Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli.
A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism.
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D001482:Base Composition; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics*; D003062:Codon / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D044643:Glucan 1,4-beta-Glucosidase; D005959:Glucosidases / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics*
D003062:Codon; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D005959:Glucosidases; D001617:beta-Glucosidase; D002480:Cellulase; D044643:Glucan 1,4-beta-Glucosidase