The study shows how expression processes can be further simplified and improved, with significantly increased protein solubility and Inducer concentration perfectly matched to the protein of interest simply and quickly to maximize yields.
More precise regulation of gene expression
The peer-reviewed study Tunable expression rate control of a growth-decoupled T7 expression system by L-arabinose only was lead authored by enGenes scientist and PhD candidate Patrick Stargardt, together with enGenes CEO Dr. Juergen Mairhofer and Prof. Gerald Striedner from the Department of Biotechnology at Vienna’s University of Natural Resources and Life Sciences (BOKU), who is also a co-founder of enGenes.
The study was motivated to achieve more precise regulation of gene expression as a key factor in the successful production of complex membrane proteins (MPs), enzymes or other proteins toxic to the host cell.
Bacteriophage-inspired production concepts
The team set out to investigate whether the enGenes X-Press production platform based on Escherichia coli expression could be adapted to emerging bacteriophage-inspired growth-decoupled recombinant protein production concepts.
This would involve allowing genes under control of a normally Isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter to be induced solely with L-arabinose in a newly constructed E.coli expression host strain BL21-AI<gp2>.
Cellular level regulation
The team succeeded in showing that the new host strain was able to regulate protein production rates very precisely at cellular level in an L-arabinose concentration-dependent manner, while simultaneously allowing for reallocation of metabolic resources due to L-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2.
“We were able to successfully characterize the system under relevant fed-batch like conditions in microscale cultivation at up to 800 µL and generate data proofing on relevant increase in specific yields for six different E. coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting,” explained Patrick Stargardt.
“We were very pleased to observe that in all cases tested, BL21-AI<gp2> outperformed the parental strain BL21-AI, operated in growth-associated production mode. We were able to achieve up to improve to 2.7-fold increases in yields of specific MP-GFP fusion proteins,” he observed.
Horizon 2020 and R4B collaboration
The study opens the way for new X-Press based processes that allow fine tuning of MP production or expression of multi-enzyme pathways in cases, for example, where a particular stoichiometry has to be met to optimize product flux.
The project was part-funded from the EU’s Horizon 2020 research and innovation programme.
It also aligns with the objectives of the Rafts4Biotech (R4B) European project seeking to build upon the discovery of bacterial lipid rafts to exploit them as a biotechnological tool to foster innovative industrial applications and allow the production of a broad range of novel compounds.
Dynamic host cell engineering
“This research exercise demonstrates enGenes Biotech’s highly dynamic capabilities in host cell engineering and problem solving, with real benefits for cost-effective production,” commented Dr. Mairhofer.
The benefit for our existing and potential customers, partners, and collaborators is to widen the range of options for improving protein yields as we progressively improve our production platforms to the latest concepts,” he concluded.
About enGenes Biotech
enGenes Biotech GmbH (enGenes) is a contract research, development and manufacturing company that provides leading edge technologies and production services focused on recombinant proteins in bacteria. The company’s mission is to provide cost-effective and scalable production of recombinant proteins at a fraction of current cost, allied to a vision of developing a world-class portfolio of cutting-edge protein production technologies, relevant to a broad spectrum of application fields.
enGenes has developed advanced technologies to drive more cost-effective recombinant protein production processes, including its proprietary enGenes-X-press™ E. coli platform that achieves outstanding yields of soluble and active recombinant protein. enGenes-X-press™ has been successfully applied for the manufacturing of enzymes and biopharmaceutical products that failed to give economically feasible yields in a conventional expression host.
enGenes Biotech offers development and manufacturing services tailored around the needs of pharmaceutical and industrial biotech companies. The services include expression strain and vector development, fermentation process development and optimization, downstream process development, production of purified protein, technology transfer and scale-up support with technology out-licensing and co-development opportunities.
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