DESIGN OF AN EXPRESSION SYSTEM FOR DETECTING FOLDED PROTEIN DOMAINS AND MAPPING MACROMOLECULAR INTERACTIONS BY NMR

Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain o f streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small s ize, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis of the fusion protei n by H-1-N-15 correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, N-15-labeling of peptides and their testing for DNA bindi ng are facilitated. Several examples are presented that demonstrate th e usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using N-15-labeled GB1-peptide fusions and unlabeled target.