Abstract
A serine protease produced by Thermomonospora fusca YX (TfpA) is heat-stable (up to 85°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 pPICZαA (inducible) and pGAPZαA (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°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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We would like to thank Dr. David Wilson for providing the gene of tfpA and Diana Irwin for technical help.
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Kim, T., Lei, X.G. Expression and characterization of a thermostable serine protease (TfpA) from Thermomonospora fusca YX in Pichia pastoris. Appl Microbiol Biotechnol 68, 355–359 (2005). https://doi.org/10.1007/s00253-005-1911-8
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DOI: https://doi.org/10.1007/s00253-005-1911-8