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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

Western Blot Analysis of the Expressed and Purified ORF2.2 Protein in E. coliThe ORF2 protein band is seen at about 55 kDa. Lane 1, the prestained protein ladder; Lane 2, the noninduced control; Lane 3, Inducing for fourhours; Lane 4, flow-through; Lane:5, wash; Lane: 6 to 8, the first, the second, and the third eluates.
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fig12173: Western Blot Analysis of the Expressed and Purified ORF2.2 Protein in E. coliThe ORF2 protein band is seen at about 55 kDa. Lane 1, the prestained protein ladder; Lane 2, the noninduced control; Lane 3, Inducing for fourhours; Lane 4, flow-through; Lane:5, wash; Lane: 6 to 8, the first, the second, and the third eluates.

Mentions: 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, which was in agreement with the expected molecular weight; however, no band was detected in non induced culture. SDS-PAGE of the purified ORF2.2 protein showed that the abundant target protein appeared in the eluate of the elution buffer at pH of 4.5 (Figure 2). The yield of the purified protein was about 1 mg/L of culture media. Western blotting analysis was performed and the presence of the truncated ORF2 protein in E. coli BL21 (DE3) was confirmed (Figure 3).


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)

Western Blot Analysis of the Expressed and Purified ORF2.2 Protein in E. coliThe ORF2 protein band is seen at about 55 kDa. Lane 1, the prestained protein ladder; Lane 2, the noninduced control; Lane 3, Inducing for fourhours; Lane 4, flow-through; Lane:5, wash; Lane: 6 to 8, the first, the second, and the third eluates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig12173: Western Blot Analysis of the Expressed and Purified ORF2.2 Protein in E. coliThe ORF2 protein band is seen at about 55 kDa. Lane 1, the prestained protein ladder; Lane 2, the noninduced control; Lane 3, Inducing for fourhours; Lane 4, flow-through; Lane:5, wash; Lane: 6 to 8, the first, the second, and the third eluates.
Mentions: 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, which was in agreement with the expected molecular weight; however, no band was detected in non induced culture. SDS-PAGE of the purified ORF2.2 protein showed that the abundant target protein appeared in the eluate of the elution buffer at pH of 4.5 (Figure 2). The yield of the purified protein was about 1 mg/L of culture media. Western blotting analysis was performed and the presence of the truncated ORF2 protein in E. coli BL21 (DE3) was confirmed (Figure 3).

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