کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2819392 1569922 2008 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Molecular cloning and sequence analysis of a novel zinc-metalloprotease gene from the Salinivibrio sp. strain AF-2004 and its extracellular expression in E. coli
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی ژنتیک
پیش نمایش صفحه اول مقاله
Molecular cloning and sequence analysis of a novel zinc-metalloprotease gene from the Salinivibrio sp. strain AF-2004 and its extracellular expression in E. coli
چکیده انگلیسی

In this work the first protease gene encoding a novel zinc-metalloprotease from the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 has been cloned, sequenced and reported to the GenBank. We have generated a library containing about 10,000 transformants whose screening yielded one clone harboring plasmid pBluescript with 3.6 kb inserted fragment (pBlueSVP2) with positive caseinolytic activity. Nucleotide sequence analysis of the selected clone revealed a single open reading frame (ORF) of 1833 bp encoding 611 amino acids. The deduced amino acid sequence includes a zinc-metalloprotease HEXXH-E consensus motif which is highly conserved in the M4 family of proteases. The primary amino acid sequence alignment search in the database revealed a moderate homology between the deduced amino acid sequence and the known zinc-metalloproteases including vibriolysin from Vibrio vulnificus and Pseudomonas aeruginosa elastase. The full length of SVP2 gene was subcloned into pQE-80L (pQEVP1) and transformed into Escherichia coli BL21 (DE3) for recombinant overexpression of the protease. Following induction by IPTG, active enzyme was found within cells and in the extracellular medium, where it slowly accumulated to high levels. Mass spectrometric fingerprinting of trypsin digested rSVP2 analysis identified the processed mature protease which starts at Ala-200 of a SVP2 full length protein. Although this result suggested a mature protein of 412 amino acids (44.8 kDa), electrospray-ionisation mass spectrometry revealed that the molecular mass of purified rSVP2 was only 34.2 kDa, which indicates a further cleavage site at the C-terminal.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Gene - Volume 408, Issues 1–2, 31 January 2008, Pages 196–203
نویسندگان
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