The study, Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness, is published in Biotechnology Journal (see Resources). The paper demonstrates that protein production is now not only possible by intracellular means but that the X-press strain can also be used to allow improved secretion to the extracellular space, thus constituting a valuable host for extracellular production of recombinant protein with Escherichia coli.
Simpler purification, superior purity
“Secretion of proteins to the cell-free supernatant has major implications for manufacturing as it allows simple purification and superior purity with fewer purification steps compared to classical approaches, where cells are disrupted and the recombinant protein is purified from a complex lysate,” commented study co-author Prof. Oliver Spadiut, Head of Integrated Bioprocess Development at TU Wien.
The original collaboration between enGenes and TU Wien, dating back to 2018, was established to undertake a project funded by the Vienna Business Agency to apply enGenes-X-press™ technology to integrative bioprocess development for biosimilar production (see Resources). The team was also able to establish a follow-up funding in form of a “FFG BRIDGE Young Scientist” project (Leaky E. coli) funded by the Austrian Research Promotion Agency FFG.
Versatile production platform
“This study is a further demonstration of the broad versatility of the X-press platform technology,” commented enGenes CEO Dr. Juergen Mairhofer.
“Extracellular production of proteins allows very cost-effective production, combined with the power to achieve very good yields, empowering our clients to get the best biomanufacturing process available, reduce their COGS (cost of goods sold) and improve quality, all the while simplifying upstream and downstream processes,” Dr. Mairhofer explained.
The study was inspired by growing interest in continuous cultivation of Escherichia coli, in which secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing.
It demonstrates that induced decoupling of growth and heterologous gene expression in the E. coli X‐press strain (derived from BL21 (DE3)) facilitates extracellular recombinant protein production.
The joint enGenes/TU Wien research team investigated effects of the process parameters temperature and specific glucose consumption rate (qS) on growth, productivity, lysis and leakiness, to identify the parameter space allowing extracellular protein production.
The team used two model proteins, Protein A from Staphylococcus aureus (SpA), a protein well-known for the purification of monoclonal antibodies, and a camelid single domain antibody (VHH) comparing performance to the industrial standard strain BL21 (DE3).
They found that inducible growth repression in the X‐press strain greatly mitigated the effect of metabolic burden under different process conditions and that temperature and qS could be used to control productivity and leakiness. In the X‐press strain, extracellular SpA and VHH titers reached up to 349 and 19.6 mg g−1, respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum.
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.
Click on Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness to read original Biotechnology Journal article.
Click on enGenes deploys X-press technology to modular prokaryotic protein expression services to learn more about original collaborative project.