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Applied and Environmental Microbiology, February 2007, p. 1189-1198, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.02427-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Novel High-Throughput Screen Reveals Yeast Genes That Increase Secretion of Heterologous Proteins{triangledown}

Alane E. Wentz and Eric V. Shusta*

Department of Chemical and Biological Engineering, University of Wisconsin—Madison, Madison, Wisconsin

Received 16 October 2006/ Accepted 9 December 2006

The yeast Saccharomyces cerevisiae is an attractive host for the production of heterologous proteins. However, low-yield production of many proteins (from micrograms to 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 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 those coding for cell wall proteins (CCW12, CWP2, and 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 7.9-fold), with CCW12 and ERO1 being the most generalizable enhancers of scFv/scTCR secretion.

* Corresponding author. Mailing address: Department of Chemical and Biological Engineering, University of Wisconsin—Madison, 1415 Engineering Drive, Madison, WI 53706. Phone: (608) 265-5103. Fax: (608) 262-5434. E-mail: shusta{at}engr.wisc.edu.

{triangledown} Published ahead of print on 22 December 2006.

Applied and Environmental Microbiology, February 2007, p. 1189-1198, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.02427-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Gasser, B., Sauer, M., Maurer, M., Stadlmayr, G., Mattanovich, D. (2007). Transcriptomics-Based Identification of Novel Factors Enhancing Heterologous Protein Secretion in Yeasts. Appl. Environ. Microbiol. 73: 6499-6507 [Abstract] [Full Text]  


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