Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin |
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| Authors: | Guillemette JGuy; Matsushima-Hibiya Yuko; Atkinson Tom; Smith Michael |
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| Affiliation: | Department of Biochemistry, Biotechnology Laboratory, and the Protein Engineering Network of Centers of Excellence, University of British Columbia 2146 Health Sciences Mall, Vancouver, British Columbia V6T 1W5
2Present address: Department of Genetics SK-50, University of Washington Seattle WA 98195, USA |
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| Abstract: | A gene for expression of horse heart myoglobin in Escherichiacoli has been constructed in one step from long synthetic oligonucleotides.The synthetic gene contains an efficient translation initiationsignal and used codons that are commonly found in E.coli. Uniquerestriction sites are placed throughout the gene. It has beeninserted in a phagemid vector and is expressed from the lacpromoter in E.coli at high efficiency, the soluble heme proteinrepresenting  10% of soluble protein. Two versions of horse heartmyoglobin were produced with aspartic acid or asparagine atresidue 122. Comparison of chromatographic mobilities of thesetwo proteins with authentic horse heart myoglobin identifiedaspartic acid as the correct residue 122. The availability ofthis gene, which is designed to facilitate oligonucleotide mutagenesisor cassette mutagenesis, will allow systematic structure—functionanalysis of horse heart myoglobin. |
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| Keywords: | gene synthesis/ myoglobin/ protein engineering/ site-directed mutagenesis |
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