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Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli.

Farshadpour F, Taherkhani R, Makvandi M, Rajabi Memari H, Samarbafzadeh AR - Jundishapur J Microbiol (2014)

Bottom Line: The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours.The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting.The yield of the purified protein was about 1 mg/L of culture media.

View Article: PubMed Central - PubMed

Affiliation: Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran ; Department of Medical Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran.

ABSTRACT

Background: Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential.

Objectives: The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate.

Materials and methods: The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.

Results: The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media.

Conclusions: In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified protein was obtained, which can be a potential vaccine candidate and as an antigen in ELISA to diagnose HEV infections.

No MeSH data available.


Related in: MedlinePlus

Polymerase Chain Reaction Amplification and Restriction Enzyme Analyses of Different Plasmids by NdeI and XhoI Restriction Enzymes and Comparison of Undigested and Digested Patterns of Plasmids With orf2.1 and orf2.2 on Agarose Gel ElectrophoresisPCR amplification and restriction enzyme analyses of plasmids pBluescript II SK-ORF2.1, pET30a-ORF2.1, pET30a-ORF2.2, and pET30a+ without ORF2.1 by NdeI and XhoI restriction enzymes. Lane 1, the 1 kb DNA marker; Lane 2, the undigested plasmid pET30a+; Lane 3, the digested plasmid pET30a+; Lane 4, the undigested pBluescript II SK-ORF2.1; Lane 5, the digested pBluescript II SK-ORF2.1; Lane 6, the undigested plasmid pET30a-ORF2.1; Lane 7, the digested plasmid pET30a- ORF2.1; Lane 8, the amplified orf2.1 gene by PCR (with T7 promoter and T7 terminator primers); Lane 9, the undigested plasmid pET30a-ORF2.2; Lane 10, the digested plasmid pET30a-ORF2.2; Lane 11, the amplified orf2.2 gene by PCR (with T7 promoter and T7 terminator primers); Lane 13, the 1kb DNA marker; Lane 14, the amplified orf2.1 gene by PCR; and Lane 15, the amplified orf2.2 gene by PCR.
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fig12171: Polymerase Chain Reaction Amplification and Restriction Enzyme Analyses of Different Plasmids by NdeI and XhoI Restriction Enzymes and Comparison of Undigested and Digested Patterns of Plasmids With orf2.1 and orf2.2 on Agarose Gel ElectrophoresisPCR amplification and restriction enzyme analyses of plasmids pBluescript II SK-ORF2.1, pET30a-ORF2.1, pET30a-ORF2.2, and pET30a+ without ORF2.1 by NdeI and XhoI restriction enzymes. Lane 1, the 1 kb DNA marker; Lane 2, the undigested plasmid pET30a+; Lane 3, the digested plasmid pET30a+; Lane 4, the undigested pBluescript II SK-ORF2.1; Lane 5, the digested pBluescript II SK-ORF2.1; Lane 6, the undigested plasmid pET30a-ORF2.1; Lane 7, the digested plasmid pET30a- ORF2.1; Lane 8, the amplified orf2.1 gene by PCR (with T7 promoter and T7 terminator primers); Lane 9, the undigested plasmid pET30a-ORF2.2; Lane 10, the digested plasmid pET30a-ORF2.2; Lane 11, the amplified orf2.2 gene by PCR (with T7 promoter and T7 terminator primers); Lane 13, the 1kb DNA marker; Lane 14, the amplified orf2.1 gene by PCR; and Lane 15, the amplified orf2.2 gene by PCR.

Mentions: The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of the recombinant plasmid pET30a-ORF2.2. The size of PCR products on 1.2% agarose gel electrophoresis for orf 2.1 and orf 2.2 genes with plasmid universal primers were 1943 and 1750 bp, respectively, which were consistent with the expected size (Figure 1). The result of sequencing analysis with plasmid universal primers showed that the synthesized gene agreed with what had been designed. A series of expression conditions that differed in induction time, IPTG concentration, and induction temperature were tested to optimize the protein expression. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. After being induced with IPTG, the target protein was highly expressed and purified by Ni2+-chelate affinity chromatography.


Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli.

Farshadpour F, Taherkhani R, Makvandi M, Rajabi Memari H, Samarbafzadeh AR - Jundishapur J Microbiol (2014)

Polymerase Chain Reaction Amplification and Restriction Enzyme Analyses of Different Plasmids by NdeI and XhoI Restriction Enzymes and Comparison of Undigested and Digested Patterns of Plasmids With orf2.1 and orf2.2 on Agarose Gel ElectrophoresisPCR amplification and restriction enzyme analyses of plasmids pBluescript II SK-ORF2.1, pET30a-ORF2.1, pET30a-ORF2.2, and pET30a+ without ORF2.1 by NdeI and XhoI restriction enzymes. Lane 1, the 1 kb DNA marker; Lane 2, the undigested plasmid pET30a+; Lane 3, the digested plasmid pET30a+; Lane 4, the undigested pBluescript II SK-ORF2.1; Lane 5, the digested pBluescript II SK-ORF2.1; Lane 6, the undigested plasmid pET30a-ORF2.1; Lane 7, the digested plasmid pET30a- ORF2.1; Lane 8, the amplified orf2.1 gene by PCR (with T7 promoter and T7 terminator primers); Lane 9, the undigested plasmid pET30a-ORF2.2; Lane 10, the digested plasmid pET30a-ORF2.2; Lane 11, the amplified orf2.2 gene by PCR (with T7 promoter and T7 terminator primers); Lane 13, the 1kb DNA marker; Lane 14, the amplified orf2.1 gene by PCR; and Lane 15, the amplified orf2.2 gene by PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216576&req=5

fig12171: Polymerase Chain Reaction Amplification and Restriction Enzyme Analyses of Different Plasmids by NdeI and XhoI Restriction Enzymes and Comparison of Undigested and Digested Patterns of Plasmids With orf2.1 and orf2.2 on Agarose Gel ElectrophoresisPCR amplification and restriction enzyme analyses of plasmids pBluescript II SK-ORF2.1, pET30a-ORF2.1, pET30a-ORF2.2, and pET30a+ without ORF2.1 by NdeI and XhoI restriction enzymes. Lane 1, the 1 kb DNA marker; Lane 2, the undigested plasmid pET30a+; Lane 3, the digested plasmid pET30a+; Lane 4, the undigested pBluescript II SK-ORF2.1; Lane 5, the digested pBluescript II SK-ORF2.1; Lane 6, the undigested plasmid pET30a-ORF2.1; Lane 7, the digested plasmid pET30a- ORF2.1; Lane 8, the amplified orf2.1 gene by PCR (with T7 promoter and T7 terminator primers); Lane 9, the undigested plasmid pET30a-ORF2.2; Lane 10, the digested plasmid pET30a-ORF2.2; Lane 11, the amplified orf2.2 gene by PCR (with T7 promoter and T7 terminator primers); Lane 13, the 1kb DNA marker; Lane 14, the amplified orf2.1 gene by PCR; and Lane 15, the amplified orf2.2 gene by PCR.
Mentions: The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of the recombinant plasmid pET30a-ORF2.2. The size of PCR products on 1.2% agarose gel electrophoresis for orf 2.1 and orf 2.2 genes with plasmid universal primers were 1943 and 1750 bp, respectively, which were consistent with the expected size (Figure 1). The result of sequencing analysis with plasmid universal primers showed that the synthesized gene agreed with what had been designed. A series of expression conditions that differed in induction time, IPTG concentration, and induction temperature were tested to optimize the protein expression. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. After being induced with IPTG, the target protein was highly expressed and purified by Ni2+-chelate affinity chromatography.

Bottom Line: The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours.The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting.The yield of the purified protein was about 1 mg/L of culture media.

View Article: PubMed Central - PubMed

Affiliation: Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran ; Department of Medical Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran.

ABSTRACT

Background: Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential.

Objectives: The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate.

Materials and methods: The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.

Results: The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media.

Conclusions: In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified protein was obtained, which can be a potential vaccine candidate and as an antigen in ELISA to diagnose HEV infections.

No MeSH data available.


Related in: MedlinePlus