High-level expression and in vivo processing of chimeric ubiquitin fusion proteins in Saccharomyces cerevisiae

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/α 1 -proteinase inhibitor (α 1 -PI) genes produced fusion proteins that were cleaved quantitatively and precisely in vivo , by an endogenous ubiquitin-specific proteinase, to yield γ-IFN and α 1 -PI containing authentic amino termini. In contrast, γ-IFN and α 1 -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.