Chemical synthesis and in vivo hyperexpression of a modular gene coding for Escherichia coli translational initiation factor IF1
- 1 June 1987
- journal article
- research article
- Published by Springer Science and Business Media LLC in Molecular Genetics and Genomics
- Vol. 208 (1-2), 63-69
- https://doi.org/10.1007/bf00330423
Abstract
An artificial gene encoding the Escherichia coli translational initiation factor IF1 was synthesized based on the primary structure (71 amino acid residues) of the protein. Codons for individual amino acids were selected on the basis of the preferred codon usage found in the structural genes for the initiation factor IF2 of E. coli and Bacillus stearothermophilus, both of which can be expressed at high levels in E. coli cells. We gave the IF1 gene a modular structure by introducing specific restriction enzyme sites into the sequence, resulting in units of three to ten codons. This was conceived to facilitate site-directed mutagenesis of the gene and thus to obtain IF1 with specific amino acid alterations at desired positions. The IF1 gene was assembled by shot-gun ligation of 9 synthetic oligodeoxyri-bonucleotides ranging in size from 31 to 65 nucleotides and cloned into an expression vector to place the gene under the control of an inducible promoter. Upon induction, E. coli cells harbouring the artificial gene were found to produce large amounts (≥60 mg/100 g cells) of a protein indistinguishable from natural IF1 in both chemecal and biological properties.Keywords
This publication has 26 references indexed in Scilit:
- The unusual translational initiation codon AUU limits the expression of the infC (initiation factor IF3) gene of Escherichia coliMolecular Genetics and Genomics, 1987
- Mechanism of translational initiation in prokaryotesFEBS Letters, 1986
- Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNANature, 1986
- Mechanism of protein biosynthesis in prokaryotic cellsFEBS Letters, 1984
- Effect of Escherichia coli initiation factors on the kinetics of N-AcPhe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow studyBiochemistry, 1983
- Plasmid vectors for high-efficiency expression controlled by the promoter of coliphage lambdaGene, 1981
- [58] DNA sequence analysis by primed synthesisMethods in enzymology, 1980
- Structure‐function relationships in Escherichia coliinitiation factorsFEBS Letters, 1979
- Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfateAnalytical Biochemistry, 1971
- Isolation and Properties of Crystalline Initiation Factor F1 from Escherichia coli RibosomesEuropean Journal of Biochemistry, 1971