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Contact details

Georgios A. Spyroulias, PhD.
Department of Pharmacy
University of Patras
Panepistimioupoli-Rion,
GR-26504 Patras, GREECE
Tel:    +30.2610.969950 (office)
+30.2610.969951 (terra silico)
+30.2610.969952 (terra vitro)
Fax:    +30.2610.969950
Email:  G.A.Spyroulias@upatras.gr

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Now is: 2017-09-20 07:35
protein_expressionThe efficient production of highly pure protein samples that are soluble, folded, active and monodisperse for NMR or X-ray studies is the bottleneck of structural biology field. Until now, in any structural proteomics project, realistic protein size for NMR investigations is among 15-20 kDa. Additionally, the purification protocols need generally the co-expression of at least one protein or peptide tag and the screening of experimental conditions for the production of enriched proteins in stable NMR-active (13C, 15N), and/or NMR silent (2H) nuclei. Introduction of stable isotopes into proteins is a prerequisite for: (a) significantly increasing of sensitivity and resolution and reducing the complexity of NMR spectra and the time requirements for NMR-based structure elucidation of proteins/polypeptides, (b) allowing the efficient use of heteronuclear multidimensional NMR experiments, and (c) providing alternative approaches to the spectral assignment process enabling the collection of conformational constraints in de novo determination of protein structure. Samples for NMR studies are most often expressed in Escherichia coli (E.coli) grown in isotopically labeled media or cell-free expression systems.

Overexpression in E. coli , labeling (2H, 13C, 15N), purification and physicochemical characterization of recombinant metal-free and/or metal-loaded proteins, is now performed in our lab, using a variety of molecular biology protocols and physicochemical techniques. A large number of diverse expression vectors are currently used for protein production yielding proteins with a number of different tags that facilitates protein isolation and purification. Up to now, many polypeptides and metal-binding proteins with the number of the amino acids ranging from 70 to up to 200, have been successfully expressed and characterized and found suitable for NMR studies.