Article

Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae

  1. Andy Rakestraw1,
  2. K. Dane Wittrup2,*

Article first published online: 6 DEC 2005

DOI: 10.1002/bit.20780

Issue

Biotechnology and Bioengineering

Biotechnology and Bioengineering

Volume 93, Issue 5, pages 896–905, 5 April 2006

Additional Information

How to Cite

Rakestraw, A. and Wittrup, K. D. (2006), Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae. Biotechnol. Bioeng., 93: 896–905. doi: 10.1002/bit.20780

Author Information

  1. 1

    Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts

  2. 2

    Department of Chemical Engineering, Massachusetts Institute of Technology, Building 66-552, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139; telephone: 617-253-4578; fax: 617-258-5766

*Department of Chemical Engineering, Massachusetts Institute of Technology, Building 66-552, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139; telephone: 617-253-4578; fax: 617-258-5766.

Publication History

  1. Issue published online: 15 MAR 2006
  2. Article first published online: 6 DEC 2005
  3. Manuscript Accepted: 19 OCT 2005
  4. Manuscript Received: 13 JUN 2005

Funded by

  • NSF BES. Grant Number: 00-02235
  • NIH. Grant Numbers: GM008334, CA96504

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Keywords:

  • heterologous protein expression;
  • unfolded-protein response (UPR);
  • vacuolar degradation;
  • ER folding chaperones;
  • yeast surface display

Abstract

In this study, secretory processing of cell-surface displayed Aga2p fusions to bovine pancreatic trypsin inhibitor (BPTI) and the single chain Fv (scFv) antibody fragment D1.3 are examined. BPTI is more efficiently processed than D1.3 both when secreted and surface-displayed, and D1.3 expression imparts a greater amount of secretory stress on the cell as assayed by a reporter of the unfolded protein response (UPR). Surprisingly, simultaneous expression of the two proteins in the same cell somewhat improves BPTI surface display while decreasing D1.3 surface display with minimal effect on UPR activation. Furthermore, co-expression leads to the accumulation of punctate vacuolar aggregates of D1.3 and increased secretion of the D1.3–Aga2p fusion into the supernatant. Overexpression of the folding chaperones protein disulfide isomerase (PDI) and BiP largely mitigates the D1.3 surface expression decrease, suggesting that changes in vacuolar and cell surface targeting may be due, in part, to folding inefficiency. Titration of constitutive UPR expression across a broad range progressively decreases surface display of both proteins as UPR increases. D1.3-Aga2p traffic through the late secretory pathway appears to be strongly affected by overall secretory load as well as folding conditions in the ER. © 2005 Wiley Periodicals, Inc.

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