The Core will express labeled proteins in E. coli to facilitate structural determination by NMR spectroscopy and X-ray crystallography. For X-ray crystallography studies, a methionine auxotrophic E. coli strain will be grown inmedia containing selenomethionine, in place of methionine (for phasing crystal structures). For NMR, 15N-, and/or 13C-labeling of proteins will be carried out by growing bacteria in minimal medium supplemented with 15NH4Cl and/or 13C-glucose as the sole nitrogen and/or carbon source. Note: Isotopes will be supplied by the users. The labeled proteins will be purified and used for subsequent structural determination, as part of the interface between the Core and Structural Biology Center.
The facility also offers to produce homogeneously labeled proteins. Such a protein will be obtained when a synthetic peptide bearing a labeled amino acid in a defined position, is combined with a recombinant N-terminal intein fusion protein. The N-terminal residue of the peptide will be cysteine, which assists in the cleavage of the protein-intein junction while it links itself covalently to the protein. The labeled protein can be purified by an appropriate chromatographic system. This method can be used to introduce biotin, fluorophores, spin labels, photoaffinity labels, amino acid phosphonates or sulfonates, etc. to the protein of interest. Note: Special amino acids will be supplied by the user. Peptides bearing such non-biogenic amino acids will be produced by solid-phase peptide synthesis, preferentially by Fmoc chemistry. If Fmoc chemistry is not suitable we will synthesize the peptide by Boc-chemistry. Both methods are routinely used in our laboratory. The peptide will be deprotected and HPLC purified prior to its combination with recombinant protein.
Scheme to show how a protein can be specifically labeled within the C-terminal region. The length of the peptide can vary between 1 to 30 amino acids and can bear any labeled amino acid.
Specialty Protein Services Provided