Rare codon priority and its position specificity at the 5′ of the gene modulates heterologous protein expression in Escherichia coli

Thangadurai, Chinnathambi; Suthakaran, Pichaimuthu; Barfal, Pankaj; Balaiah, Anandaraj; Pradhan, Satya Narayan; Ramalingam, Subramanian; Murugan, Vadivel

doi:10.1016/j.bbrc.2008.09.024

Cited by 22 publications

(13 citation statements)

References 27 publications

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“…Alternatively, targeted substitution to introduce low-frequency codons at the 5′-end of a coding sequence enhanced heterologous expression levels for streptokinase from the src gene of Streptococcus equisimilis in E. coli [56] .Arguably similar to the recent findings by Tuller et al, inclusion of rare codons at key positions allowed for stabilization of the ribosomal initiation complex, or concomitantly, reduced mRNA secondary structure, thereby yielding higher levels of recombinant protein [41]. They reported that the positioning of low-frequency codons to the 5′-end of an ORF is a universally conserved phenomenon and is a putative mechanism for regulating gene expression.…”

Section: Role Of Rare Codons In Gene Translationmentioning

confidence: 99%

Codon usage: Nature's roadmap to expression and folding of proteins

Angov

2011

Biotechnology Journal

206

152

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Biomedical and biotechnological research relies on processes leading to the successful expression and production of key biological products. High-quality proteins are required for many purposes, including protein structural and functional studies. Protein expression is the culmination of multistep processes involving regulation at the level of transcription, mRNA turnover, protein translation, and post-translational modifications leading to the formation of a stable product. Although significant strides have been achieved over the past decade, advances toward integrating genomic and proteomic information are essential, and until such time, many target genes and their products may not be fully realized. Thus, the focus of this review is to provide some experimental support and a brief overview of how codon usage bias has evolved relative to regulating gene expression levels.

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Section: Role Of Rare Codons In Gene Translationmentioning

confidence: 99%

Codon usage: Nature's roadmap to expression and folding of proteins

Angov

2011

Biotechnology Journal

206

152

View full text Add to dashboard Cite

show abstract

“…So using the codons with high level 216 of frequency does not help to reach more rhG-CSF expression. The 217 other factors such as secondary structure or special codons in some 218regions may be more important[29,30]. The 'ramp hypothesis' 219 may explain this finding.…”

mentioning

confidence: 69%

“…This is especially important for the expression 27 of the genes with eukaryotic origin in Escherichia coli. The N-terminus of human granulocyte colony 28 stimulating factor (hG-CSF) contains amino acids whose coding sequences belong to the rare codons 29 in E. coli. In this study, the effect of rare codons on hG-CSF expression level was evaluated through intro- 30 ducing silent mutations in the 5 0 -end of the coding sequence.…”

mentioning

confidence: 99%

The effect of rare codons following the ATG start codon on expression of human granulocyte-colony stimulating factor in Escherichia coli

Karimi

Nezafat

Negahdaripour

et al. 2015

Protein Expression and Purification

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“…Previous studies supported that positions of rare codons played an important role in target protein expression . By studying the effect of rare codon “AGG” on streptokinase expression, Thangadurai et al assumed that the specific position of rare codons in the downstream of the start codon modulated the protein expression. In this study, we made a similar conclusion.…”

Section: Discussionmentioning

confidence: 99%

Examination of alternate codon bias solutions for expression and purification of recombinant mechano‐growth factor in Escherichia coli

Feng

Wan

Qian

et al. 2015

Biotech and App Biochem

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Mechano-growth factor (MGF), an alternative splicing variant of insulin-like growth factor-1 (IGF-1) gene, promotes cell proliferation and inhibits cell differentiation. It also plays an important role in tumor development. It is important to optimize the production process and achieve MGF protein because there is no commercial MGF protein available. In this study, the human MGF gene is cloned into pGEX-4T-1 and the recombinant human MGF (rhMGF) protein could be expressed in Rosetta (DE3) by isopropyl β-D-1-thiogalactopyranoside induction but not in BL21 (DE3). Mutation from rare codons to Escherichia coli preferred ones is performed. We obtain MGF(Mut54-56) and MGF(Mut-total) fragments through site-directed mutagenesis and overlapping PCR. Both pGEX-4T-1/MGF(Mut54-56)- and pGEX-4T-1/MGF(Mut-total)-transformed BL21 (DE3) can be induced to express rhMGF protein. To optimize the production technology, expression and purification of rhMGF are analyzed and compared in Rosetta (DE3) and BL21 (DE3). Results indicate that rhMGF expression in BL21 (DE3) is significantly higher than that in Rosetta (DE3). The protein yield of pGEX-4T-1/MGF(Mut-total) in BL21 (DE3) is higher than that of pGEX-4T-1/MGF(Mut54-56). We test the biological activity of MGF protein purified by affinity chromatography in C2C12 cell line and find that rhMGF promotes cell proliferation significantly. In conclusion, we establish a method to produce rhMGF economically with high biological activity in BL21 (DE3).

show abstract

Rare codon priority and its position specificity at the 5′ of the gene modulates heterologous protein expression in Escherichia coli

Cited by 22 publications

References 27 publications

Codon usage: Nature's roadmap to expression and folding of proteins

Codon usage: Nature's roadmap to expression and folding of proteins

The effect of rare codons following the ATG start codon on expression of human granulocyte-colony stimulating factor in Escherichia coli

Examination of alternate codon bias solutions for expression and purification of recombinant mechano‐growth factor in Escherichia coli

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