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Chemistry A563

Thomas Tolbert received his B.S. degree in chemistry in 1991 from Purdue University. Subsequently he began graduate studies at Massachusetts Institute of Technology in the laboratory of James R. Williamson, and earned a Ph.D. in 1998 after developing methods for selective isotopic labeling of RNA and NMR studies of the HIV TAR RNA. Prior to beginning his independent career at Indiana University Dr. Tolbert did postdoctoral studies in the laboratory of Chi-Huey Wong at the Scripps Research Institute where he worked on site-specific protein modification and glycoprotein synthesis.

Research in the Tolbert lab focuses on two main areas the biochemistry of N-linked glycoprotein and oligosaccharide interactions and chemoselective modification of proteins. In our glycobiology studies, we are interested in how N-linked glycosylation can alter protein stability and activity. Because N-linked glycoproteins are naturally produced as heterogeneous mixtures, this research involves a combination of genetic engineering and in vitro enzymatic synthesis to produce homogeneous glycoconjugates. We are currently applying these approaches to glycoproteins of the immune system such as interleukin-1 receptor antagonist and antibody fragments to better understand the role of glycosylation in immune responses. In our protein modification studies, we are developing and applying chemoselective ligation techniques to site-selective modification of expressed proteins and glycoproteins. By incorporating synthetic molecules onto proteins we can produce chimeric molecules with unique properties and use them to facilitate biochemical and structural studies.

Research

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.

Publications

Enzyme-Catalyzed Synthesis of a Hybrid N-Linked Oligosaccharide using N-Acetylglucosaminyltransferase I, R. Chen, M. A. Pawlicki, B. S. Hamilton, and T. J. Tolbert, Adv. Synth. Catal., 350, 1689-1695 (2008).

Increasing Solubility of Proteins and Peptides by Site-Specific Modification with Betaine, J. Xiao, A. Burn, and T. J. Tolbert, Bioconjugate Chemistry, 19, 1113-1118 (2008).

Expression and characterization of human glycosylated interleukin-1 receptor antagonist in Pichia pastoris, B. S. Hamilton, Y. Brede, and T. J. Tolbert, Protein Expression and Purification, 59, 64-68 (2008).

A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as TEV protease cleavable fusion protein, T. J. Tolbert, D. Franke, and C.-H. Wong, Bioorganic & Medicinal Chemistry, 13, 909-915 (2005).

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).

Awards

• 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.

Highlights