Our Science – Waugh Website

David S. Waugh, Ph.D.

Macromolecular Crystallography Laboratory Head, Protein Engineering Section Senior Investigator Building 538 Room 209A Frederick, MD 21702-1201 Phone:   301-846-1842 Fax:   301-846-7148 E-Mail:   waughd@ncifcrf.gov Link: Other Homepage

Biography

Dr. Waugh earned his Ph.D. in biochemistry from Indiana University in 1989 under the direction of Dr. Norman Pace. He was a postdoctoral fellow in Dr. Robert Sauer's laboratory at the Massachusetts Institute of Technology before becoming director of the Macromolecular Engineering Laboratory at Hoffmann-La Roche in 1991. In 1996, Dr. Waugh established the Protein Engineering Section at the NCI-Frederick Cancer Research and Development Center.

Research

High-Throughput Protein Expression and Purification; Structural Genomics

Our goal is to create a unified technological infrastructure for high-throughput protein expression and purification that will be suitable for large-scale structural biology initiatives. Central to our approach is the use of multiple genetically engineered affinity tags. We are currently trying to establish what combination of affinity tags is most appropriate for our purposes and how to use them with maximal efficiency. At the same time, we are striving to develop more reliable methods for removing affinity tags, which is another critical element of our strategy. The feasibility of this approach is being evaluated in the context of two structural proteomics projects.

One of the greatest technical obstacles that we face is 'the inclusion body problem'-i.e., the tendency of proteins to accumulate in an insoluble, inactive form. Because refolding of proteins seems incompatible with high-throughput applications, some means of circumventing the formation of inclusion bodies is needed. Sometimes this can be accomplished by fusing an aggregation-prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners-maltose-binding protein (MBP), glutathione S-transferase, and thioredoxin-to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a much more effective solubilizing agent than the other two fusion partners. Moreover, in some cases we were able to demonstrate that fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. This chaperone-like quality distinguishes MBP from other affinity domains and greatly enhances its value as a fusion partner. Accordingly, MBP fusion proteins have become a cornerstone of our strategy for protein expression. We are using a variety of experimental approaches to try to understand how MBP influences the folding of its fusion partners. At the same time, to improve its utility as a fusion partner, we are attempting to endow MBP with an engineered affinity for additional ligands.

Affinity tags would probably be used more often if it were not so difficult to remove them. This is usually accomplished by endoproteolysis of a fusion protein at a designed site. The main difficulty with this approach stems from the intrinsically promiscuous specificity of the proteases that are commonly used to cleave fusion proteins. This problem is compounded by the fact that it is prohibitively expensive to purchase enough of any of these reagents to cleave fusion proteins on a scale amenable for structural studies. To overcome these problems, we produce our own supply of TEV protease, the catalytic domain of the nuclear inclusion protease from tobacco etch virus. TEV protease cleaves the amino acid sequence ENLYFQG between Q and G with high specificity; in contrast to factor Xa, enteropeptidase and thrombin, there have never been any reports of cleavage at noncanonical sites in fusion proteins by TEV protease. The production of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility, but we have been able to solve both problems by making synonymous codon replacements and producing the protease in the form of an MBP fusion protein. A more troublesome shortcoming of TEV protease is that it cleaves itself at a specific site, generating a truncated protease with greatly diminished activity. We have been able to rectify this problem as well by introducing amino acid substitutions that prevent autoinactivation without impeding the ability of the protease to cleave canonical target sequences.

The technological framework that we are assembling for high-throughput protein expression and purification is being field tested in the context of two structural genomics projects. In collaboration with the Northeast Structural Genomics Consortium (http://www.nesg.org), we are attempting to solve the structures of evolutionarily conserved proteins from eukaryotic model organisms to gain a more comprehensive view of existing protein folds and to improve our understanding of how protein structures have evolved. The essential virulence factors in Yersinia pestis, which causes bubonic plague in humans, are the focus of the other structural genomics project. The long-term goal of this research is to facilitate the design or discovery of effective countermeasures for this potential agent of bioterrorism.

This page was last updated on 6/12/2008.