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High-level expression of a novel liver-targeting fusion interferon with preferred Escherichia coli codon preference and its anti-hepatitis B virus activity in vivo.

Lu X, Wang J, Jin X, Zhu J - BMC Biotechnol. (2015)

Bottom Line: IFN-CSP has significant inhibition effects on HBV-DNA replication in HepG2.2.15 cells.The in vivo tissue distribution were assayed and compared with native IFN α2b.The results showed that the E. coli expression system was very efficient to produce target protein.

View Article: PubMed Central - PubMed

Affiliation: School of Basic Courses, Guangdong Pharmaceutical University, 280 Wai Huan Dong Road, Guangzhou Higher Education Mega Center, Guangzhou, People's Republic of China. luxuemei605@163.com.

ABSTRACT

Background: In our previous study, a novel liver-targeting fusion interferon (IFN-CSP) combining IFN α2b with plasmodium region I peptide was successfully constructed. IFN-CSP has significant inhibition effects on HBV-DNA replication in HepG2.2.15 cells. The aim of the present investigation was focused on how to produce high levels of recombinant IFN-CSP and its in vivo anti-hepatitis B virus (HBV) activity.

Methods: A modified DNA fragment encoding IFN-CSP was synthesized according to Escherichia coli (E. coli) preferred codon usage and transformed into E. coli BL21 (DE3) for protein expression. The induction conditions were systematically examined by combining one-factor experiments with an orthogonal test (L(9)(3)(4)). The antigenicity of the purified protein was characterized by western blot analysis. The in vivo tissue distribution were assayed and compared with native IFN α2b. HBV-transgenic mice were used as in vivo model to evaluate the anti-HBV effect of the recombinant IFN-CSP.

Results: The results showed that the E. coli expression system was very efficient to produce target protein.

Conclusion: Our current research demonstrates for the first time that IFN-CSP gene can be expressed at high levels in E. coli through codon and expression conditions optimization. The purified recombinant IFN-CSP showed liver-targeting potentiality and anti-HBV activity in vivo. The present study further supported the application of IFN-CSP in liver-targeting anti-HBV medicines.

No MeSH data available.


Related in: MedlinePlus

Effect of temperature on the expression level of IFN-CSP in E. coli BL21/pET21b-IFN-CSP. a: SDS-PAGE analyses of IFN-CSP expression under different temperatures. Lane M: Protein molecular weight marker; Lane 1: E. coli BL21/pET21b-IFN-CSP before induction; Lanes 2–7: E. coli BL21/pET21b-IFN-CSP was cultured at 17, 22, 27, 32, 37 and 42 °C, respectively. b: Percentage and concentration of IFN-CSP were calculated by the target bands in SDS-PAGE (A)
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Fig4: Effect of temperature on the expression level of IFN-CSP in E. coli BL21/pET21b-IFN-CSP. a: SDS-PAGE analyses of IFN-CSP expression under different temperatures. Lane M: Protein molecular weight marker; Lane 1: E. coli BL21/pET21b-IFN-CSP before induction; Lanes 2–7: E. coli BL21/pET21b-IFN-CSP was cultured at 17, 22, 27, 32, 37 and 42 °C, respectively. b: Percentage and concentration of IFN-CSP were calculated by the target bands in SDS-PAGE (A)

Mentions: It is well-known that temperature has a great impact on the recombinant protein expression. Effects of temperature on IFN-CSP expression were investigated at 17, 22, 27, 32, 37 and 42 °C, respectively. The results (Fig. 4) revealed that IFN-CSP achieved a high percentage in total proteins and a high concentration in the temperature range of 32–37 °C.Fig. 4


High-level expression of a novel liver-targeting fusion interferon with preferred Escherichia coli codon preference and its anti-hepatitis B virus activity in vivo.

Lu X, Wang J, Jin X, Zhu J - BMC Biotechnol. (2015)

Effect of temperature on the expression level of IFN-CSP in E. coli BL21/pET21b-IFN-CSP. a: SDS-PAGE analyses of IFN-CSP expression under different temperatures. Lane M: Protein molecular weight marker; Lane 1: E. coli BL21/pET21b-IFN-CSP before induction; Lanes 2–7: E. coli BL21/pET21b-IFN-CSP was cultured at 17, 22, 27, 32, 37 and 42 °C, respectively. b: Percentage and concentration of IFN-CSP were calculated by the target bands in SDS-PAGE (A)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4464711&req=5

Fig4: Effect of temperature on the expression level of IFN-CSP in E. coli BL21/pET21b-IFN-CSP. a: SDS-PAGE analyses of IFN-CSP expression under different temperatures. Lane M: Protein molecular weight marker; Lane 1: E. coli BL21/pET21b-IFN-CSP before induction; Lanes 2–7: E. coli BL21/pET21b-IFN-CSP was cultured at 17, 22, 27, 32, 37 and 42 °C, respectively. b: Percentage and concentration of IFN-CSP were calculated by the target bands in SDS-PAGE (A)
Mentions: It is well-known that temperature has a great impact on the recombinant protein expression. Effects of temperature on IFN-CSP expression were investigated at 17, 22, 27, 32, 37 and 42 °C, respectively. The results (Fig. 4) revealed that IFN-CSP achieved a high percentage in total proteins and a high concentration in the temperature range of 32–37 °C.Fig. 4

Bottom Line: IFN-CSP has significant inhibition effects on HBV-DNA replication in HepG2.2.15 cells.The in vivo tissue distribution were assayed and compared with native IFN α2b.The results showed that the E. coli expression system was very efficient to produce target protein.

View Article: PubMed Central - PubMed

Affiliation: School of Basic Courses, Guangdong Pharmaceutical University, 280 Wai Huan Dong Road, Guangzhou Higher Education Mega Center, Guangzhou, People's Republic of China. luxuemei605@163.com.

ABSTRACT

Background: In our previous study, a novel liver-targeting fusion interferon (IFN-CSP) combining IFN α2b with plasmodium region I peptide was successfully constructed. IFN-CSP has significant inhibition effects on HBV-DNA replication in HepG2.2.15 cells. The aim of the present investigation was focused on how to produce high levels of recombinant IFN-CSP and its in vivo anti-hepatitis B virus (HBV) activity.

Methods: A modified DNA fragment encoding IFN-CSP was synthesized according to Escherichia coli (E. coli) preferred codon usage and transformed into E. coli BL21 (DE3) for protein expression. The induction conditions were systematically examined by combining one-factor experiments with an orthogonal test (L(9)(3)(4)). The antigenicity of the purified protein was characterized by western blot analysis. The in vivo tissue distribution were assayed and compared with native IFN α2b. HBV-transgenic mice were used as in vivo model to evaluate the anti-HBV effect of the recombinant IFN-CSP.

Results: The results showed that the E. coli expression system was very efficient to produce target protein.

Conclusion: Our current research demonstrates for the first time that IFN-CSP gene can be expressed at high levels in E. coli through codon and expression conditions optimization. The purified recombinant IFN-CSP showed liver-targeting potentiality and anti-HBV activity in vivo. The present study further supported the application of IFN-CSP in liver-targeting anti-HBV medicines.

No MeSH data available.


Related in: MedlinePlus