Anaerobic E. coli fermentations as a means to better-folded recombinant proteins

Document Type


Publication Date



Inclusion bodies are frequently formed inside the cytoplasm of Escherichia coli as a result of high-level expression of heterologous proteins. One of the most promising approaches to this problem is exploitation of folding modulators including molecular chaperones. Molecular chaperones not only prevent the formation of aggregates by facilitating the folding process, but also are capable of disassociation and refolding of inclusion bodies. The present study aimed to investigate the effect of anaerobic conditions on the expression of chaperones and subsequently on the quality of recombinant proteins expressed in E. coli. For this reason E. coli strain BL21*(DE3) was employed as the host cell for expression of a Chemotaxis protein tagged with a green fluorescent protein (CheY-GFP). Among various terminal electron acceptors used for the activation of anaerobic respiration of E. coli BL21*(DE3) fumarate and Trimethylamine N-oxide slightly improved the growth rate and biomass yield, Nitrite showed an adverse effect and nitrate and DMSO had no effect on the growth. It was concluded that the lack of a global anaerobic transcriptional regulator gene, fnr, in E. coli BL21*(DE3) was the reason for the inability of bacteria to metabolise anaerobically. To investigate the effect of oxygen limitation on protein folding, a shift from aerobic to anaerobic conditions was applied on cultures of E. coli BL21*(DE3)-pET20-CheY-GFP and the solubility of recombinant proteins was measured before and after the shift. There was no significant change in the solubility of proteins produced during aerobic conditions. In contrast, although growth was ceased upon exposure to anaerobic conditions, both fluorescence and soluble fraction of recombinant proteins were increased indicating that oxygen limitation contributes to the solubility and the refolding of insoluble recombinant proteins.

This document is currently not available here.