Optimized production of a truncated form of the recombinant neuraminidase of influenza virus in Escherichia coli as host with suitable functional activity.

Background: To discover effective drugs for treating Influenza (a disease with high annual mortality), large amounts of recombinant neuraminidase (NA) with suitable catalytic activity are needed. However, the functional activity of the full-length form of this enzyme in the bacterial host (as producing cells with a low cost) in a soluble form is limited. Thus, in the present study, a truncated form of the neuraminidase (derived from California H1N1 influenza strain) was designed, then biosynthesized in Escherichia coli BL21 (DE3), Shuffle T7, and SILEX systems. E. coli BL21 (DE3) was selected as a best host for statistical optimization. Using central composite design methodology, neuraminidase expression level was measured at 20 different runs considering most effective factors including; concentration of isopropyl-β-D-thiogalactopyranoside (IPTG), temperature, and induction time.
Result: The recombinant neuraminidase was purified using Ni-affinity chromatography in soluble form. The neuraminidase expression was confirmed by western blot technique with a molecular mass of 48 kDa. The optimum expression condition was at temperature (30°C), induction time (3 h), and concentration of IPTG (0.6 mM) resulting in maximum neuraminidase expression (7.6 µg/mL) with P < 0.05. The analysis of variance with the significant value of R ² (0.97) indicated that the quadratic model utilized for this prediction was highly significant (p < 0.0001). Applying the optimized condition led to a ~ 2.2-fold increase in NA expression level (from 3.4 to 7.6 µg/ml). The kinetic parameters were also confirmed by fluorescent signals (by 2'-(4-Methylumbelliferyl)-α-D-N acetyl neuraminic acid substrate) with specific activity; ~3.5 IU/mg and Km: 86.49 ± 0.1 µ, close to the Vibrio Cholera neuraminidase with specific activity; 4 IU/mg. The neuraminidase inhibition test confirmed the inhibition of the neuraminidase activity by the drug inhibitor (Oseltamivir) compared to the control sample.
Conclusion: The high quality and proper functional activity of the truncated neuraminidase described in this research show that E. coli can be a suitable host for a wide range of applications with less cost and risk compared to eukaryotic expression systems.
Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.
(© 2024. The Author(s).)