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Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

Mualif SA, Teow SY, Omar TC, Chew YW, Yusoff NM, Ali SA - PLoS ONE (2015)

Bottom Line: These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids.To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector.We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

View Article: PubMed Central - PubMed

Affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.

ABSTRACT
Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of expression vector pSA-HNef-6His.The nef gene was PCR amplified from pNL4.3 plasmid, restricted with NdeI/ SacI, and ligated into pSA-6His vector at the same sites.
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pone.0130446.g001: Schematic representation of expression vector pSA-HNef-6His.The nef gene was PCR amplified from pNL4.3 plasmid, restricted with NdeI/ SacI, and ligated into pSA-6His vector at the same sites.

Mentions: pSA-HNef-6His vector (Fig 1) used to produce HIV-1 Nef protein was constructed by modifying the pSA-Hp24-6His vector [13]. The HIV-1 p24 coding sequence was removed by restricting the plasmid with NdeI and SacI, and replaced with HIV-1 nef coding sequence at the same sites. The resulting 6.517kb vector was verified by DNA restriction and sequence analyses (data not shown). The mRNA sequence encoding the nef gene is transcribed from an inducible T7 promoter and the resulting protein has a C-terminal 6His tag for IMAC-mediated purification.


Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

Mualif SA, Teow SY, Omar TC, Chew YW, Yusoff NM, Ali SA - PLoS ONE (2015)

Schematic representation of expression vector pSA-HNef-6His.The nef gene was PCR amplified from pNL4.3 plasmid, restricted with NdeI/ SacI, and ligated into pSA-6His vector at the same sites.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492947&req=5

pone.0130446.g001: Schematic representation of expression vector pSA-HNef-6His.The nef gene was PCR amplified from pNL4.3 plasmid, restricted with NdeI/ SacI, and ligated into pSA-6His vector at the same sites.
Mentions: pSA-HNef-6His vector (Fig 1) used to produce HIV-1 Nef protein was constructed by modifying the pSA-Hp24-6His vector [13]. The HIV-1 p24 coding sequence was removed by restricting the plasmid with NdeI and SacI, and replaced with HIV-1 nef coding sequence at the same sites. The resulting 6.517kb vector was verified by DNA restriction and sequence analyses (data not shown). The mRNA sequence encoding the nef gene is transcribed from an inducible T7 promoter and the resulting protein has a C-terminal 6His tag for IMAC-mediated purification.

Bottom Line: These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids.To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector.We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

View Article: PubMed Central - PubMed

Affiliation: Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia.

ABSTRACT
Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

No MeSH data available.


Related in: MedlinePlus