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Genome‐scale analysis of library sorting (GALibSo): Isolation of secretion enhancing factors for recombinant protein production in Pichia pastoris
Author(s) -
Stadlmayr Gerhard,
Benakovitsch Katrin,
Gasser Brigitte,
Mattanovich Diethard,
Sauer Michael
Publication year - 2009
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22573
Subject(s) - pichia pastoris , biology , gene , complementary dna , cdna library , heterologous , cell sorting , microbiology and biotechnology , dna microarray , protein microarray , secretory protein , secretion , recombinant dna , genomic library , microarray , gene expression , genetics , flow cytometry , biochemistry , peptide sequence
A method combining fluorescence activated cell sorting (FACS) and DNA microarray assisted clone identification was developed and termed Genome‐Scale Analysis of Library Sorting (GALibSo). Genes enhancing the production of secreted heterologous proteins in Pichia pastoris were identified out of a cDNA library by cell surface display and FACS. The trends of gene enrichment during consecutive FACS rounds were monitored by DNA microarrays. In a case study a P. pastoris cDNA library was co‐expressed in a strain secreting the Fab fragment of a monoclonal antibody against human immunodeficiency virus type 1 as a model protein. Three genes were identified, increasing the relative expression level of the surface‐displayed model protein up to 45%. While one of these genes had a positive effect on three out of four tested proteins, the product specific effect of the other two suggested that the effects of the co‐expressed secretion enhancing factors are partly dependent on the protein to be produced. The microarray based monitoring of the enrichment of genes causing enhanced protein secretory capacity led to novel insights into the limitation of protein secretion. Biotechnol. Bioeng. 2010; 105: 543–555. © 2009 Wiley Periodicals, Inc.