Synonymous codon changes at the 5′-end of the gene strongly impact the heterologous protein expression in Escherichia coli
Applied Biochemistry and Microbiology, ISSN: 0003-6838, Vol: 53, Issue: 3, Page: 296-303
2017
- 4Citations
- 8Captures
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Article Description
This study describes the impact of 5′-end codon modulation on the expression of a heterologous gene, human granulocyte colony stimulating factor (GCSF), in Escherichia coli. Fourteen different constructs (pGCSF-01 to pGCSF-14) carrying single or multiple synonymous substitutions at +2, +3 and further down from +4 to +7 codons, were prepared and their expression was monitored in E. coli BL21 Codon-Plus (DE3) RIPL using a strong T7 lac-promoter based expression system. A single nucleotide change at +2 Thr codon (ACC→ACA) either alone or in combination with +3 Pro codon (CCC/CCT/CCA) resulted in the expression enhancement of an otherwise poorly expressed native-GCSF, to a level that corresponded to 45–50% of the total E. coli BL21 CodonPlus (DE3) RIPL cellular proteins. The differences in GCSF expression amongst different constructs could be attributed to the preferential or non-preferential codon usage, reduced number of G/C nucleotides and the stability of mRNA secondary structure formed near the 5′-end coding region. The expression of GCSF achieved was in the form of biologically inactive inclusion bodies that were solubilized using mild concentration of a non-ionic surfactant and refolded by a simplified, step-dialysis approach. Biological activity of the purified GCSF, assessed in induced neutropenic mice, was similar to the commercially available preparation of the GCSF analog (filgrastim).
Bibliographic Details
http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=85028608843&origin=inward; http://dx.doi.org/10.1134/s0003683817030024; http://link.springer.com/10.1134/S0003683817030024; http://link.springer.com/content/pdf/10.1134/S0003683817030024.pdf; http://link.springer.com/article/10.1134/S0003683817030024/fulltext.html; https://dx.doi.org/10.1134/s0003683817030024; https://link.springer.com/article/10.1134/S0003683817030024
Pleiades Publishing Ltd
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