Cloning and Expression of a Yeast Ubiquitin-Protein Cleaving Activity in Escherichia Coli

Abstract

We have cloned the gene coding for a ubiquitin-protein hydrolase from the yeast Saccharomyces cerevisiae. The gene (YUH1) was isolated from a yeast genomic library by screening with an oligonucleotide probe designed from the amino acid sequence of the purified hydrolase. The YUH1 gene encodes a 26 kD protein and contains no introns. The YUH1 gene product can be overexpressed in active form in Escherichia coli and purified by a two column procedure. The purified hydrolase is capable of cleaving ubiquitin-protein fusions in vitro specifically at the ubiquitin fusion junction and requires no high energy cofactors. Fusions can also be cleaved in E. coli in strains expressing the hydrolase. Gene disruptions in haploid yeast strains have no apparent phenotypic change and ubiquitin-protein hydrolase activity in extracts is normal, indicating the existence of additional genes for ubiquitin-protein hydrolase. In vitro and in vivo cleavage of ubiquitin-protein fusions may be a useful method of producing proteins with defined ammo-termini.

Cite this article

Miller, H., Henzel, W., Ridgway, J. et al. Cloning and Expression of a Yeast Ubiquitin-Protein Cleaving Activity in Escherichia Coli. Nat Biotechnol 7, 698–704 (1989). https://doi.org/10.1038/nbt0789-698

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