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.
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.
Karbalaei-Heidari
Hamid Reza
HR
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. heidary@ibb.ut.ac.ir
Ziaee
Abed-Ali
AA
Amoozegar
Mohammad Ali
MA
Cheburkin
Yuri
Y
Budisa
Nediljko
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D045726:Metalloproteases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D014737:Vibrionaceae / Q000145:classification / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism*
D045726:Metalloproteases; D015032:Zinc