Enhanced expression of the yeast Ure2 protein in Escherichia coli: the effect of synonymous codon substitutions at a selected place in the gene.
The expression of the yeast Ure2 protein and its two N- and C-terminal HA-(YPYPVDYA) epitope and His-tag fusions has been enhanced in E. coli by selected silent mutagenesis of the URE2 gene. The two Arg-AGA codons at positions 253 and 254 of the URE2 gene coding sequence were exchanged by CGT codons accordingly. This has allowed an increased yield (up to 100-fold) of the full-length protein synthesized. Western blotting with HA-epitope-specific antibodies using N- and C-terminal Ure2p-HA(epitope)-His-tag fusion constructs confirmed the integrity of the recombinant proteins. The N-(C-) terminal tagged proteins were shown to possess biological activity of the natural Ure2 protein.
Komar
A A
AA
Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette, France.
Guillemet
E
E
Reiss
C
C
Cullin
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D005979:Glutathione Peroxidase; D016296:Mutagenesis; D016366:Open Reading Frames; D010957:Plasmids; D011328:Prions; D012097:Repressor Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D003062:Codon; D004274:DNA, Recombinant; D005656:Fungal Proteins; D011328:Prions; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae