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Over-expression and characterization of NS3 and NS5A of Hepatitis C virus genotype 3a.

Anwar MI, Iqbal M, Yousef MS, Rahman M - Microb. Cell Fact. (2013)

Bottom Line: Yield of the purified NS3 obtained is four fold higher than previous reports.CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein.Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. hamzamgondal@gmail.com.

ABSTRACT

Background: Hepatitis C virus (HCV) is a common and leading cause for liver cirrhosis and hepatocellular carcinoma. Current therapies to treat HCV infection are shown to be partially effective and poorly tolerated. Therefore, ample efforts are underway to rationally design therapies targeting the HCV non-structural proteins. Most of the work carried out in this direction has been focusing mainly on HCV genotype 1. Two direct-acting antiviral agents (DAAs) Telaprevir and Boceprevir are being used against genotype 1a infection in combination therapy with interferon and ribavirin. Unfortunately these DAAs are not effective against genotype 3a. Considering the wide spread infection by HCV genotype 3a in developing countries especially South Asia, we have focused on the recombinant production of antiviral drug targets NS3 and NS5A from HCV genotype 3a. These protein targets are to be used for screening of inhibitors.

Results: High-level expression of NS3 and NS5A was achieved at 25°C, using ~1 and 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. Yields of the purified NS3 and NS5A were 4 and 1 mg per liter culture volume, respectively. Although similar amounts of purified NS3 were obtained at 25 and 14°C, specificity constant (Kcat/Km) was somewhat higher at expression temperature of 25°C. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy revealed that both NS3 and NS5A contain a mixture of alpha-helix and beta-sheet secondary structures. For NS3 protein, percentages of secondary structures were similar to the values predicted from homology modeling.

Conclusions: NS3 and NS5A were over-expressed and using Nickel-affinity method both proteins were purified to ~ 95% purity. Yield of the purified NS3 obtained is four fold higher than previous reports. CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein. Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal. The production of NS3 and NS5A in milligram quantities will allow their characterization by biophysical and biochemical means that will help in designing new strategies to fight against HCV infection.

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Characterization of His6-NS3 [A] and NS5A-T-His6 [B] over-expressed at 32°C, 25°C and 14°C by circular dichroism spectroscopy. CD experiments were performed as described in Methods.
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Figure 5: Characterization of His6-NS3 [A] and NS5A-T-His6 [B] over-expressed at 32°C, 25°C and 14°C by circular dichroism spectroscopy. CD experiments were performed as described in Methods.

Mentions: Previous studies report expression of NS3 and NS5A from different HCV genotypes using a range of temperatures. For example, expression of NS3/HCV genotype 1a/2b/3a has been achieved at 30°C and 22°C [38-40], and NS5A has been expressed at room temperature in most of studies [21,41,43,44]. In this study, we systematically investigated the affect of temperature (32°C, 25°C and 14°C) on expression and purification yield of His6-NS3 and His6-NS5A-T/HCV genotype 3a. Both proteins can be expressed at all tested temperatures and purified by single step Ni-NTA affinity chromatography to ~ 95% purity as judged by Coomassie blue staining (Figure 4). His6-NS5A-T is further polished by gel filtration to produce homogenous and mono-disperse protein (Figure 4B, lane 4). Higher purification yields 4.0 and 1.0 mg per liter culture volume were obtained at both 25°C, 14°C and 25°C for His6-NS3 and His6-NS5A-T, respectively (Table 1). Optimized expression conditions yielded four fold purified His6-NS3 than achieved earlier [40] whereas His6-NS5A-T yield was somewhat similar as reported in earlier studies for NS5A or NS5A-T/HCV genotype 1 [41,44]. Higher purification yield of NS3/3a might be due to its sequence variability from NS3 sequences of other genotypes (Figure 1A). Identity of purified NS3 and NS5A was confirmed by peptide mass finger printing based mass-spectrometry method. CD analyses of the purified His6-NS3 and His6-NS5A-T produced at different temperatures (32°C, 25°C and 14°C) revealed that both proteins are folded polypeptides (Figure 5).


Over-expression and characterization of NS3 and NS5A of Hepatitis C virus genotype 3a.

Anwar MI, Iqbal M, Yousef MS, Rahman M - Microb. Cell Fact. (2013)

Characterization of His6-NS3 [A] and NS5A-T-His6 [B] over-expressed at 32°C, 25°C and 14°C by circular dichroism spectroscopy. CD experiments were performed as described in Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842787&req=5

Figure 5: Characterization of His6-NS3 [A] and NS5A-T-His6 [B] over-expressed at 32°C, 25°C and 14°C by circular dichroism spectroscopy. CD experiments were performed as described in Methods.
Mentions: Previous studies report expression of NS3 and NS5A from different HCV genotypes using a range of temperatures. For example, expression of NS3/HCV genotype 1a/2b/3a has been achieved at 30°C and 22°C [38-40], and NS5A has been expressed at room temperature in most of studies [21,41,43,44]. In this study, we systematically investigated the affect of temperature (32°C, 25°C and 14°C) on expression and purification yield of His6-NS3 and His6-NS5A-T/HCV genotype 3a. Both proteins can be expressed at all tested temperatures and purified by single step Ni-NTA affinity chromatography to ~ 95% purity as judged by Coomassie blue staining (Figure 4). His6-NS5A-T is further polished by gel filtration to produce homogenous and mono-disperse protein (Figure 4B, lane 4). Higher purification yields 4.0 and 1.0 mg per liter culture volume were obtained at both 25°C, 14°C and 25°C for His6-NS3 and His6-NS5A-T, respectively (Table 1). Optimized expression conditions yielded four fold purified His6-NS3 than achieved earlier [40] whereas His6-NS5A-T yield was somewhat similar as reported in earlier studies for NS5A or NS5A-T/HCV genotype 1 [41,44]. Higher purification yield of NS3/3a might be due to its sequence variability from NS3 sequences of other genotypes (Figure 1A). Identity of purified NS3 and NS5A was confirmed by peptide mass finger printing based mass-spectrometry method. CD analyses of the purified His6-NS3 and His6-NS5A-T produced at different temperatures (32°C, 25°C and 14°C) revealed that both proteins are folded polypeptides (Figure 5).

Bottom Line: Yield of the purified NS3 obtained is four fold higher than previous reports.CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein.Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. hamzamgondal@gmail.com.

ABSTRACT

Background: Hepatitis C virus (HCV) is a common and leading cause for liver cirrhosis and hepatocellular carcinoma. Current therapies to treat HCV infection are shown to be partially effective and poorly tolerated. Therefore, ample efforts are underway to rationally design therapies targeting the HCV non-structural proteins. Most of the work carried out in this direction has been focusing mainly on HCV genotype 1. Two direct-acting antiviral agents (DAAs) Telaprevir and Boceprevir are being used against genotype 1a infection in combination therapy with interferon and ribavirin. Unfortunately these DAAs are not effective against genotype 3a. Considering the wide spread infection by HCV genotype 3a in developing countries especially South Asia, we have focused on the recombinant production of antiviral drug targets NS3 and NS5A from HCV genotype 3a. These protein targets are to be used for screening of inhibitors.

Results: High-level expression of NS3 and NS5A was achieved at 25°C, using ~1 and 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. Yields of the purified NS3 and NS5A were 4 and 1 mg per liter culture volume, respectively. Although similar amounts of purified NS3 were obtained at 25 and 14°C, specificity constant (Kcat/Km) was somewhat higher at expression temperature of 25°C. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy revealed that both NS3 and NS5A contain a mixture of alpha-helix and beta-sheet secondary structures. For NS3 protein, percentages of secondary structures were similar to the values predicted from homology modeling.

Conclusions: NS3 and NS5A were over-expressed and using Nickel-affinity method both proteins were purified to ~ 95% purity. Yield of the purified NS3 obtained is four fold higher than previous reports. CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein. Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal. The production of NS3 and NS5A in milligram quantities will allow their characterization by biophysical and biochemical means that will help in designing new strategies to fight against HCV infection.

Show MeSH
Related in: MedlinePlus