Chemical synthesis and expression of a cassette adapted ubiquitin gene.
A gene encoding the yeast ubiquitin was chemically synthesized and expressed in yeast under regulatory control of the copper metallothionein (CUP1) promoter. The gene was assembled in a one-step ligation reaction from eight oligonucleotide fragments ranging in length from 50 to 64 nucleotides. To facilitate mutagenesis and gene fusion studies, eight unique 6-base-cutting restriction enzyme sites were placed in the reading frame which did not alter the encoded protein sequence or force the utilization of rare codons. In a copper-resistant yeast strain (CUP1r), expression of the gene was induced by copper to approximately 5% of the total yeast proteins, as determined by Coomassie-stained polyacrylamide gels. The protein, purified from yeast, reacted with ubiquitin-specific antibodies and was found to be biologically active in supporting ubiquitin-dependent protein degradation in vitro.[1]References
- Chemical synthesis and expression of a cassette adapted ubiquitin gene. Ecker, D.J., Khan, M.I., Marsh, J., Butt, T.R., Crooke, S.T. J. Biol. Chem. (1987) [Pubmed]
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