Strickler J E, Berka T R, Gorniak J, Fornwald J, Keys R, Rowland J J, Rosenberg M, Taylor D P
Department of Macromolecular Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19046.
J Biol Chem. 1992 Feb 15;267(5):3236-41.
In contrast to the Gram-negative bacteria, Gram-positive bacteria such as Streptomyces lack a mucopolysaccharide cell wall which allows them to produce and secrete a variety of proteins directly into their environment. In an effort to understand and eventually exploit the synthesis and secretion of proteins by Streptomyces, we identified and characterized two naturally occurring abundantly produced proteins in culture supernatants of Streptomyces lividans and Streptomyces longisporus. We purified these 10-kDa proteins and obtained partial amino acid sequence information which was then used to design oligonucleotide probes in order to clone their genes. Analysis of the sequence data indicated that these proteins were related to each other and to several other previously characterized Streptomyces protein protease inhibitors. We demonstrate that both proteins are protein protease inhibitors with specificity for trypsin-like enzymes. The presumptive signal peptidase cleavage sites and subsequent aminopeptidase products of each protein are characterized. Finally, we show that the cloned genes contain all of the information necessary to direct synthesis and secretion of the proteins by Streptomyces spp. or Escherichia coli.
与革兰氏阴性菌不同，革兰氏阳性菌如链霉菌缺乏粘多糖细胞壁，这使得它们能够直接将多种蛋白质合成并分泌到周围环境中。为了理解并最终利用链霉菌蛋白质的合成与分泌机制，我们鉴定并表征了在变铅青链霉菌和长孢链霉菌培养上清液中自然大量产生的两种蛋白质。我们纯化了这些10 kDa的蛋白质，并获得了部分氨基酸序列信息，随后利用这些信息设计寡核苷酸探针以克隆它们的基因。序列数据分析表明，这些蛋白质彼此相关，并且与其他几种先前表征的链霉菌蛋白蛋白酶抑制剂相关。我们证明这两种蛋白质都是对胰蛋白酶样酶具有特异性的蛋白蛋白酶抑制剂。对每种蛋白质的推定信号肽切割位点和随后的氨肽酶产物进行了表征。最后，我们表明克隆的基因包含指导链霉菌属或大肠杆菌合成和分泌这些蛋白质所需的所有信息。
