Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin.
Abstract: 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 approximately 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.
Publication Date: 1991-06-01 PubMed ID: 1891466DOI: 10.1093/protein/4.5.585Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The researchers have successfully created a synthetic gene that allows the bacteria E. coli to produce horse heart myoglobin, a protein. They used long synthetic oligonucleotides to construct the gene which has been designed to encourage further DNA mutation experiments for a better understanding of how myoglobin behaves.
Construction of the Synthetic Gene
- The researchers constructed a gene that would allow Escherichia coli to express horse heart myoglobin. A single, linear set of steps was used to build this gene from long synthetic oligonucleotides, which are short DNA or RNA molecules.
- The synthetic gene was constructed with an efficient translation initiation signal, a feature that boosts the gene’s effectiveness in code translation.
- The researchers used codons frequently found in E. coli. Codons are a sequence of three DNA or RNA nucleotides that corresponds with a specific amino acid or termination signal during protein synthesis.
- The gene was additionally equipped with unique restriction sites, which are locations on the DNA where specific proteins (restriction enzymes) can cut the DNA strand.
Expression and Analysis of the Gene
- The constructed gene was then inserted into a phagemid vector – a type of plasmid (small, circular, double-stranded DNA molecule within a cell) that uses a bacteriophage (virus that parasitizes a bacterium by infecting it) life cycle to amplify its copy number.
- The gene expression was driven by the lac promoter – a DNA sequence that initiates transcription of the gene – from E. coli at a high efficiency. In fact, the quantity of the resulting heme protein comprised about 10% of the total soluble protein.
- Two variants of horse heart myoglobin were generated: one with aspartic acid and one with asparagine at residue 122.
- Comparing chromatographic movements (a method of separating components) of these two proteins with authentic horse heart myoglobin confirmed that aspartic acid was the accurate residue at the 122nd position.
Purpose and Future Research
- The access to this synthetic gene, which is purposively designed to ease oligonucleotide mutagenesis or cassette mutagenesis (methods of artificial mutation), will foster systematic structure-function investigations of horse heart myoglobin.
- This research can contribute to a deeper understanding of the behavior and function of myoglobin and other related proteins.
Cite This Article
APA
Guillemette JG, Matsushima-Hibiya Y, Atkinson T, Smith M.
(1991).
Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin.
Protein Eng, 4(5), 585-592.
https://doi.org/10.1093/protein/4.5.585 Publication
Researcher Affiliations
- Department of Biochemistry, University of British Columbia, Vancouver.
MeSH Terms
- Amino Acid Sequence
- Animals
- Base Sequence
- Escherichia coli / genetics
- Genes, Synthetic
- Genetic Vectors
- Horses
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- Myocardium / chemistry
- Myoglobin / chemical synthesis
- Myoglobin / genetics
- Myoglobin / isolation & purification
Citations
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