Chemical synthesis and in vivo hyperexpression of a modular gene coding for Escherichia coli translational initiation factor IF1

Molecular & General Genetics : MGG
R A CalogeroC O Gualerzi

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 oligodeoxyribonucleotides 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 (greater than or equal to 60 mg/100 g cells) of a protein indistinguishable from natural IF1 in both chemical and biological properties.

References

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Citations

Aug 6, 2003·Journal of Molecular Biology·D PetrelliAnna La Teana
Aug 6, 2003·Journal of Molecular Biology·Claudio O GualerziCynthia L Pon
Sep 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·R A CalogeroClaudio O Gualerzi
Aug 14, 2001·The EMBO Journal·D PetrelliClaudio O Gualerzi
Jan 20, 2004·RNA·Anna M GiuliodoriCynthia L Pon
Jun 22, 2000·Journal of Molecular Biology·K D Dahlquist, J D Puglisi
Jun 3, 2017·Nucleic Acids Research·He ChengguangClaudio O Gualerzi

Related Concepts

DNA Sequence
DNA, Bacterial
Alkalescens-Dispar Group
Genes, Bacterial
Synthetic Genes
Peptide Chain Initiation, Translational
Gene Products, Protein
Structure-Activity Relationship
Eukaryotic Initiation Factor-1
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