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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

Expression of HIV-1 P24 from pSA-HP24-6His vectors with or without rare tRNA genes array.To evaluate the effect of the introduction of rare tRNA genes array in pSA-HP24-6His vector, the NiCo21(DE3) E. coli were individually transformed with pSA-HP24-6His, and pSA-HP24-6His-RIL and P24 expression was compared with NiCo21(DE3) E.coli co-transformed with pSA-Hp24-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HP24-6His or pSA-HP24-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HP24-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A), immuno-blotting (B), and band densitometery (C). The P24 expression in NiCo21 transformed with pSA-HP24-6His-RIL was comparable with NiCo21 co-transformed with pSA-HP24-6His and pACYC-RIL.
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pone.0130446.g008: Expression of HIV-1 P24 from pSA-HP24-6His vectors with or without rare tRNA genes array.To evaluate the effect of the introduction of rare tRNA genes array in pSA-HP24-6His vector, the NiCo21(DE3) E. coli were individually transformed with pSA-HP24-6His, and pSA-HP24-6His-RIL and P24 expression was compared with NiCo21(DE3) E.coli co-transformed with pSA-Hp24-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HP24-6His or pSA-HP24-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HP24-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A), immuno-blotting (B), and band densitometery (C). The P24 expression in NiCo21 transformed with pSA-HP24-6His-RIL was comparable with NiCo21 co-transformed with pSA-HP24-6His and pACYC-RIL.

Mentions: In order to further confirm the hypothesis that recombinant protein(s) can be produced in similar or better amounts from a single vector concomitantly expressing rare tRNA and a heterologous gene compared to traditional two-vector system, we expressed HIV-1 p24 gene. HIV-1 p24 gene contains (a) three rare codons (AGG, AGA, CGA) for arginine at positions 44, 59, 62, 94, 105, 116, 124, 129, 135, 191; (b) one rare codon (CTA) for leucine at positions 18, 134, 173; (c) one rare codon (ATA) for isoleucine at positions 66, 77, 96, 103, 115; and (d) one rare codon (CCC) for proline at positions 122, 186. We have previously shown that pACYC-RIL plasmid significantly helped with improving the overall yields of P24/CA [13]. Here we replaced the nef gene in pSA-HNef-6His-RIL with p24 and compared the expression profile of P24/CA from pSA-HP24-6His, pSA-HP24-6His together with pACYC-RIL, and pSA-HP24-6His-RIL (Fig 7). The helper plasmids pRARE2/pRARE2 lysS were omitted because in the previous work they were found not to offer any advantage over pACYC-RIL helper plasmid [13]. As anticipated, almost 2 fold more P24/CA was expressed in cultures containing pSA-HP24-6His+pACYC-RIL and pSA-HP24-6His-RIL. The SDS-PAGE (Fig 8A), Western Blot (Fig 8B), and densitometric (Fig 8C) analyses showed that relatively more p24/CA was produced from the single plasmid (pSA-HP24-6His-RIL) compared to traditional two plasmid (pSA-HP24-6His+pACYC-RIL) system.


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)

Expression of HIV-1 P24 from pSA-HP24-6His vectors with or without rare tRNA genes array.To evaluate the effect of the introduction of rare tRNA genes array in pSA-HP24-6His vector, the NiCo21(DE3) E. coli were individually transformed with pSA-HP24-6His, and pSA-HP24-6His-RIL and P24 expression was compared with NiCo21(DE3) E.coli co-transformed with pSA-Hp24-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HP24-6His or pSA-HP24-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HP24-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A), immuno-blotting (B), and band densitometery (C). The P24 expression in NiCo21 transformed with pSA-HP24-6His-RIL was comparable with NiCo21 co-transformed with pSA-HP24-6His and pACYC-RIL.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130446.g008: Expression of HIV-1 P24 from pSA-HP24-6His vectors with or without rare tRNA genes array.To evaluate the effect of the introduction of rare tRNA genes array in pSA-HP24-6His vector, the NiCo21(DE3) E. coli were individually transformed with pSA-HP24-6His, and pSA-HP24-6His-RIL and P24 expression was compared with NiCo21(DE3) E.coli co-transformed with pSA-Hp24-6His and rare tRNA expressing pACYC-RIL vectors. The cultures were grown overnight at 30°C in LB broth containing Amp (for bacteria harboring pSA-HP24-6His or pSA-HP24-6His-RIL) or Amp+Cam (for bacteria harboring pSA-HP24-6His + pACYC-RIL). The cultures were then diluted 100-fold in the same medium and grown to mid-log phase (A600 ~0.5–0.6), at which point IPTG was added to a final concentration of 0.05mM. The induced cells were grown for another 12 hours at 22°C and stored on ice. Three microliters of samples were mixed with 4X loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining (A), immuno-blotting (B), and band densitometery (C). The P24 expression in NiCo21 transformed with pSA-HP24-6His-RIL was comparable with NiCo21 co-transformed with pSA-HP24-6His and pACYC-RIL.
Mentions: In order to further confirm the hypothesis that recombinant protein(s) can be produced in similar or better amounts from a single vector concomitantly expressing rare tRNA and a heterologous gene compared to traditional two-vector system, we expressed HIV-1 p24 gene. HIV-1 p24 gene contains (a) three rare codons (AGG, AGA, CGA) for arginine at positions 44, 59, 62, 94, 105, 116, 124, 129, 135, 191; (b) one rare codon (CTA) for leucine at positions 18, 134, 173; (c) one rare codon (ATA) for isoleucine at positions 66, 77, 96, 103, 115; and (d) one rare codon (CCC) for proline at positions 122, 186. We have previously shown that pACYC-RIL plasmid significantly helped with improving the overall yields of P24/CA [13]. Here we replaced the nef gene in pSA-HNef-6His-RIL with p24 and compared the expression profile of P24/CA from pSA-HP24-6His, pSA-HP24-6His together with pACYC-RIL, and pSA-HP24-6His-RIL (Fig 7). The helper plasmids pRARE2/pRARE2 lysS were omitted because in the previous work they were found not to offer any advantage over pACYC-RIL helper plasmid [13]. As anticipated, almost 2 fold more P24/CA was expressed in cultures containing pSA-HP24-6His+pACYC-RIL and pSA-HP24-6His-RIL. The SDS-PAGE (Fig 8A), Western Blot (Fig 8B), and densitometric (Fig 8C) analyses showed that relatively more p24/CA was produced from the single plasmid (pSA-HP24-6His-RIL) compared to traditional two plasmid (pSA-HP24-6His+pACYC-RIL) system.

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