Optimization of medium composition for propagation of recombinant Escherichia coli

Sabina Lipničanová,Miroslav Ondrejovič,Andrej Godány,Daniela Chmelová

doi:10.2478/nbec-2019-0011

Sabina Lipničanová, Miroslav Ondrejovič + Show 2 more

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https://doi.org/10.2478/nbec-2019-0011

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Abstract

AbstractRecombinant protein production in heterologous hosts often seems a simpler and more effective way than its production by natural producer. The secretion of recombinant protein inEscherichia colihas many advantages comparing to than in insect or mammalian cells. The important factor for high-level recombinant protein production is the sufficient amount ofE. colibiomass. Therefore, the aim of this study was to optimize the composition of propagation medium resulting in the maximum biomass yield of recombinantE. colias the part of fermentation strategy for neuraminidase (NA) production. Three independent variables including glucose, asparagine and phosphate concentrations, and four dependent variables, such as biomass yield, residual concentrations of glucose or asparagine and pH of the propagation medium after fermentation, were chosen to the optimization by Response Surface Methodology (RSM). The optimal conditions for the maximum biomass yield expressed as dry cell weight (DCW) (16.57±0.55 g DCW.L−1) were as follows: glucose concentration of 39.37 mM, asparagine concentration of 62.68 mM and phosphate concentration of 14.80 mM. For this model, the predicted values for the responses are close to the experimental values. The yield of desired pET15b-neu plasmid fromE. colicells cultivated in optimized propagation medium was almost 23 % higher than in commonly used Luria-Bertani (LB) medium suggesting that asparagine may be involved in the induction of plasmid amplification.

Highlights

The recombinant production of desired protein can facilitate product isolation and impact its quality and activity (Horga et al 2018)
The aim of this study was to optimize the composition of propagation medium by Response Surface Methodology (RSM) to improve E. coli biomass yield leading to highlevel production of recombinant NA as the perspective tool for design of novel antiviral compounds
The results of the present study confirm that the key factors for the propagation of Escherichia coli BL21 (DE3)pLys are the concentration of glucose as carbon source, concentration of asparagine as nitrogen source and concentration of phosphates

Summary

Introduction

The recombinant production of desired protein can facilitate product isolation and impact its quality and activity (Horga et al 2018). Bacteria have been widely used as the hosts for high-level expression of protein (Nikerel et al 2006). The use of gram-negative bacterial host Escherichia coli has several advantages, namely the simplicity of system, fast growth linked to high cell density and cost-effective cultivation to produce recombinant protein at a relative high level (Fakruddin et al 2012; Rosano and Ceccarelli 2014). The production of recombinant protein by E. coli host cells is two-step process. The cells are cultivated in a simple, defined medium with a carbon source that suppresses gene expression and results in the accumulation of biomass (Potvin et al 2012; Celik et al 2012). The induction of recombinant protein synthesis is Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 12:20 UTC

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