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粘质沙雷氏菌新型中性蛋白酶——蛋白酶解素的克隆、测序、表达及特性分析,该酶代表了一类前体N端区域较短的新型嗜热菌蛋白酶样蛋白酶。

Cloning, sequencing, expression, and characterization of protealysin, a novel neutral proteinase from Serratia proteamaculans representing a new group of thermolysin-like proteases with short N-terminal region of precursor.

作者信息

Demidyuk Ilya V, Kalashnikov Alexander E, Gromova Tatiana Yu, Gasanov Eugene V, Safina Dina R, Zabolotskaya Maria V, Rudenskaya Galina N, Kostrov Sergey V

机构信息

Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia.

出版信息

Protein Expr Purif. 2006 Jun;47(2):551-61. doi: 10.1016/j.pep.2005.12.005. Epub 2006 Jan 5.

Abstract

The gene of Serratia proteamaculans proteinase, protealysin, was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a precursor of 341 amino acids (AAs) with a significant homology to thermolysin-like proteinases (TLPs). The molecular weight of the purified mature active recombinant protein was 32 kDa, the N-terminal amino acid sequence was AKTSTGGEVI. The optimum pH for azocasein hydrolysis by protealysin was seven and it was completely inhibited by o-phenanthroline. The enzyme hydrolyzed 3-(2-furyl)acryloyl-glycyl-L-leucine amide, the standard substrate for TLPs, with k(cat)/K(m) ratio of (2.52 +/- 0.02) x 10(2) M(-1) s(-1). Protealysin maturation removes 50 AA from the N-terminus of the precursor. The removed region had no similarity with the preprosequence of thermolysin (232 AA) but was homologous to some other TLPs. These proteins shared a conserved 7-AA motif near the initial methionine. Such motif was also found in some nonproteolytic putative proteins; two of them were eukaryotic.

摘要

粘质沙雷氏菌蛋白酶(蛋白酶解酶)基因被克隆、测序并在大肠杆菌中表达。该基因编码一个由341个氨基酸(AA)组成的前体,与嗜热菌蛋白酶样蛋白酶(TLP)具有显著同源性。纯化的成熟活性重组蛋白分子量为32 kDa,N端氨基酸序列为AKTSTGGEVI。蛋白酶解酶水解偶氮酪蛋白的最适pH为7,邻菲罗啉可完全抑制其活性。该酶水解TLP的标准底物3-(2-呋喃基)丙烯酰甘氨酰-L-亮氨酸酰胺,催化常数与米氏常数的比值为(2.52±0.02)×10² M⁻¹ s⁻¹。蛋白酶解酶成熟过程中从前体N端去除了50个AA。去除的区域与嗜热菌蛋白酶(232个AA)的前原序列没有相似性,但与其他一些TLP同源。这些蛋白质在起始甲硫氨酸附近有一个保守的7个AA基序。在一些非蛋白水解性推定蛋白中也发现了这样的基序;其中两个是真核生物的。

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