Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin

A gene for expression of horse heart myoglobin in Escherichia coli has been constructed in one step from long synthetic oligonucleotides. The synthetic gene contains an efficient translation initiation signal and used codons that are commonly found in E.coli. Unique restriction sites are placed thro...

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Bibliographic Details
Published in:Protein engineering 1991-06, Vol.4 (5), p.585-592
Main Authors: Guillemette, J.Guy, Matsushima-Hibiya, Yuko, Atkinson, Tom, Smith, Michael
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
Biological and medical sciences
Biotechnology
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
gene synthesis
Genes, Synthetic
Genetic engineering
Genetic technics
Genetic Vectors
Horses
Methods. Procedures. Technologies
Molecular Sequence Data
Mutagenesis, Site-Directed
Myocardium - chemistry
myoglobin
Myoglobin - chemical synthesis
Myoglobin - genetics
Myoglobin - isolation & purification
protein engineering
site-directed mutagenesis
Synthetic digonucleotides and genes. Sequencing
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Summary:A gene for expression of horse heart myoglobin in Escherichia coli has been constructed in one step from long synthetic oligonucleotides. The synthetic gene contains an efficient translation initiation signal and used codons that are commonly found in E.coli. Unique restriction sites are placed throughout the gene. It has been inserted in a phagemid vector and is expressed from the lac promoter in E.coli at high efficiency, the soluble heme protein representing ∼10% of soluble protein. Two versions of horse heart myoglobin were produced with aspartic acid or asparagine at residue 122. Comparison of chromatographic mobilities of these two proteins with authentic horse heart myoglobin identified aspartic acid as the correct residue 122. The availability of this gene, which is designed to facilitate oligonucleotide mutagenesis or cassette mutagenesis, will allow systematic structure—function analysis of horse heart myoglobin.
ISSN:1741-0126
0269-2139
1741-0134
1460-213X
DOI:10.1093/protein/4.5.585