publications of the research group recombinant protein expression
(RPEX)http://hdl.handle.net/10033/6207772024-03-28T13:36:34Z2024-03-28T13:36:34ZRecombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1.Weber, JanLi, ZhaopengRinas, Ursulahttp://hdl.handle.net/10033/6230632021-10-07T01:51:22Z2021-08-26T00:00:00ZRecombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1.
Weber, Jan; Li, Zhaopeng; Rinas, Ursula
Background: Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden.
Results: Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate.
Conclusions: Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated "metabolic burden". Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.
2021-08-26T00:00:00ZA SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.Bertoglio, FedericoFühner, ViolaRuschig, MaximilianHeine, Philip AlexanderAbassi, LeilaKlünemann, ThomasRand, UlfertMeier, DorisLangreder, NoraSteinke, StephanBallmann, RicoSchneider, Kai-ThomasRoth, Kristian Daniel RalphKuhn, PhilippRiese, PeggySchäckermann, DorinaKorn, JaninKoch, AllanChaudhry, M ZeeshanEschke, KathrinKim, YeonsuZock-Emmenthal, SusanneBecker, MarliesScholz, MargittaMoreira, Gustavo Marçal Schmidt GarciaWenzel, Esther VeronikaRusso, GiulioGarritsen, Hendrikus S PCasu, SebastianGerstner, AndreasRoth, GünterAdler, JuliaTrimpert, JakobHermann, AndreasSchirrmann, ThomasDübel, StefanFrenzel, Andrévan den Heuvel, JoopČičin-Šain, LukaSchubert, MarenHust, Michaelhttp://hdl.handle.net/10033/6230302021-09-15T01:55:10Z2021-07-07T00:00:00ZA SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.
Bertoglio, Federico; Fühner, Viola; Ruschig, Maximilian; Heine, Philip Alexander; Abassi, Leila; Klünemann, Thomas; Rand, Ulfert; Meier, Doris; Langreder, Nora; Steinke, Stephan; Ballmann, Rico; Schneider, Kai-Thomas; Roth, Kristian Daniel Ralph; Kuhn, Philipp; Riese, Peggy; Schäckermann, Dorina; Korn, Janin; Koch, Allan; Chaudhry, M Zeeshan; Eschke, Kathrin; Kim, Yeonsu; Zock-Emmenthal, Susanne; Becker, Marlies; Scholz, Margitta; Moreira, Gustavo Marçal Schmidt Garcia; Wenzel, Esther Veronika; Russo, Giulio; Garritsen, Hendrikus S P; Casu, Sebastian; Gerstner, Andreas; Roth, Günter; Adler, Julia; Trimpert, Jakob; Hermann, Andreas; Schirrmann, Thomas; Dübel, Stefan; Frenzel, André; van den Heuvel, Joop; Čičin-Šain, Luka; Schubert, Maren; Hust, Michael
The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.
2021-07-07T00:00:00ZDiscovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor of Soluble Adenylyl Cyclase (sAC, ADCY10).Fushimi, MakotoBuck, HannesBalbach, MelanieGorovyy, AnnaFerreira, JacobRossetti, ThomasKaur, NavpreetLevin, Lonny RBuck, JochenQuast, Jonathanvan den Heuvel, JoopSteegborn, ClemensFinkin-Groner, EfratKargman, StaciaMichino, MayakoFoley, Michael AMiller, MichaelLiverton, Nigel JHuggins, David JMeinke, Peter Thttp://hdl.handle.net/10033/6230102021-09-02T02:41:32Z2021-07-14T00:00:00ZDiscovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor of Soluble Adenylyl Cyclase (sAC, ADCY10).
Fushimi, Makoto; Buck, Hannes; Balbach, Melanie; Gorovyy, Anna; Ferreira, Jacob; Rossetti, Thomas; Kaur, Navpreet; Levin, Lonny R; Buck, Jochen; Quast, Jonathan; van den Heuvel, Joop; Steegborn, Clemens; Finkin-Groner, Efrat; Kargman, Stacia; Michino, Mayako; Foley, Michael A; Miller, Michael; Liverton, Nigel J; Huggins, David J; Meinke, Peter T
Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.
2021-07-14T00:00:00ZReproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.Schubert, MarenNimtz, ManfredBertoglio, FedericoSchmelz, StefanLukat, Peervan den Heuvel, Joophttp://hdl.handle.net/10033/6229142021-07-01T01:43:54Z2021-05-21T00:00:00ZReproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.
Schubert, Maren; Nimtz, Manfred; Bertoglio, Federico; Schmelz, Stefan; Lukat, Peer; van den Heuvel, Joop
he expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.
2021-05-21T00:00:00Z