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High-Level Expression and In Vivo Processing of Chimeric Ubiquitin Fusion Proteins in Saccharomyces Cerevisiae

Bio/Technology volume 7, pages 705–709 (1989)Cite this article

Abstract

We have developed a system for the regulatable production of high levels of correctly processed heterologous proteins in the yeast Saccharomyces cerevisiae. An expression vector, pBS24Ub, that contains a synthetic gene for yeast ubiquitin (Ub) was constructed. The gene was expressed under the control of a glucose regulatable alcohol dehydrogenase-2/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) hybrid promoter. Inclusion of unique restriction sites at the 3′-end of the synthetic gene allowed for the precise in-frame insertion of heterologous genes. Expression of chimeric Ub/human γ-interferon (γ-IFN) and Ub/α1-proteinase inhibitor (α1-PI) genes produced fusion proteins that were cleaved quantitatively and precisely in vivo, by an endogenous ubiquitin-specific proteinase, to yield γ-IFN and α1-PI containing authentic amino termini. In contrast, γ-IFN and α1-PI, like many other heterologous proteins usually retain their initiation codon-derived methionine residues when expressed directly in bacteria or yeast. The in vivo ubiquitin fusion approach may provide a general method for circumventing problems associated with this additional methionine residue, for pharmaceutical proteins, and for other recombinant polypeptides where amino-terminal authenticity is desirable or critical.

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Author notes

  1. Elizabeth A. Sabin

    Present address: Dept. of Veterinary Microbiology and Immunology, University of California, Davis, CA, 95616

  2. Philip J. Barr: Corresponding author.

Authors and Affiliations

  1. Chiron Corporation, 4560 Horton Street, Emeryville, California, 94608

    Chun Ting Lee-Ng, Jeffrey R. Shuster & Philip J. Barr

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  1. Elizabeth A. Sabin
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  2. Chun Ting Lee-Ng
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  3. Jeffrey R. Shuster
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Sabin, E., Lee-Ng, C., Shuster, J. et al. High-Level Expression and In Vivo Processing of Chimeric Ubiquitin Fusion Proteins in Saccharomyces Cerevisiae. Nat Biotechnol 7, 705–709 (1989). https://doi.org/10.1038/nbt0789-705

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