A gene encoding a thermophilic alkaline serine proteinase from Thermus sp. strain Rt41A and its expression in Escherichia coli.
The extreme thermophile Thermus sp. strain Rt41A produces an extracellular alkaline serine proteinase during growth. This enzyme is stable for more than 24 h at 70 degrees C and has a pH optimum of 8.0. The proteinase gene was identified using primers designed to amplify a region between two highly conserved amino acid motifs in subtilisin-like proteinases and the PCR product was used to identify a genomic fragment containing the gene. The amino acid sequence deduced from the Rt41A gene contained a region identical to that obtained by amino-terminal sequencing of purified Rt41A proteinase. Comparison of the entire derived peptide sequence with other subtilisin-like serine proteinases revealed significant homologies, especially with aqualysin I from Thermus aquaticus YT-1 and with exoprotease A from Vibrio alginolyticus. The Rt41A proteinase was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase as an aid for purification and to overcome difficulties experienced with other plasmid vectors which produced inactive protein. The enzyme is inactive as synthesized and activation was shown to be temperature-dependent, with shorter incubation times required at higher temperatures; removal of the hydrophobic signal peptide from the start of the gene reduced the time required for activation to less than a third of that required if the signal peptide was present.
Munro
G K
GK
School of Biology and Biochemistry, University of Bath, UK.
McHale
R H
RH
Saul
D J
DJ
Reeves
R A
RA
Bergquist
P L
PL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002614:Chelating Agents / Q000737:chemistry; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010664:Phenylmethylsulfonyl Fluoride / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics; D013696:Temperature; D013824:Thermus / Q000235:genetics*; D013997:Time Factors
D002614:Chelating Agents; D011993:Recombinant Fusion Proteins; D010664:Phenylmethylsulfonyl Fluoride; C095755:AprA protein, Thermus; D012697:Serine Endopeptidases; D013381:Subtilisins