Background:

• Postdoctoral Research Associate, The Scripps Research Institute
• 2000 Presidential Green Chemistry Challenge Award (Co-recipient) honoring C.-H. Wong for "Enzymes in Large-Scale Organic Synthesis", presented by the United States Environmental Protection Agency and the National Partnership for Reinventing Government.

Our research utilizes an interdisciplinary approach applying both chemistry and molecular biology to develop new synthetic techniques and research tools that are then used to study biological systems and to discover and produce treatments for diseases. Two current research projects that illustrate this interdisciplinary approach, a N-linked glycoprotein synthesis project and a protein chemistry/proteomics project are outlined below.

Glycoprotein Synthesis

Glycoproteins play many important roles in biology and medicine, covering the surfaces of cells, mediating cell-cell recognition events, and forming a large fraction of FDA approved protein therapeutics. Unfortunately the study of the structure and function of glycoproteins has lagged behind the study of other biomolecules due to difficulties in working with them. Biological systems produce glycoproteins as heterogeneous mixtures, and this presents practical problems in the purification of glycoproteins and the characterization of their biological effects. To overcome some of the difficulties in studying glycoproteins, we are utilizing genetic engineering of yeast and in vitro enzymatic synthesis to produce homogeneous glycoproteins. Homogeneous glycoproteins produced in genetically engineered yeast strains will be used in biochemical and structural studies of the function of protein glycosylation.

Protein Chemistry/Proteomics

Chemoselective ligations, which target specific chemical moieties on proteins, can be utilized to site-specifically label, immobilize, and modify recombinant proteins. We are developing a group of site-specific protein labeling reagents and immobilization techniques that can be used with a wide variety of proteins during in vitro biochemical, structural, and proteomic studies. In addition, chemical modification of protein and peptide therapeutics can be used to produce imaging agents, drug delivery agents, and to extend in vivo half-life of the protein drugs. We are also trying to improve the beneficial properties of protein therapeutics used to treat cancer and HIV by site-specifically incorporating synthetic modifications into existing therapeutic proteins.

Selected Publications:

Conjugation of glycopeptide thioesters to expressed protein fragments: semisynthesis of glycosylated interleukin-2, T. J. Tolbert and C.-H. Wong, Methods Mol. Biol., 283, 255-266. (2004).

Subtilisin-catalyzed glycopeptide condensation, T. J. Tolbert and C.-H. Wong, Methods Mol. Biol., 283, 267-280. (2004).

New methods for proteomic research: preparation of proteins with N-terminal cysteines for labeling and conjugation, T. J. Tolbert and C.-H. Wong, Angew. Chem., Int. Ed., 41, 2171-2174 (2002).

Intein-Mediated Synthesis of Proteins Containing Carbohydrates and Other Molecular Probes, T. J. Tolbert and C.-H. Wong, J. Am. Chem. Soc., 122, 5421-5428 (2000).

3D C (CC)H TOCSY experiment for assigning protons and carbons in uniformly 13C- and selectively 2H-labeled RNA, K. T. Dayie, T. J. Tolbert and J. R. Williamson, J. Magn. Reson., 130, 97-101 (1998).

Preparation of Specifically Deuterated and 13C-Labeled RNA for NMR Studies Using Enzymic Synthesis, T. J. Tolbert and J. R. Williamson, J. Am. Chem. Soc., 119, 12100-12108 (1997).

Preparation of Specifically Deuterated RNA for NMR Studies Using a Combination of Chemical and Enzymic Synthesis, T. J. Tolbert and J. R. Williamson, J. Am. Chem. Soc., 118, 7929-7940 (1996).