ISSN:
1617-4623
Schlagwort(e):
Translational initiation
;
Synthetic gene
;
Sitedirected mutagenesis
;
Genetic engineering
;
Oligonucleotides
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
Notizen:
Summary 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.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/BF00330423
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