Recombinant production of bioactive human TNF-α by SUMO-fusion system – High yields from shake-flask culture

Hoffmann, A.; Müller, Mathias Q.; Gloser, Manja; Sinz, Andrea; Rudolph, Rainer; Pfeifer, Sven

doi:10.1016/j.pep.2010.03.022

Cited by 21 publications

(13 citation statements)

References 27 publications

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“…The target protein TNF-alpha was produced by SUMO-fusion strategy as published previously [21]. The product identity was confirmed by mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

“…Following incubation for 20 h at 37°C and 220 rpm, cells were harvested, resuspended in NPI-20 (20 mM Na 2 HPO 4 , 150 mM NaCl, 20 mM imidazole pH 8.0) and disrupted as described previously [21]. The lysate was cleared by centrifugation and the supernatant was applied on an ÄktaXpress system to a 5-ml HisTrap HP column equilibrated with buffer NPI-20.…”

Section: Methodsmentioning

confidence: 99%

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New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

et al. 2012

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A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.

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“…The target protein TNF-alpha was produced by SUMO-fusion strategy as published previously [21]. The product identity was confirmed by mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

et al. 2012

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“…TCP-1 gene was first introduced into our preserved pET-14b/EGFP plasmid by PCR-mediated site-directed mutagenesis with the primer pair 5′-ttttcgcattgcggaggtaccgtgagcaagggcgaggag-3′ and 5′-aggactaggcgtacaagcgggccccatatggctgccgcgcgg-3′. The cDNA of mature human TNFα was kindly provided by Prof. Sven Pfeifer [18]. The TNFα fragment was amplified by PCR with primer pair 5′-catggtaccgtgcgtagcagcagccgtaccc-3′and 5′-catggatcccagcgcaataatgcc aaaatacacc-3′ flanked by KpnI and BamHI (underlined) restriction enzyme sites.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…The activities of TNFα and TCP-1/TNFα were determined in L929 and Colon 26 cells as previously described [18]. Briefly, 2 × 10 4 cells/well were seeded in a 96-well plate with 100 μL growth medium.…”

Section: Tnfα Activity Assaymentioning

confidence: 99%

Vascular-targeted TNFα improves tumor blood vessel function and enhances antitumor immunity and chemotherapy in colorectal cancer

Lü

et al. 2015

Journal of Controlled Release

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“…Recently, IFN‐γ was generated in a functionally active form following SUMO expression and purification, although it was expressed as an inclusion body previously . TNF‐α was also generated as a mature and active product for use in drug development assays . Finally, the activity of chemokines can be drastically altered by truncation or extension at the N‐terminus (see for instance).…”

Section: Tags For Functional Activitymentioning

confidence: 99%

To fuse or not to fuse: What is your purpose?

et al. 2013

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Since the dawn of time, or at least the dawn of recombinant DNA technology (which for many of today's scientists is the same thing), investigators have been cloning and expressing heterologous proteins in a variety of different cells for a variety of different reasons. These range from cell biological studies looking at protein-protein interactions, post-translational modifications, and regulation, to laboratory-scale production in support of biochemical, biophysical, and structural studies, to large scale production of potential biotherapeutics. In parallel, fusion-tag technology has grown-up to facilitate microscale purification (pull-downs), protein visualization (epitope tags), enhanced expression and solubility (protein partners, e.g., GST, MBP, TRX, and SUMO), and generic purification (e.g., His-tags, streptag, and FLAG TM -tag). Frequently, these latter two goals are combined in a single fusion partner. In this review, we examine the most commonly used fusion methodologies from the perspective of the ultimate use of the tagged protein.That is, what are the most commonly used fusion partners for pull-downs, for structural studies, for production of active proteins, or for large-scale purification? What are the advantages and limitations of each? This review is not meant to be exhaustive and the approach undoubtedly reflects the experiences and interests of the authors. For the sake of brevity, we have largely ignored epitope tags although they receive wide use in cell biology for immunopreciptation.

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Recombinant production of bioactive human TNF-α by SUMO-fusion system – High yields from shake-flask culture

Cited by 21 publications

References 27 publications

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

Vascular-targeted TNFα improves tumor blood vessel function and enhances antitumor immunity and chemotherapy in colorectal cancer

To fuse or not to fuse: What is your purpose?

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