Overexpression and characterization of thermostable serine protease in Escherichia coli encoded by the ORF TTE0824 from Thermoanaerobacter tengcongensis.
A novel extracellular serine protease derived from Thermoanaerobacter tengcongensis, designated tengconlysin, was successfully overexpressed in Escherichia coli as a soluble protein by recombination of an N-terminal Pel B leader sequence instead of the original presequence and C-terminal 6x histidine tags. The purified protein was activated by 0.1% sodium dodecyl sulfate (SDS) treatment but not by thermal treatment. The molecular weight of tengconlysin estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration chromatography was 37.9 and 36.2 kDa, respectively, suggesting that the enzyme is monomeric. The N-terminal sequence of mature tengconlysin was LDTAT, suggesting that it is a preproprotein containing a 29 amino acid presequence (predicted from the SigP program) and a 117 amino acid prosequence in the N-terminus. The C-terminal putative propeptide (position 469-540 in the preproprotein) did not inhibit the protease activity. The optimum temperature for tengconlysin activity was 90 degrees C in the presence of 1 mM calcium ions and the optimum pH ranged from 6.5 to 7.0. Activity inhibition studies suggest that the protease is a serine protease. The protease was stable in 0.1% SDS and 1-4 M urea at 70 degrees C in the presence of calcium ions and was activated by the denaturing agents.
Koma
D
D
Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka, 536-8553, Japan. koma@omtri.city.osaka.jp
Yamanaka
H
H
Moriyoshi
K
K
Ohmoto
T
T
Sakai
K
K
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000737:chemistry; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000235:genetics; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D014508:Urea; D012697:Serine Endopeptidases; D002118:Calcium