David V A, Deutch A H, Sloma A, Pawlyk D, Ally A, Durham D R
BioMolecular Research Department, Washington Research Center, W.R. Grace and Co.-Conn., Columbia, MD 21044.
Gene. 1992 Mar 1;112(1):107-12. doi: 10.1016/0378-1119(92)90310-l.
The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long 'pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.
从构建于大肠杆菌中的解蛋白弧菌DNA文库中分离出编码革兰氏阴性海洋微生物解蛋白弧菌胞外中性蛋白酶弧菌溶素（NprV）的基因（nprV）。重组大肠杆菌产生的一种蛋白酶，在非变性聚丙烯酰胺凝胶上与解蛋白弧菌纯化的中性蛋白酶迁移位置相同，并且对中性蛋白酶底物N-[3-(2-呋喃基)丙烯酰基]-L-丙氨酰苯丙氨酸酰胺表现出酶特异性。克隆的nprV基因的核苷酸（nt）序列显示一个开放阅读框，编码609个氨基酸（aa），包括一个推定的信号肽序列，其后是一个由172个氨基酸组成的长“前体”序列。从解蛋白弧菌培养物中纯化的NprV的N端氨基酸序列，确定了从nt序列推导的氨基酸序列中成熟蛋白的起始位置。将成熟的NprV与嗜热解蛋白芽孢杆菌（嗜热菌蛋白酶）和嗜热脂肪芽孢杆菌的中性蛋白酶序列进行比较分析，发现了广泛的保守氨基酸同源区域，特别是在活性位点残基、锌结合残基和钙结合位点方面。通过将编码“前体”和成熟酶的DNA置于芽孢杆菌启动子和信号序列下游，在枯草芽孢杆菌中过量表达了NprV。
