Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites

doi:10.1016/0020-711X(89)90231-0

International Journal of Biochemistry

Volume 21, Issue 9, 1989, Pages 987-996

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Abstract

1.
1. Expression plasmids containing non-overlapping tandemly repeated ribosome binding sites (RBS) were constructed in order to stabilize mRNA and enhance translation.
2.
2. Two synthetic genes (human calcitonin tetramer gene and a fusion gene human γ-interferon-human calcitonin) were cloned in these vectors and the effect of multiplicity of Shine-Dalgarno (S/D) sequence on heterologous gene expression was studied.
3.
3. It was found that duplication and triplication of RBS had no effect on the stability of mRNA but led to a strong decrease in the level of recombinant protein and mRNA in the cell.
4.
4. Plasmids bearing four times repeated S/D sequences gave longer-lived mRNAs and maintained a level of protein and mRNA very close to the values obtained with a single S/D containing plasmids.

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There are more references available in the full text version of this article.

Cited by (6)

Unusual effect of clusters of rare arginine (AGG) codons on the expression of human interferon α1 gene in Escherichia coli
1997, International Journal of Biochemistry and Cell Biology
The human interferon (hIFNα₁) gene contains 11 arginine (Arg) codons AGG or AGA, which are extremely rare for bacteria, four of which are organized in tandems. The two AGG tandems (corresponding to Arg¹² Arg¹³ and Arg¹⁶³ Arg¹⁶⁴) are known to inhibit the translation of hIFNα₁ mRNA and therefore they are considered to be responsible for the poor expression of hIFNα₁ gene in bacterial cells. To study the effect of these two tandems on the expression of hIFNα₁ in E. coli, four new gene variants were designed to contain preferential Arg codons (CGT) substituted for the rare AGG codons in either the first, the second or both AGG tandems. We found that, whereas the yield of hIFNα₁ protein per cell remained unchanged, the level of hIFNα₁ mRNA decreased gradually (by a factor of two) with the consecutive substitution of the first, second and both AGG tandems. These results indicated, first, that the AGG clusters might have a stabilizing effect on the mRNA, and second, that mRNAs devoid of such clusters were translated at a higher rate in vivo. The protein products of the four genes (having the same amino acid sequence) showed different specific antiviral activity. The most active was the product of gene hIFNα_1(c) in which the second AGG tandem (corresponding to Arg¹⁶³, Arg¹⁶⁴) was preserved while the least active was the protein of gene hIFNα_1(d) (devoid of both AGG clusters). The role of the AGG tandems in folding of the gene product is discussed.
Effect of tandemly repeated AGG triplets on the translation of CAT-mRNA in E. coli
1992, FEBS Letters
It has been shown that tandems of rare arginine codons AGG have a strong inhibitory effect on translation of mRNA in E. coli [5]. This has been explained by the rate-limiting interaction of these codons with the less abundant tRNA^AGG [6]. In this study tandemly repeated AGG triplets were introduced into the chloramphenicol acetyltransferase (CAT) gene either upstream of the initiation ATG codon or downstream of it (both in frame and out of frame) and the expression of the modified genes was investigated. We report that the addition of AGG clusters resulted in a substantial inhibitory effect on CAT gene expression independently of their localization in mRNA. This inhibitory effect is explained by a competition of the tandem AGGAGG with the natural Shine—Dalgarno (SD) sequence (consensus AAGGAGGU) for the 3′-end of the 16S small ribosomal RNA (rRNA).
Increasing Signal Specificity of the TOL Network of Pseudomonas putida mt-2 by Rewiring the Connectivity of the Master Regulator XylR
2012, PLoS Genetics
Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli
2011, BMC Biotechnology
Human calcitonin tetrameric gene: Comparative expression in yeast and transgenic potato plants
1999, Biotechnology and Biotechnological Equipment
Gene expression evidence indicates that nucleotides 507-513 and 1434-1440 in 16S rRNA are organized in close proximity on the Escherichia coli 30S ribosomal subunit
1997, European Journal of Biochemistry

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International Journal of Biochemistry

Abstract

References (16)

Studies on transformation of E. coli with plasmids

J. mol. Biol.

Determination of plasmid copy-number by the “boiling” method

Analyt. Biochem.

Chemical synthesis and expression in E. coli of a human Val⁸-c .Icitonin gene by fusion to a synthetic human interferon-gamma gene

FEBS Lett.

Sequencing end-labelled DNA with base-specific chemical cleavages

Meth. Enzym.

Large scale purification of plasmid DNA

Prep. Biochem.

A comprehensive set of sequence analysis programs for VAX

Nucl. Acids Res.

Expression of an interferon-2 gene variant in E. coli using tandemly repeated ribosomal binding sites

DNA

Radioimmunoassay for calcitonin

Clin. Chem.

Cited by (6)

Unusual effect of clusters of rare arginine (AGG) codons on the expression of human interferon α1 gene in Escherichia coli

Effect of tandemly repeated AGG triplets on the translation of CAT-mRNA in E. coli

Increasing Signal Specificity of the TOL Network of Pseudomonas putida mt-2 by Rewiring the Connectivity of the Master Regulator XylR

Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

Human calcitonin tetrameric gene: Comparative expression in yeast and transgenic potato plants

Gene expression evidence indicates that nucleotides 507-513 and 1434-1440 in 16S rRNA are organized in close proximity on the Escherichia coli 30S ribosomal subunit