Expression and characterization of a thermostable serine protease (TfpA) from Thermomonospora fusca YX in Pichia pastoris.
A serine protease produced by Thermomonospora fusca YX (TfpA) is heat-stable (up to 85 degrees C) and has a broad pH activity range and strong resistance to detergents. The objective of this study was to determine if the methylotropic yeast Pichia pastoris could express TfpA extracellularly. A 1.0-kb DNA fragment (tfpA) encoding the pro-peptide and mature protein of TfpA was cloned into expression vectors pPICZalphaA (inducible) and pGAPZalphaA (constitutive) and introduced into P. pastoris by electroporation. Expression of r-TfpA was greater in the inducible system than in the constitutive one, producing 135 U ml(-1) medium supernatant 6 days after methanol induction. The r-TfpA was not glycosylated (21.7 kDa), and had pH and temperature optima of 8.5 and 80 degrees C, respectively, using azocasein as a substrate. In conclusion, P. pastoris can be used as a host to produce extracellular r-TfpA, and expression efficiency may be improved by optimizing fermentation conditions and modifying factors related to protein expression and stability.
Kim
Taewan
T
Department of Animal Science, Cornell University, Morrison Hall 252, Ithaca, NY 14853, USA.
Lei
Xin Gen
XG
D000190:Actinomyces / Q000201:enzymology*; D002364:Caseins / Q000378:metabolism; D018274:Electroporation; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D002364:Caseins; D011994:Recombinant Proteins; C026018:azocasein; D012697:Serine Endopeptidases