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Isolation of Pichia pastoris PIR genes and their utilization for cell surface display and recombinant protein secretion
Author(s) -
Pal Khasa Yogender,
Conrad Sara,
Sengul Mustafa,
Plautz Sarah,
Meagher Michael M.,
Inan Mehmet
Publication year - 2011
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1832
Subject(s) - pichia pastoris , biology , fusion protein , recombinant dna , yeast , green fluorescent protein , saccharomyces cerevisiae , pichia , microbiology and biotechnology , complementary dna , signal peptide , gene , biochemistry
Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two p roteins with i nternal r epeats ( PIR ) genes, i.e. PpPIR1 and PpPIR2 , from the methylotrophic yeast Pichia pastoris has been reported. The PIR1 and PIR2 genes' open reading frames were found to contain 1068 and 972 bases, respectively. The sequence homology search showed a homologous conserved repeat of PIR yeast block (SQIGDGQIQATT) in both proteins. The PIR yeast block was present eight times in the PpPir1p and four times in the PpPir2p proteins. Both proteins showed conserved glutamine (Q) and aspartic acid (D) in the repeated sequences, indicating a possible alkali‐sensitive β1,3‐glucan ester linkage. The fusion constructs of PpPir1‐2p and enhanced green fluorescent protein (EGFP) were developed for yeast cell surface display. The immunofluorescence assay showed uniform localization of EGFP protein on the P. pastoris cell surface in all fusion constructs. Furthermore, new vectors were developed for recombinant protein secretion in P. pastoris , utilizing the pre‐pro signal of PpPir1p protein. Efficient processing of the signal sequence was observed from EGFP and human α1‐antitrypsin (AAT) fusion constructs and recombinant protein secretion was obtained in the culture supernatant. The DNA sequences of the P. pastoris PpPIR1 and PpPIR2 genes have been submitted to GenBank under Accession Nos HM446634 and HM446635, respectively. Copyright © 2010 John Wiley & Sons, Ltd.