E. coli HMS174(DE3) is a sustainable alternative to BL21(DE3)

Abstract

BackgroundEscherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG. We recently published a few studies regarding lactose induction in BL21(DE3) strains. BL21(DE3) can only take up the glucose-part of the disaccharide when fed with lactose. However, initially additional glucose has to be supplied as otherwise the ATP-related lactose uptake barely happens. Yet, as lactose is an inexpensive compound compared to glucose and IPTG, a new induction strategy by a lactose-only feed during induction seems attractive. Thus, we investigated this idea in the galactose metabolizing strain HMS174(DE3).ResultsWe show that strain HMS174(DE3) can be cultivated on lactose as sole carbon source during induction. We demonstrate that strain HMS174(DE3) exhibits higher product and biomass yields compared to BL21(DE3) when cultivated in a lactose fed-batch. More importantly, HMS174(DE3) cultivated on lactose even expresses more product than BL21(DE3) in a standard IPTG induced glucose fed-batch at the same growth rate. Finally, we demonstrate that productivity in HMS174(DE3) lactose-fed batch cultivations can easily be influenced by the specific lactose uptake rate (qs,lac). This is shown for two model proteins, one expressed in soluble form and one as inclusion body.ConclusionsAs strain HMS174(DE3) expresses even slightly higher amounts of target protein in a lactose fed-batch than BL21(DE3) in a standard cultivation, it seems a striking alternative for recombinant protein production. Especially for large scale production of industrial enzymes cheap substrates are essential. Besides cost factors, the strategy allows straight forward adjustment of specific product titers by variation of the lactose feed rate.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1016-6) contains supplementary material, which is available to authorized users.

Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance

“…In conclusion, all biological experiments ultilising the pET system, will need to consider the presence of genomic mutations driven by the protein over-expression process. We suggest that this can be overcome by genome sequencing veri cation of the production strain, using alternative expression strains such as HMS174(DE3) [45], using the weaker inducer lactose [46,47], controlled-down regulation of T7 RNAP production [19,20], making use of alternative mutant expression strains, using a cell-free system [48] or a combination of new strains and weaker induction.…”

Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance

“…In conclusion, all biological experiments ultilising the pET system, will need to consider the presence of genomic mutations driven by the protein over-expression process. We suggest that this can be overcome by genome sequencing veri cation of the production strain, using alternative expression strains such as HMS174(DE3) [45], using the weaker inducer lactose [46,47], controlled-down regulation of T7 RNAP production [19,20], making use of alternative mutant expression strains, using a cell-free system [48] or a combination of new strains and weaker induction. In light of our new results, we suggest two possible adaptations to the existing protein over-expression protocols with the aim of reducing the total number of unproductive LW phenotypes.…”

Recombinant production of a hard‐to‐express membrane‐bound cytochrome P450 in different yeasts—Comparison of physiology and productivity

“…The homogenized samples as well as a His‐6‐ tagged protein (flavanone 3‐hydroxylase FHT (Hausjell, Weissensteiner, Molitor, Halbwirth, & Spadiut, ) with known concentration, used as standard, were incubated 1:1 with a 2x concentrated Lämmli buffer (Laemmli, ) at 100 °C for 15 minutes. In case of the strep‐tagged CH3H expressed by S. cerevisiae a commercial standard (Precision Plus Protein™ Unstained Protein Standards, Strep‐tagged, Biorad, Vienna, Austria) was used.…”