Our laboratory is well-equipped for the overexpression of recombinant proteins using bacterial expression systems, including E. coli and V. natriegens. We focus on producing a wide range of protein samples for NMR spectroscopy, including human, bacterial, and intrinsically disordered proteins.

Protein production is initiated typically by transforming plasmids containing the desired gene construct into various bacterial strains, followed by optimizing expression conditions in a rich medium. Subsequently, the cultures are transferred to a minimal medium to facilitate uniform or selective isotopic labeling which is essential for biomolecular NMR experiments.

Purification of protein samples involves the use of affinity, ion-exchange and size-exclusion chromatography on an FPLC system, aided by purification and solubilization tags incorporated in the target protein sequence prior expression. Throughout the process, protein purity is monitored using SDS-PAGE.

Additionally, our laboratory specializes in peptide synthesis using a solid-phase peptide synthesizer equipped with a microwave chamber. Using Fmoc-protected chemistry, we synthesize peptides up to 35 amino acids in length, producing yields in the hundreds of milligrams.

­NMR spectroscopy spans a broad field, and in our group, we put heavy emphasis on the development of new NMR methodology as well as its application to study structural properties and function of biomolecules.

Standard biomolecular NMR methods include tools for obtaining resonance assignments, deriving three-dimensional molecular structures, assessing protein dynamics and probing interactions between target biomolecules and their ligands. All of these applications typically demand high amounts of isotopically labeled samples which we produce in house. For example, chemical shift mapping can be used to study molecular interactions at an atomic resolution and therefore help to decipher binding sites of drugs on a protein target, estimate dissociation constants of a complex or define protein-protein interfaces. In addition, protein dynamics is often neglected when investigating ligand binding, where in reality it can have significant effect on the entropy of binding, offering more informative approach towards understanding protein functions.

Analogously, protein electrostatics can be used to provide insights into protein-protein interactions, specificity of protein ligands, conformation changes and more. We study how the electrostatic potential of a disordered protein changes under the influence of increasing salt using NMR.