Reference: Wentz AE and Shusta EV (2007) A novel high-throughput screen reveals yeast genes that increase secretion of heterologous proteins. Appl Environ Microbiol 73(4):1189-98

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Abstract


The yeast Saccharomyces cerevisiae is an attractive host for the production of heterologous proteins. However, low yield production of many proteins (micrograms-milligrams/liter) leaves considerable room for optimization. By engineering the yeast cell via traceable genome-wide libraries, genes that can enhance protein expression level because of their roles in the protein transcription, translation, folding and trafficking processes can be readily identified. This report details a novel approach that combines yeast cDNA overexpression libraries with yeast surface display to allow the rapid flow cytometric screening of engineered yeast for gene products that improve the display of heterologous proteins. After optimization of the screening conditions, a genome-wide scan yielded five yeast gene products that promoted increased display levels of a single-chain T-cell receptor (scTCR). The display-enhancing genes included cell wall proteins (CCW12, CWP2, SED1), a ribosomal subunit protein (RPP0), and an endoplasmic reticulum-resident protein (ERO1). Under the premise that yeast surface display levels could be used as a predictor of secretion efficiency, each display-enhancing gene product was tested for its ability to affect secretion levels of multiple scTCR and single-chain antibodies (scFv). All of the selected yeast gene products were shown to promote increased secretion of active protein (1.5-fold to 7.4-fold), with CCW12 and ERO1 being the most generalizable enhancers of scFv/scTCR secretion.

Reference Type
Journal Article
Authors
Wentz AE, Shusta EV
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