Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli.
The prtV gene, encoding a collagenase of Vibrio parahaemolyticus, was expressed in Escherichia coli and purified by affinity chromatography. The transformant E. coli BL21(DE3)(pPRT2) secreted the recombinant PrtV, and the highest enzyme activity was detected in the culture supernatant after 5 h IPTG induction. The molecular mass of purified PrtV was 62 kDa as determined by gel filtration, which was similar to that obtained by SDS-PAGE (64 kDa). This suggested that PrtV was a monomer protein having no subunit structure. The isoelectric point of PrtV was 8.52. In addition, PrtV contained a 27 amino acid signal peptide, and the amino acid composition of the PrtV showed satisfactory agreement with that predicted from the DNA sequence. The optimum temperature and pH of PrtV were 40 degrees C and pH 7.5, respectively. The activity of PrtV was inhibited by chelators such as EDTA, EGTA and 1,10-phenanthroline; however, its activity was restored by the addition of various metal ions (Co2+, Mn2+, Ca2+, Cu2+, Ni2+ and Zn2+), indicating that PrtV is a metalloprotease. PrtV degraded both type I collagen and synthetic substrate FALGPA well, showing that PrtV is indeed a collagenase.
Yu
Mei-Shiuan
MS
Lee
Chia-Yin
CY
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002614:Chelating Agents / Q000494:pharmacology; D002846:Chromatography, Affinity; D003094:Collagen / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008670:Metals / Q000494:pharmacology; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014736:Vibrio parahaemolyticus / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008670:Metals; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C031821:2-furanacryloyl-leucyl-glycyl-prolyl-alanine; D003094:Collagen; D003012:Microbial Collagenase