Journal Article

Cloning, disruption and protein secretory phenotype of the GAS1 homologue of Pichia pastoris

Hans Marx, Michael Sauer, David Resina, Marina Vai, Danilo Porro, Francisco Valero, Pau Ferrer and Diethard Mattanovich

in FEMS Microbiology Letters

Volume 264, issue 1, pages 40-47
Published in print November 2006 |
Published online September 2006 | e-ISSN: 1574-6968 | DOI: https://dx.doi.org/10.1111/j.1574-6968.2006.00427.x

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Abstract

The aim of the study was the identification, cloning and disruption of the GAS1 homologue of Pichia pastoris. Gas1p is a glycoprotein anchored to the outer layer of the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. Gas1p is a β-1,3-glucanosyltransglycosylase (EC 2.4.1.-). This cross-linking enzyme highly affects the structure and permeability of the yeast cell wall. The gene coding for the GAS1 homologue of P. pastoris was cloned by PCR, and its functionality was proven in a Saccharomyces cerevisiae GAS1 null mutant. Based on the nucleotide sequence information of the P. pastoris GAS1 homologue, a disruption cassette was constructed for the knockout of the GAS1 in P. pastoris. The morphology of ΔGAS1 P. pastoris was identical to that of S. cerevisiae GAS1 mutants. Finally, the impact of GAS1 disruption on secretion of three recombinant model proteins in P. pastoris, human trypsinogen, human serum albumin and Rhizopus oryzae lipase, was evaluated. While the disruption had no effect on the secretion of trypsinogen and albumin, the amount of lipase released from the cells was doubled.

Keywords: β-1,3-glucanosyltransglycosylase; cell wall; heterologous protein secretion; Pichia pastoris; gene disruption; yeast

Journal Article.  4263 words.  Illustrated.

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