Genome-scale analysis of library sorting (GALibSo): Isolation of secretion enhancing factors for recombinant protein production in Pichia pastoris.
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.
Stadlmayr
Gerhard
G
Department of Biotechnology, BOKU - University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Benakovitsch
Katrin
K
Gasser
Brigitte
B
Mattanovich
Diethard
D
Sauer
Michael
M
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D015723:Gene Library; D016681:Genome, Fungal; D015483:HIV Antibodies / Q000235:genetics / Q000378:metabolism*; D046228:Microarray Analysis; D010843:Pichia / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D011994:Recombinant Proteins