Cloning, disruption and protein secretory phenotype of the GAS1 homologue of Pichia pastoris.
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 beta-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 DeltaGAS1 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.
Marx
Hans
H
Institute of Applied Microbiology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Sauer
Michael
M
Resina
David
D
Vai
Marina
M
Porro
Danilo
D
Valero
Francisco
F
Ferrer
Pau
P
Mattanovich
Diethard
D
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D016695:Glycosyltransferases / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D010641:Phenotype; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012233:Rhizopus / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism; D014362:Trypsinogen / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D014362:Trypsinogen; D016695:Glycosyltransferases; D008049:Lipase