کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2021184 | 1069234 | 2008 | 9 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study](/preview/png/2021184.png)
The efficiency of heterologous protein production in Escherichia coli (E. coli) can be diminished by biased codon usage. Approaches normally used to overcome this problem include targeted mutagenesis to remove rare codons or the addition of rare codon tRNAs in specific cell lines. Recently, improvements in technology have enabled cost-effective production of synthetic genes, making this a feasible alternative. To explore this option, the expression patterns in E. coli of 30 human short-chain dehydrogenase/reductase genes (SDRs) were analyzed in three independent experiments, comparing the native and synthetic (codon-optimized) versions of each gene. The constructs were prepared in a pET-derived vector that appends an N-terminal polyhistidine tag to the protein; expression was induced using IPTG and soluble proteins were isolated by Ni–NTA metal-affinity chromatography. Expression of the native and synthetic gene constructs was compared in two isogenic bacterial strains, one of which contained a plasmid (pRARE2) that carries seven tRNAs recognizing rare codons. Although we found some degree of variability between experiments, in normal E. coli synthetic genes could be expressed and purified more readily than the native version. In only one case was native gene expression better. Importantly, in most but not all cases, expression of the native genes in combination with rare codon tRNAs mimicked the behavior of the synthetic genes in the native strain. The trend is that heterologous expression of some proteins in bacteria can be improved by altering codon preference, but that this effect can be generally recapitulated by introducing rare codon tRNAs into the host cell.
Journal: Protein Expression and Purification - Volume 59, Issue 1, May 2008, Pages 94–102