Optimization of an Inclusion Body-Based Production of the Influenza Virus Neuraminidase in Escherichia coli.

Sabina Lipničanová,Miroslav Ondrejovič,Stanislav Miertuš,Barbora Legerská,Daniela Chmelová

doi:10.3390/biom12020331

Abstract

Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the H1N1 influenza virus produced this overexpressed enzyme in the insoluble fraction of cells in the form of inclusion bodies. The aim of this work was to investigate the effect of independent variables (propagation time, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). The maximum yield of NA (112.97 ± 2.82 U/g) was achieved under optimal conditions, namely, a propagation time of 7.72 h, IPTG concentration of 1.82 mM and gene expression time of 7.35 h. This study demonstrated that bacterially expressed NA was enzymatically active.

Highlights

(propagation time, isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM)
The aim of this study was to investigate the influence of factors such as propagation time, isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, expression time and temperature, which affect the NA yield in an insoluble fraction, in the form of inclusion bodies
The optimization of recombinant proteins expressed in this form is relatively rare, but may represent a promising strategy for various insoluble proteins produced by E. coli [35,43–46]

Summary

Introduction

(propagation time, isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). Influenza viruses cause seasonal epidemics that mainly affect the adult population. Vaccination currently remains the most effective tool for influenza infection prevention [10]. It is possible to protect the host and prevent the multiplication of viruses in the body by the inhibition of this enzyme [19]. Some NA inhibitors are already available on the market, such as oseltamivir (Tamiflu), zanamivir (Relenza), peramivir (Rapivab) and laninamivir (Inavir) [20–23]. There is a need to have a broad spectrum of different types of NA to effectively study new compounds with the potential to inhibit viral NA. NAs are commonly obtained from the surface of the virus, this process requires expensive laboratory

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