TRANSFORMING GROWTH FACTOR-BETA-1 - NMR SIGNAL ASSIGNMENTS OF THE RECOMBINANT PROTEIN EXPRESSED AND ISOTOPICALLY ENRICHED USING CHINESE-HAMSTER OVARY CELLS

The transforming growth factor betas are a homologous family of multifunctional cytokines that regulate cell growth and differentiation. As a prelude to studies of the solution structure and dynamics of TGF-beta1, we report virtually complete assignment of H-1 and N-15 resonances for this 25-kDa homodimeric protein. Recombinant TGF-beta1 was expressed in Chinese hamster ovary cells. The cells were grown either in a completely N-15-enriched medium or in a medium containing selectively C-13,N-15-labeled amino acids to obtain either uniformly or specifically labeled protein, respectively. Two- and three-dimensional heteronuclear edited magnetic resonance spectra of the uniformly N-15-labeled protein and three samples selectively labeled with C-13 and N-15 yielded assignments for 96% of the backbone amide and Calpha protons and 87% of the side chain protons. To our knowledge, this is the first report of the use of an animal cell expression system to obtain extensive isotopic enrichment in order to sequentially assign a protein. The methodology described herein for the isotopic enrichment and resonance assignments of TGF-beta1 should be generally applicable to other eukaryotic proteins expressed by animal cells.