Abstract
BackgroundPrecise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with l-arabinose in a newly constructed Escherichia coli expression host BL21-AI<gp2>, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production.ResultsHere, we show that BL21-AI<gp2> is able to precisely regulate protein production rates on a cellular level in an l-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to l-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting.ConclusionsIn all cases tested, BL21-AI<gp2> outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.
Highlights
Precise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell
We focus on the characterization of a novel host strain which is capable of reallocation of metabolic resources by decoupling growth from recombinant protein production (RPP), as was shown recently with the enGenes-X-press technology [18], and allow expression rate control in plasmid-based systems on the cellular level by titration of only one inducer (l-arabinose or Isopropyl β-d-1-thiogalactopyranoside (IPTG))
As recently shown by our group in a growth decoupled expression system, induction of recombinant Green fluorescent protein (GFP) expression from a pET-derived vector by the sole addition of l-arabinose, resulted in a comparable expression rate of soluble GFP but at the same time drastically reduced inclusion body formation, compared to induction with l-arabinose and IPTG or IPTG only
Summary
Precise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with l-arabinose in a newly constructed Escherichia coli expression host BL21-AI, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production. Tunable control of gene expression for the overproduction of recombinant proteins in Escherichia coli (E. coli) is a promising strategy to further optimize soluble recombinant protein production (RPP) levels. This approach is of particular interest for membrane proteins (MP) or other difficult-to-express proteins (e.g. proteins rich in disulfide bonds) that tend to overwhelm host cell capacities [1]. Published research by Kim et al found further mutations responsible for a reduction in cellular toxicity caused by MP overexpression in C41/C43 strains, one occurring in lacI in the λDE3 chromosomal, which resulted in an even more reduced level of expressed T7 RNAP [9, 10]
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