Applied protein engineering for biotechnology development
My work seeks to develop highly useful biotechnologies through engineering proteins and enzymes for specific applications. So far, these applications include new ways to purify recombinant proteins, bacterial biosensors that incorporate human drug targets, and new capabilities in drug discovery and drug delivery.
The development of affinity tag technology in the mid 1980's provided a means to purify virtually any arbitrary recombinant protein through a single general and simple method. This method is now ubiquitous in the laboratory, where dozens of different tags, kits and accessories are now commercially available.
Surprisingly, this method has not been adopted for large-scale bioprocessing, largely due to the expense of removing the affinity tag after the target protein is purified. Thus the promise of this method has been severely limited due to basic limitations in the proteins involved. We are currently developing new protein elements that effectively make the affinity tags self-cleaving after purification, and therefore enable many new tag-based protein purification methods. These methods retain the simplicity and generality of conventional affinity tag technology, and can potentially make tag technology a ubiquitous and critical platform in protein bioseparations. Although our early methods are very effective for proteins expressed in E. coli, we must adapt this technology for proteins expressed in Pichia and CHO, as well as transgenic and other expression hosts. Further, the tags we use must be attractive to the biotech industry, and address its needs for scale, simplicity and expense. Thus our focus is on the development of new tags that meet these needs. For example, these tags must bind at high capacity, and have chemistries that lend themselves to disposable technologies, and the self-cleaving reaction must be more controllable and reliable in every expression host. Our approaches range from simple chemistry to complex protein re-design, and require a variety of backgrounds.
Biosensing and Drug Discovery
The nuclear hormone receptors (NHRs) are involved in vital functions of the cell, such as development, differentiation, homeostasis, reproduction and metabolism. Accordingly, NHRs comprise one of the most important classes of drug targets, and about 4% of all currently marketed therapeutics interfere with the activity of one or more of these proteins. Importantly, they are also targets for naturally occurring and synthetic endocrine disrupting compounds, and interference with their function has been connected to breast cancer, decreased fertility, and other disorders in humans and in animals. We have developed a very simple reporter system in E. coli that can sense human hormone-like compounds, and report their presence and activity through changes in growth phenotype. Our sensor is based on a chimeric fusion protein where genetically inserting various hormone ligand-binding domains yields sensors with altered specificity. We have now constructed sensors using the alpha and beta subtypes of the human estrogen receptor, as well as the human thyroid receptor and insect ecdysone receptor. These designs are not only able to detect hormone-like compounds for these targets, but they are also able to discriminate between estrogen agonistic and antagonistic activities, distinguish subtype-selective estrogen receptor modulators, and report rough estimates of binding affinity. Our current goal is to understand the structure, energy and kinetics of our designed proteins, and generate new rules for engineering allosteric protein chimeras. In the short term we hope to modify this system for screening large natural-product libraries, and libraries derived from combinatorial synthesis techniques. The ability of this system to work effectively with three examples of human nuclear hormone receptors suggests that it will be generally useful for identifying natural product ligands that modulate this family or targets.
- Wu WY, Fong BA, Gilles AG, Wood DW. (2009) Recombinant protein purification by self-cleaving elastin-like polypeptide fusion tag., Curr Protoc Protein Sci. ;Chapter 26:Unit 26.4.1-18.
- Hartman I, Gillies AR, Arora S, Andaya C, Royapet N, Welsh WJ, Wood DW, Zauhar RJ. (2009) Application of screening methods, shape signatures and engineered biosensors in early drug discovery process., Pharm Res. 2009 Oct;26(10):2247-58. Epub
- Fong BA, Wu WY, Wood DW. (2009), Optimization of ELP-intein mediated protein purification by salt substitution., Protein Expr Purif. ;66(2):198-202. Epub Staii C, Wood DW, Scoles G. (2008), Verification of biochemical activity for proteins nanografted on gold surfaces., J Am Chem Soc.;130(2):640-6.
- Skretas G, Meligova AK, Villalonga-Barber C, Mitsiou DJ, Alexis MN, Micha-Screttas M, Steele BR, Screttas CG, Wood DW. (2007) Engineered chimeric enzymes as tools for drug discovery: generating reliable bacterial screens for the detection, discovery, and assessment of estrogen receptor modulators., J Am Chem Soc.;129(27):8443-57. Epub