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Expression and characterization of a thermostable serine protease (TfpA) from Thermomonospora fusca YX in Pichia pastoris

Expression and characterization of a thermostable serine protease (TfpA) from Thermomonospora fusca YX in Pichia pastoris

Applied Microbiology and Biotechnology 68(3): 355-359

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.

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Accession: 012071389

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PMID: 15821916

DOI: 10.1007/s00253-005-1911-8

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