Expression und Reinigung rekombinanter Proteine
Eine Reihe biophysikalischer Methoden erforden große Mengen an rekombinant exprimiertem und gereinigtem Protein. Hinzu kommt, dass zur Lösung der dreidimensionalen Struktur von Proteinen mittels NMR, die Herstellung isotopenmarkierter Proteine nötig ist. Aufgrund dessen, werden von uns effiziente rekombinante Expressionssysteme verwendet, die Zielprotein-abhängig optimiert weren. Zu den Expressionssystemen gehören neben E.coli auch Hefen, humane bzw. tierische Zellinien und Insektenzellen. Die so exprimierten Proteine werden anschließend mit Hilfe biochemischer Methoden wie Affinitäts-, Ionenaustausch-, Größenausschluss- oder Hydrophobe Interaktionschromatographie gereinigt bzw. tag-basiereten Reinigungsverfahren unterzogen.
- Amariei DA, Pozhydaieva N, David B, Schneider P, Classen T, Gohlke H, Weiergräber OH, Pietruszka J
Enzymatic C3-Methylation of Indoles Using Methyltransferase PsmD─Crystal Structure, Catalytic Mechanism, and Preparative Applications
ACS Catal 12, 14130-14139 (2022)
https://doi.org/10.1021/acscatal.2c04240
- Brown H, Chung M, Üffing A, Batistatou N, Tsang T, Doskocil S, Mao W, Willbold D, Bast RC Jr, Lu Z, Weiergräber OH, Kritzer JA
Structure-Based Design of Stapled Peptides That Bind GABARAP and Inhibit Autophagy
J Am Chem Soc 144, 14687-14697 (2022)
https://doi.org/10.1021/jacs.2c04699
- Huwa N, Weiergräber OH, Fejzagić AV, Kirsch C, Schaffrath U, Classen T
The Crystal Structure of the Defense Conferring Rice Protein OsJAC1 Reveals a Carbohydrate Binding Site on the Dirigent-like Domain
Biomolecules 12, 1126 (2022)
https://doi.org/10.3390/biom12081126
- Weiergräber OH, Petrović D, Kislat A, Pattky M, Fabig J, Batra-Safferling R, Schulte am Esch J, Hänel K, Huhn C, Strodel B, Homey B, Willbold D
Structure and Dynamics of Human Chemokine CCL16—Implications for Biological Activity
Biomolecules 12, 1588 (2022)
https://doi.org/10.3390/biom12111588
- Cukkemane A, Becker N, Zielinski M, Frieg B, Lakomek NA, Heise H, SchröderGF, Willbold D, Weiergräber OH
Conformational heterogeneity coupled with β-fibril formation of a scaffold protein involved in chronic mental illnesses
Transl Psychiatry 11, 639 (2021)
https://doi.org/10.1038/s41398-021-01765-1
- Freischem S, Grimm I, López-Pérez A, Willbold D, Klenke B, Vuong C, DingleyAJ, Weiergräber OH
Interaction Mode of the Novel Monobactam AIC499 Targeting Penicillin Binding Protein 3 of Gram-Negative Bacteria
Biomolecules 11, 1057 (2021)
https://doi.org/10.3390/biom11071057
- Huwa N, Weiergräber OH, Kirsch C, Schaffrath U, Classen T
Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1
Int J Mol Sci 22, 5639 (2021)
https://doi.org/10.3390/ijms22115639
- López-Pérez A, Freischem S, Grimm I, Weiergräber O, Dingley AJ, López-AlbercaMP, Waldmann H, Vollmer W, Kumar K, Vuong C
Discovery of Pyrrolidine-2,3-diones as Novel Inhibitors of P. aeruginosa PBP3
Antibiotics 10, 529 (2021)
https://doi.org/10.3390/antibiotics10050529
- Bleffert F, Granzin J, Gohlke H, Batra-Safferling R, Jaeger KE, Kovacic F
Pseudomonas aeruginosa esterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization.
Acta Crystallography F75, 270-277 (2019)
https://doi.org/10.1107/S2053230X19002152
http://scripts.iucr.org/cgi-bin/paper?S2053230X19002152
- Klinker S, Stindt S, Gremer L, Bode JG, Gertzen CGW, Gohlke H, Weiergräber OH, Hoffmann S, Willbold D
Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication
J Biol Chem 294, 7388-7402 (2019)
https://doi.org/10.1074/jbc.RA119.007656
- Kukuk L, Dingley AJ, Granzin J, Nagel-Steger L, Thiagarajan-RosenkranzP, Ciupka D, Hänel K, Batra-Safferling R, Pacheco V, Stoldt M, PfefferK, Beer-Hammer S, Willbold D, Koenig BW
Structure of the SLy1 SAM homodimer reveals a new interface for SAM domain self-association
Scientific Reports 9, 54 (2019)
https://doi.org/10.1038/s41598-018-37185-3
- Viennet T, Bungert-Pümke S, Elter S, Viegas A, Fahlke C, Etzkorn M
Reconstitution and NMR characterization of the ion-channel accessory subunit barttin in detergents and lipid-bilayer nanodiscs
Frontiers in Molecular Biosciences 6, 13 (2019)
https://doi.org/10.3389/fmolb.2019.00013
- Santur K, Sevenich M, Schwarten M, Nischwitz V, Willbold D, Mohrlüder J
In Vitro Reconstitution of the Highly Active and Natively Folded Recombinant Human Superoxide Dismutase 1 Holoenzyme
ChemistrySelect 3, 7627-7632 (2018)
https://doi.org/10.1002/slct.201801319
https://onlinelibrary.wiley.com/doi/abs/10.1002/slct.201801319
- Dammers C, Reiss K, Gremer L, Lecher J, Ziehm T, Stoldt M, Schwarten M, Willbold D.
Pyroglutamate-modified amyloid-β(3-42) shows α-helical intermediates before amyloid formation.
Biophys. J. 112, 1621-1633 (2017)
- Schlesinger R, Cousin A, Granzin J, Batra-Safferling R.
Expression and purification of arrestin in yeast Saccharomyces cerevisiae.
Methods Cell Biol 142, 159-172 (2017)
https://doi.org/10.1016/bs.mcb.2017.07.003
- Bronder AM, Bieker A, Elter S, Etzkorn M, Haeussinger D, Oesterhelt F
Oriented membrane protein reconstitution into tethered lipid membranes for AFM Force Spectroscopy
Biophys. J. 111, 1925-1934 (2016)
http://dx.doi.org/10.1016/j.bpj.2016.08.051
- Hänel K, Möckel L, Brummel M, Peiris K, Hartmann R, Dingley AJ, Willbold D, Loidl-Stahlhofen A
Expression and purification of soluble HIV-2 viral protein R (Vpr) using a sandwich-fusion protein strategy
Prot. Expr. Purific. 95, 156-161 (2014)
http://www.sciencedirect.com/science/article/pii/S1046592813002763
- Mirecka EA, Gremer L, Schiefer S, Oesterhelt F, Stoldt M, Willbold D, Hoyer W
Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide
J. Biotechnol. 191, 221-227 (2014)
http://dx.doi.org/10.1016/j.jbiotec.2014.06.006
- Marbach, J; Zentis, P; Ellinger, P; Müller, H; Birkmann, E
Expression and characterization of fully posttranslational modified cellular prion protein in Pichia pastoris
Biol. Chem. 394, 1475-1483 (2013)
- Glück JM
Ligand interaction analysis of membrane-anchored proteins.
Schriften des Forschungszentrums Jülich Reihe Gesundheit, Band 31, (2011)
http://wwwzb1.fz-juelich.de/contentenrichment/inhaltsverzeichnisse/2010/verlag/gesund31_i.pdf
- Glück JM, Hoffmann S, Koenig BW, Willbold D
Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli
PLoS ONE 5, e10081 (2010)
full text open access
The plasmids in this publication can be received via https://www.addgene.org/Dieter_Willbold/
- Wiesehan K, Funke SA, Fries M, Willbold D
Purification of recombinantly expressed and cytotoxic human amyloid-beta peptide.
J. Chrom. B 856, 229-233 (2007)
- Wittlich M, Wiesehan K, Koenig BW, Willbold D
Expression, purification, and membrane reconstitution of a CD4 fragment comprising the transmembrane and cytoplasmic domains of the receptor.
Protein Expr. Purif. 55, 198-207 (2007)
- Koenig BW, Rogowski M, Louis JM
A rapid method to attain isotope labeled small soluble peptides for NMR studies.
J. Biomol. NMR 26, 193-202 (2003)
- Preusser A, Jonas G and Willbold D
Purification of recombinantly expressed human cluster determinant 4 cytoplasmatic domain.
J. Chromatogr. B 786, 39-44 (2003)