Patrick Stargardt1, Gerald Striedner2, Juergen Mairhofer3. 1. enGenes Biotech GmbH, Mooslackengasse 17, 1190, Vienna, Austria. 2. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria. 3. enGenes Biotech GmbH, Mooslackengasse 17, 1190, Vienna, Austria. juergen.mairhofer@engenes.cc.
Abstract
BACKGROUND: 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<gp2>, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production. RESULTS: Here, 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. CONCLUSIONS: In 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.
BACKGROUND: 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<gp2>, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production. RESULTS: Here, 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. CONCLUSIONS: In 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.
Authors: Samuel Wagner; Mirjam M Klepsch; Susan Schlegel; Ansgar Appel; Roger Draheim; Michael Tarry; Martin Högbom; Klaas J van Wijk; Dirk J Slotboom; Jan O Persson; Jan-Willem de Gier Journal: Proc Natl Acad Sci U S A Date: 2008-09-16 Impact factor: 11.205
Authors: Samuel Wagner; Louise Baars; A Jimmy Ytterberg; Anja Klussmeier; Claudia S Wagner; Olof Nord; Per-Ake Nygren; Klaas J van Wijk; Jan-Willem de Gier Journal: Mol Cell Proteomics Date: 2007-04-19 Impact factor: 5.911