、蛋白水解特性分析以及外毒素S 49 kDa形式（exoS）结构基因的克隆。外毒素S的49 kDa形式是从SDS-聚丙烯酰胺凝胶中纯化得到的。确定了能使外毒素S的49 kDa形式高效被胰蛋白酶消化的条件。通过对外毒素S的49 kDa形式的氨基末端和胰蛋白酶肽段进行氨基酸序列测定，生成了简并寡核苷酸，这些寡核苷酸用于扩增编码外毒素S的49 kDa形式的氨基末端序列和内部序列的DNA。这些DNA片段被用于从铜绿假单胞菌388菌株的黏粒文库中克隆外毒素S的49 kDa形式（exoS）的整个结构基因。外毒素S的49 kDa形式（ExoS）预计是一种由453个氨基酸组成的蛋白质。预测的氨基酸序列表明ExoS从铜绿假单胞菌分泌时没有氨基末端序列的切割。BESTFIT分析确定了ExoS与大肠杆菌不耐热肠毒素活性位点之间的三个比对区域。一个同源区域似乎在细菌ADP-核糖基转移酶家族的几个成员中是共有的。
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