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

Effect of rare tRNA supplementation on Nef expression in NiCo21(DE3) E. coli.(A) ORF coding for HIV-1 nef gene was subjected to rare codon analysis using an online tool ‘Rare Codon Calculator (RaCC)’ http://nihserver.mbi.ucla.edu/RACC/. Three rare codons encoding arginine and one rare codon encoding leucine are present at 17 amino acid positions in Nef ORF. (B) NiCo21(DE3) E. coli were individually transformed with rare tRNA-expressing helper plasmids (pACYC-RIL, pRARE2, pRARE2-lysS) and selected on LB+Cam. These bacteria were then made competent, transformed with pSA-HNef-6His, and selected on LB+Cam+Amp plates. Cultures were grown overnight from a single colony at 30°C in LB+Cam+Amp. The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (OD600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05 mM. The induced cells were grown for another 12 h at 22°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel analyzed by Coomassie staining. Expression of rare codon tRNA genes resulted in high level expression of Nef. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130446.g004: Effect of rare tRNA supplementation on Nef expression in NiCo21(DE3) E. coli.(A) ORF coding for HIV-1 nef gene was subjected to rare codon analysis using an online tool ‘Rare Codon Calculator (RaCC)’ http://nihserver.mbi.ucla.edu/RACC/. Three rare codons encoding arginine and one rare codon encoding leucine are present at 17 amino acid positions in Nef ORF. (B) NiCo21(DE3) E. coli were individually transformed with rare tRNA-expressing helper plasmids (pACYC-RIL, pRARE2, pRARE2-lysS) and selected on LB+Cam. These bacteria were then made competent, transformed with pSA-HNef-6His, and selected on LB+Cam+Amp plates. Cultures were grown overnight from a single colony at 30°C in LB+Cam+Amp. The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (OD600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05 mM. The induced cells were grown for another 12 h at 22°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel analyzed by Coomassie staining. Expression of rare codon tRNA genes resulted in high level expression of Nef. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction.

Mentions: Over-expression of heterologous proteins in E. coli may be considerably circumscribed by the presence of "rare" codons in the foreign mRNA that are seldom used by E. coli [23, 24]. When subjected to ‘rare’ codon analysis, the 618bp coding sequence for HIV-1 Nef was found to contain (a) three rare codons (AGG, AGA, CGA) for arginine at positions 17, 19, 21, 22, 35, 77, 105, 106, 134, 178, 184, 194, and (b) one rare codon (CTA) for leucine at positions 37, 58, 100, 145, and 189 (Fig 4A). To investigate whether Nef expression could be improved by expressing nef together with rare tRNA genes, NiCo21(DE3) were transformed with rare tRNA expressing pACYC-RIL, pRARE2, and pLysSRARE2 helper plasmids. The pACYC-RIL plasmid supplies tRNA for four rare codons (AUA, AGG, AGA, CUA), whereas pRARE2 supplies those for seven rare codons (AUA, AGG, AGA, CUA, CCC, CGG, and GGA). In addition to seven rare codons, transformation with pLysSRARE2 also results in lower background expression due to the expression of T7 lysozyme. When expressed in the presence of rare tRNA supplying helper plasmids, Nef expression strikingly improved as shown in Fig 4B. However, this also lead some Nef protein to end up in the insoluble fractions, suggesting that bacterial protein folding machinery was saturated by the enhanced expression. There was no significant change in Nef expression between the NiCo21(DE3) containing pACYC-RIL and pRARE2/ pLysSRARE2, suggesting that supplementation with four rare codons was sufficient to optimize Nef expression. In a side-by-side comparison, NiCo21(DE3)/pACYC-RIL showed relatively higher production of soluble Nef (Fig 4B).


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)

Effect of rare tRNA supplementation on Nef expression in NiCo21(DE3) E. coli.(A) ORF coding for HIV-1 nef gene was subjected to rare codon analysis using an online tool ‘Rare Codon Calculator (RaCC)’ http://nihserver.mbi.ucla.edu/RACC/. Three rare codons encoding arginine and one rare codon encoding leucine are present at 17 amino acid positions in Nef ORF. (B) NiCo21(DE3) E. coli were individually transformed with rare tRNA-expressing helper plasmids (pACYC-RIL, pRARE2, pRARE2-lysS) and selected on LB+Cam. These bacteria were then made competent, transformed with pSA-HNef-6His, and selected on LB+Cam+Amp plates. Cultures were grown overnight from a single colony at 30°C in LB+Cam+Amp. The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (OD600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05 mM. The induced cells were grown for another 12 h at 22°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel analyzed by Coomassie staining. Expression of rare codon tRNA genes resulted in high level expression of Nef. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130446.g004: Effect of rare tRNA supplementation on Nef expression in NiCo21(DE3) E. coli.(A) ORF coding for HIV-1 nef gene was subjected to rare codon analysis using an online tool ‘Rare Codon Calculator (RaCC)’ http://nihserver.mbi.ucla.edu/RACC/. Three rare codons encoding arginine and one rare codon encoding leucine are present at 17 amino acid positions in Nef ORF. (B) NiCo21(DE3) E. coli were individually transformed with rare tRNA-expressing helper plasmids (pACYC-RIL, pRARE2, pRARE2-lysS) and selected on LB+Cam. These bacteria were then made competent, transformed with pSA-HNef-6His, and selected on LB+Cam+Amp plates. Cultures were grown overnight from a single colony at 30°C in LB+Cam+Amp. The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (OD600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05 mM. The induced cells were grown for another 12 h at 22°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel analyzed by Coomassie staining. Expression of rare codon tRNA genes resulted in high level expression of Nef. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction.
Mentions: Over-expression of heterologous proteins in E. coli may be considerably circumscribed by the presence of "rare" codons in the foreign mRNA that are seldom used by E. coli [23, 24]. When subjected to ‘rare’ codon analysis, the 618bp coding sequence for HIV-1 Nef was found to contain (a) three rare codons (AGG, AGA, CGA) for arginine at positions 17, 19, 21, 22, 35, 77, 105, 106, 134, 178, 184, 194, and (b) one rare codon (CTA) for leucine at positions 37, 58, 100, 145, and 189 (Fig 4A). To investigate whether Nef expression could be improved by expressing nef together with rare tRNA genes, NiCo21(DE3) were transformed with rare tRNA expressing pACYC-RIL, pRARE2, and pLysSRARE2 helper plasmids. The pACYC-RIL plasmid supplies tRNA for four rare codons (AUA, AGG, AGA, CUA), whereas pRARE2 supplies those for seven rare codons (AUA, AGG, AGA, CUA, CCC, CGG, and GGA). In addition to seven rare codons, transformation with pLysSRARE2 also results in lower background expression due to the expression of T7 lysozyme. When expressed in the presence of rare tRNA supplying helper plasmids, Nef expression strikingly improved as shown in Fig 4B. However, this also lead some Nef protein to end up in the insoluble fractions, suggesting that bacterial protein folding machinery was saturated by the enhanced expression. There was no significant change in Nef expression between the NiCo21(DE3) containing pACYC-RIL and pRARE2/ pLysSRARE2, suggesting that supplementation with four rare codons was sufficient to optimize Nef expression. In a side-by-side comparison, NiCo21(DE3)/pACYC-RIL showed relatively higher production of soluble Nef (Fig 4B).

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