Abstract
This work assesses the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of enhanced green fluorescent protein (eGFP) by planktonic and biofilm cells of Escherichia coli JM109(DE3) transformed with a plasmid containing a T7 promoter. It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Heterologous protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the heterologous protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of heterologous protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth.
Highlights
Escherichia coli remains at the forefront of the expression systems used for the production of many recombinant proteins [1], despite the fact that it is unable to perform some post-translational modifications like glycosylation [1] and shows limited secretion capacity [2,3]
The origin of replication controls the plasmid copy number (PCN) [1] and it is often assumed that a high gene dosage is favorable for high level heterologous protein production, this is not always the case [5]
It has been shown that a high PCN may impose a metabolic burden that decreases the bacterial growth rate and originates plasmid instability, reducing the number of bacteria that are capable of high level protein synthesis [6,7,8]
Summary
Escherichia coli remains at the forefront of the expression systems used for the production of many recombinant proteins [1], despite the fact that it is unable to perform some post-translational modifications like glycosylation [1] and shows limited secretion capacity [2,3]. Plasmids are the most commonly used vectors for the expression of recombinant proteins in E. coli. Their design is crucial in order to maintain an equilibrium between the transcriptional and translational machinery of the host cell so that the deleterious effects of heterologous protein production can be managed [2,5]. The origin of replication controls the plasmid copy number (PCN) [1] and it is often assumed that a high gene dosage is favorable for high level heterologous protein production, this is not always the case [5]. It has been shown that a high PCN may impose a metabolic burden that decreases the bacterial growth rate and originates plasmid instability, reducing the number of bacteria that are capable of high level protein synthesis [6,7,8]
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