Bacterial production of site specific 13C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins.

Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.