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Production of recombinant nonstructural 1 protein in Escherichia coli for early detection of Japanese encephalitis virus infection.

Tripathi NK, Kumar JS, Biswal KC, Rao PV - Microb Biotechnol (2012)

Bottom Line: The reactivity of purified protein was confirmed by Western blotting and Enzyme linked immunosorbent assay.These results show that rNS1 protein may be used as a diagnostic reagent or for further prophylactic studies.This approach of producing rNS1 protein in E. coli with high yield may also offer promising method for production of other viral recombinant proteins.

View Article: PubMed Central - PubMed

Affiliation: Bioprocess Scale up Facility, Defence Research and Development Establishment, Jhansi Road, Gwalior-474002, India. tripathink@gmail.com

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Related in: MedlinePlus

A. Coomassie stained SDS‐PAGE. The protein band of ∼ 44.0 kDa confirmed the predicted size of the rJE NS1 protein. The protein profiles of the eluted protein in coomassie stained gel were analysed densitometrically using Quantity One image quantification software, which showed that more than 90% purity has been achieved. Lane 1, Molecular Weight Marker (kDa); lane 2, purified rJEV NS1 protein. B. Silver stained SDS‐PAGE. Lane 1, Molecular Weight Marker (kDa); lane 2, Purified rJEV NS1 protein. C. Western blot analysis of the purified rJEV protein. The sample showing reaction with protein at desired size (∼ 44 kDa) was considered positive. Lane 1, Hyper immune serum. D. Results of Dipstick elisa with rJEV NS1 protein. Here the sample showing a dot against a clear background were scored positive and those with no dot were scored negative. Lane 1, Positive control; lane 2, IgM positive CSF; lane 3, IgM positive serum; lane 4, IgM negative CSF; lane 5, IgM negative serum samples.
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f2: A. Coomassie stained SDS‐PAGE. The protein band of ∼ 44.0 kDa confirmed the predicted size of the rJE NS1 protein. The protein profiles of the eluted protein in coomassie stained gel were analysed densitometrically using Quantity One image quantification software, which showed that more than 90% purity has been achieved. Lane 1, Molecular Weight Marker (kDa); lane 2, purified rJEV NS1 protein. B. Silver stained SDS‐PAGE. Lane 1, Molecular Weight Marker (kDa); lane 2, Purified rJEV NS1 protein. C. Western blot analysis of the purified rJEV protein. The sample showing reaction with protein at desired size (∼ 44 kDa) was considered positive. Lane 1, Hyper immune serum. D. Results of Dipstick elisa with rJEV NS1 protein. Here the sample showing a dot against a clear background were scored positive and those with no dot were scored negative. Lane 1, Positive control; lane 2, IgM positive CSF; lane 3, IgM positive serum; lane 4, IgM negative CSF; lane 5, IgM negative serum samples.

Mentions: Expression of heterologous protein in E. coli allows its rapid and economical production in large amounts. In an effort to obtain target protein in host E. coli strain, IBs formation is still considered as a convenient and effective way in recombinant protein production (Singh and Panda, 2005). After cell disruption and centrifugation analysis of the lysate confirmed the presence of the major proportion of ∼ 44 kDa protein band. IBs were harvested and purified from the induced and lysed cell mass. The IBs were solubilized in buffer containing 8 M urea and purified by affinity chromatography under denaturing conditions. The purified protein was further dialysed before used for elisa. The SDS‐PAGE profile of eluted protein is shown in Fig. 2A and B. From the protein profiles of the eluted protein in SDS‐PAGE (Fig. 2A and B) and densitometry analysis using Quantity One image quantification software (Bio‐Rad, USA), more than 90% purity was found to be achieved. The cell pellet harvested from 50 ml of induced fed‐batch culture yielded ∼ 7.0 mg purified rJEV NS1 protein with ∼ 92% purity. This corresponds to a recovery of ∼ 50% as the crude cell lysate was estimated to contain ∼ 13.9 mg of the rJEV NS1 protein and 104 mg total protein, based on densitometric analysis using Quantity One software (Bio‐Rad, USA). The solubilized IBs was estimated to contain ∼ 9.0 mg of the rJEV NS1 protein with ∼ 64% recovery and ∼ 75% purity. The final product concentration of rJEV NS1 protein following affinity chromatography was significantly higher for fed‐batch cultivation as compared with batch cultivation (Table 1). Improvement in product yield about more than eight times for fed‐batch cultivation as compared with shake flask culture resulted using modified SB medium (Table 1). The final rJEV NS1 protein yield after fed‐batch cultivation was ∼ 142.16 mg l−1 (Table 1).


Production of recombinant nonstructural 1 protein in Escherichia coli for early detection of Japanese encephalitis virus infection.

Tripathi NK, Kumar JS, Biswal KC, Rao PV - Microb Biotechnol (2012)

A. Coomassie stained SDS‐PAGE. The protein band of ∼ 44.0 kDa confirmed the predicted size of the rJE NS1 protein. The protein profiles of the eluted protein in coomassie stained gel were analysed densitometrically using Quantity One image quantification software, which showed that more than 90% purity has been achieved. Lane 1, Molecular Weight Marker (kDa); lane 2, purified rJEV NS1 protein. B. Silver stained SDS‐PAGE. Lane 1, Molecular Weight Marker (kDa); lane 2, Purified rJEV NS1 protein. C. Western blot analysis of the purified rJEV protein. The sample showing reaction with protein at desired size (∼ 44 kDa) was considered positive. Lane 1, Hyper immune serum. D. Results of Dipstick elisa with rJEV NS1 protein. Here the sample showing a dot against a clear background were scored positive and those with no dot were scored negative. Lane 1, Positive control; lane 2, IgM positive CSF; lane 3, IgM positive serum; lane 4, IgM negative CSF; lane 5, IgM negative serum samples.
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Related In: Results  -  Collection

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f2: A. Coomassie stained SDS‐PAGE. The protein band of ∼ 44.0 kDa confirmed the predicted size of the rJE NS1 protein. The protein profiles of the eluted protein in coomassie stained gel were analysed densitometrically using Quantity One image quantification software, which showed that more than 90% purity has been achieved. Lane 1, Molecular Weight Marker (kDa); lane 2, purified rJEV NS1 protein. B. Silver stained SDS‐PAGE. Lane 1, Molecular Weight Marker (kDa); lane 2, Purified rJEV NS1 protein. C. Western blot analysis of the purified rJEV protein. The sample showing reaction with protein at desired size (∼ 44 kDa) was considered positive. Lane 1, Hyper immune serum. D. Results of Dipstick elisa with rJEV NS1 protein. Here the sample showing a dot against a clear background were scored positive and those with no dot were scored negative. Lane 1, Positive control; lane 2, IgM positive CSF; lane 3, IgM positive serum; lane 4, IgM negative CSF; lane 5, IgM negative serum samples.
Mentions: Expression of heterologous protein in E. coli allows its rapid and economical production in large amounts. In an effort to obtain target protein in host E. coli strain, IBs formation is still considered as a convenient and effective way in recombinant protein production (Singh and Panda, 2005). After cell disruption and centrifugation analysis of the lysate confirmed the presence of the major proportion of ∼ 44 kDa protein band. IBs were harvested and purified from the induced and lysed cell mass. The IBs were solubilized in buffer containing 8 M urea and purified by affinity chromatography under denaturing conditions. The purified protein was further dialysed before used for elisa. The SDS‐PAGE profile of eluted protein is shown in Fig. 2A and B. From the protein profiles of the eluted protein in SDS‐PAGE (Fig. 2A and B) and densitometry analysis using Quantity One image quantification software (Bio‐Rad, USA), more than 90% purity was found to be achieved. The cell pellet harvested from 50 ml of induced fed‐batch culture yielded ∼ 7.0 mg purified rJEV NS1 protein with ∼ 92% purity. This corresponds to a recovery of ∼ 50% as the crude cell lysate was estimated to contain ∼ 13.9 mg of the rJEV NS1 protein and 104 mg total protein, based on densitometric analysis using Quantity One software (Bio‐Rad, USA). The solubilized IBs was estimated to contain ∼ 9.0 mg of the rJEV NS1 protein with ∼ 64% recovery and ∼ 75% purity. The final product concentration of rJEV NS1 protein following affinity chromatography was significantly higher for fed‐batch cultivation as compared with batch cultivation (Table 1). Improvement in product yield about more than eight times for fed‐batch cultivation as compared with shake flask culture resulted using modified SB medium (Table 1). The final rJEV NS1 protein yield after fed‐batch cultivation was ∼ 142.16 mg l−1 (Table 1).

Bottom Line: The reactivity of purified protein was confirmed by Western blotting and Enzyme linked immunosorbent assay.These results show that rNS1 protein may be used as a diagnostic reagent or for further prophylactic studies.This approach of producing rNS1 protein in E. coli with high yield may also offer promising method for production of other viral recombinant proteins.

View Article: PubMed Central - PubMed

Affiliation: Bioprocess Scale up Facility, Defence Research and Development Establishment, Jhansi Road, Gwalior-474002, India. tripathink@gmail.com

Show MeSH
Related in: MedlinePlus