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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

SDS-PAGE and immunoblot analysis of recombinant Nef.NiCo21(DE3) E. coli were transformed with pSA-HNef-6His vector and grown overnight from a single colony at 30°C in LB broth supplemented with 100 μg/ml ampicillin. The cultures were 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 0.4 mM. The induced cells were grown for another 6 h at 30°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by (A) Coomassie staining and (B) immuno-blotting. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction; M1, MagicMark XP Western Protein Standard. Recombinant Nef was produced in the induced cells but not in the un-induced controls, and mainly present in the soluble fraction.
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pone.0130446.g002: SDS-PAGE and immunoblot analysis of recombinant Nef.NiCo21(DE3) E. coli were transformed with pSA-HNef-6His vector and grown overnight from a single colony at 30°C in LB broth supplemented with 100 μg/ml ampicillin. The cultures were 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 0.4 mM. The induced cells were grown for another 6 h at 30°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by (A) Coomassie staining and (B) immuno-blotting. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction; M1, MagicMark XP Western Protein Standard. Recombinant Nef was produced in the induced cells but not in the un-induced controls, and mainly present in the soluble fraction.

Mentions: E. coli NiCo21(DE3) was used to express 6His-tagged HIV-1 Nef using shaker flask culture conditions for expression. Plasmid-borne retroviral sequences are unstable in E. coli when grown at 37°C [22]. Therefore, the cultures of pSA-HNef-6His-transformed NiCo21(DE3) were grown at 30°C until the OD600 reached 0.5–0.6. Cultures were then induced with 0.4mM IPTG and incubated for another 6h at 30°C. When subjected to SDS-PAGE analysis, the expressed Nef protein appeared as an approximately 27kDa band in the IPTG-induced fractions (Fig 2A). Overexpressed Nef protein was mainly present in the soluble fraction. However, immunoblot analysis with the anti-Nef antibody revealed that some Nef was also present in the insoluble fraction (Fig 2B).


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)

SDS-PAGE and immunoblot analysis of recombinant Nef.NiCo21(DE3) E. coli were transformed with pSA-HNef-6His vector and grown overnight from a single colony at 30°C in LB broth supplemented with 100 μg/ml ampicillin. The cultures were 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 0.4 mM. The induced cells were grown for another 6 h at 30°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by (A) Coomassie staining and (B) immuno-blotting. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction; M1, MagicMark XP Western Protein Standard. Recombinant Nef was produced in the induced cells but not in the un-induced controls, and mainly present in the soluble fraction.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130446.g002: SDS-PAGE and immunoblot analysis of recombinant Nef.NiCo21(DE3) E. coli were transformed with pSA-HNef-6His vector and grown overnight from a single colony at 30°C in LB broth supplemented with 100 μg/ml ampicillin. The cultures were 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 0.4 mM. The induced cells were grown for another 6 h at 30°C and stored on ice. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by (A) Coomassie staining and (B) immuno-blotting. Lanes: M, PageRuler Prestained Protein Ladder Plus; WCL, whole cell lysate; IS, insoluble fraction; S, soluble fraction; M1, MagicMark XP Western Protein Standard. Recombinant Nef was produced in the induced cells but not in the un-induced controls, and mainly present in the soluble fraction.
Mentions: E. coli NiCo21(DE3) was used to express 6His-tagged HIV-1 Nef using shaker flask culture conditions for expression. Plasmid-borne retroviral sequences are unstable in E. coli when grown at 37°C [22]. Therefore, the cultures of pSA-HNef-6His-transformed NiCo21(DE3) were grown at 30°C until the OD600 reached 0.5–0.6. Cultures were then induced with 0.4mM IPTG and incubated for another 6h at 30°C. When subjected to SDS-PAGE analysis, the expressed Nef protein appeared as an approximately 27kDa band in the IPTG-induced fractions (Fig 2A). Overexpressed Nef protein was mainly present in the soluble fraction. However, immunoblot analysis with the anti-Nef antibody revealed that some Nef was also present in the insoluble fraction (Fig 2B).

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