Limits...
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 vectors pSA-C6His-RIL.Map shows multiple cloning site with eight unique restriction enzyme sites for facile cloning of heterologous genes.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492947&req=5

pone.0130446.g011: Schematic representation of expression vectors pSA-C6His-RIL.Map shows multiple cloning site with eight unique restriction enzyme sites for facile cloning of heterologous genes.

Mentions: To facilitate clone manipulation, we substituted nef gene with a multiple cloning site (MCS) clustered with eight unique restriction enzyme sites (Fig 11). Any two of the restriction enzyme sites can be chosen to clone gene of interest into the pSA-C6His-RIL vector. However it will be desirable to clone the gene of interest between NdeI and SacI, in order to avoid the addition of extra amino acids at N- and/or C-terminals of the resultant recombinant protein. Another possibility is to introduce desired restriction enzyme sites to the plasmid using whole-plasmid PCR. Several high fidelity polymerases suitable for whole-plasmid PCR are now available at reasonable cost. Whenever possible, we amplify the entire plasmid backbone with a set of primers containing desired restriction enzyme sites using Phusion or Q5 High-Fidelity DNA Polymerases. By using 50–100 ng plasmid vector as template, and using 18–25 PCR cycles, it is possible to obtain sufficient amount of PCR amplified plasmid, which can be used to clone the gene of interest with compatible restriction enzymes sites. To reduce the background, we add 10U of DpnI restriction enzymes and 1U of alkaline phosphatase in the reaction. This treatment effectively eliminates the bacterially-produced methylated-plasmid template, and dephosphorylates the ends of the restricted PCR amplified plasmid vector.


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 vectors pSA-C6His-RIL.Map shows multiple cloning site with eight unique restriction enzyme sites for facile cloning of heterologous genes.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130446.g011: Schematic representation of expression vectors pSA-C6His-RIL.Map shows multiple cloning site with eight unique restriction enzyme sites for facile cloning of heterologous genes.
Mentions: To facilitate clone manipulation, we substituted nef gene with a multiple cloning site (MCS) clustered with eight unique restriction enzyme sites (Fig 11). Any two of the restriction enzyme sites can be chosen to clone gene of interest into the pSA-C6His-RIL vector. However it will be desirable to clone the gene of interest between NdeI and SacI, in order to avoid the addition of extra amino acids at N- and/or C-terminals of the resultant recombinant protein. Another possibility is to introduce desired restriction enzyme sites to the plasmid using whole-plasmid PCR. Several high fidelity polymerases suitable for whole-plasmid PCR are now available at reasonable cost. Whenever possible, we amplify the entire plasmid backbone with a set of primers containing desired restriction enzyme sites using Phusion or Q5 High-Fidelity DNA Polymerases. By using 50–100 ng plasmid vector as template, and using 18–25 PCR cycles, it is possible to obtain sufficient amount of PCR amplified plasmid, which can be used to clone the gene of interest with compatible restriction enzymes sites. To reduce the background, we add 10U of DpnI restriction enzymes and 1U of alkaline phosphatase in the reaction. This treatment effectively eliminates the bacterially-produced methylated-plasmid template, and dephosphorylates the ends of the restricted PCR amplified plasmid vector.

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