Directed Evolution of ScFv/ScTCR-Specific Periplasmic Chaperones, Based on the Framework of wt Skp from E. Coli  (Primary Researcher: Ryan Myhre)

My project aims to build upon previous work that has shown the propensity of Skp, an E. coli chaperone, to improve periplasmic recombinant protein expression.  The specific aim is to develop scFv and scTCR specific, ATP independent, chaperones for maximizing soluble periplasmic expression of these valuable protein products.  I am working on developing novel chaperones from the wild type Skp framework by introducing mutations into the Skp gene.  Mutants are then co-expressed with single-chain antibody or T-cell receptor fragments and screened for variants that exhibit increased expression levels over wild type Skp.  Robust mutants are then isolated and characterized to elucidate fundamental differences from native Skp, with hopes that more robust mutants will shed some light on the mechanism of interaction between Skp and recombinant scFvs and scTCRs.

Figure to Right: A ribbon diagram representation of the Skp chaperone complex from wt E. Coli.  The figure is adapted from Walton, T.A. and M.C. Sousa, Crystal Structure of Skp, a Prefoldin-like Chaperone that Protects Soluble and Membrane Proteins from Aggregation. Mol Cell, 2004. 15(3): p. 367-74.  My work as of late has focused on mutation of the LPS binding motif (shown in purple on the diagram), with hopes of gaining some insight into the mechanism of association between Skp, recombinant protein, and cell membrane (inner and outer).

Engineering of Functional T-Cell Receptors for Therapeutic Applications (Primary Researcher: Benjamin Roy)

Antibodies are currently one of the most rapidly growing classes of therapeutic molecules, able to treat tumors and limit inflammation by virtue of specific, high affinity ligand recognition. In contrast, the cellular immune system, responsible for eradication of cancerous and virally infected cells as well as autoimmune responses and organ rejection, is much less well understood. This is primarily due to difficulties in expressing recombinant T cell receptor (TCR) material coupled with low affinity for and low expression levels of cognate pMHC. By optimizing multiple aspects of recombinant expression, we were able to solve the three-dimensional structure of the first auto-immune TCR complex, providing insight into aberrant immune recognition. My work now primarily focuses around developing a platform technology to identify TCRs with selective, low affinity for binding partners. As indicated by the successes of pMHC multimers and antibodies, such molecules could directly impact our understanding of TCR-pMHC recognition in disease (autoimmunity) and health (vaccine responses) as well as form the basis of novel reagents for immunology research and targeted immunotherapeutics.

Figure to Right:  Ribbon representation of TCR-pMHC complex, including 172.10 Va (red), 172.10 Vb (blue), I-Au (green) and 1-8 MBP (yellow).  The six CDR binding loops of 172 are numbered.  Adapted from  Maynard, J., Petersson, K., Wilson, D.H., Adams, E.J., Blondelle, S.E., Boulanger, M.J., Wilson, D.B. and Garcia, K.C. (2005b) Structure of an autoimmune t cell receptor complexed with class ii peptide-mhc: Insights into mhc bias and antigen specificity. Immunity 22, 81-92.

From Bugs to Drugs: Adapting Pathogenic Mechanisms For Biomedical and Biotechnological Applications (Primary Researcher: Tarik Khan)

Of the characterized pathogenic systems, one with great potential for biotechnological and biomedical applications is the bacterial type III secretion system (TTSS), utilized by bacteria to inject toxins into a human host cell (see Figure).  My project focuses on re-engineering this system to recognize specific cell types and to deliver desired proteins intracellularly.  Several applications of such technology include (i) delivery of therapeutic proteins, i.e. vaccines to achieve a cellular immune response or artificial zinc finger proteins to specifically bind DNA and prevent oncogene expression in cancerous cells; (ii) delivery of a protein which is presented on the cell surface as a novel cellular receptor and used to follow the fate of that particular cell in developmental or cancer metastasis studies; and (iii) a drug screening platform and basic research tool to assess the effects of proteins with intracellular activity. Previous efforts to reprogram pathogenic mechanisms have been limited but include several successes, primarily focused on bacterial toxins: enhancing immune responses during vaccination, selectively targeting tumor cells, and effecting muscle paralysis to eliminate wrinkles. In contrast, we will develop a general method for intracellular delivery of multiple proteins with a single delivery vehicle. This technology will have impact on design of therapeutic proteins as well as fundamental cell biology and virology studies.  

Scaffolds For Building Protein Crystals (Primary Researcher: Jennifer Pai)

For those who work with biological systems, the most coveted information is often a protein structure.  Once extremely difficult to obtain, structures are now being produced at an ever-accelerating pace and their arrival often represents a major breakthrough in scientific understanding. These models allow you to “see” the spatial position of potentially every atom in every amino acid in the protein, facilitating rational drug design, providing a physical context for biochemical data and revealing interactions with binding partners. Unfortunately, while the structure itself is a rich source of information, obtaining it is not so easy.  The primary bottlenecks in crystallography are (i) expression and purification of pure, homogeneous material and (ii) identification of conditions resulting in growth of diffracting crystals.  Practically, experimentalists employ the shotgun approach: try one expression host, purification method and a matrix of crystallization conditions; if unsuccessful, repeat ad infinitum.

             The empirical nature of traditional approaches emphasizes the need for new engineering tools to facilitate crystal growth.  Moreover, each protein exhibits unique properties in terms of stability and surface charges (among others) - properties which have been shown repeatedly to influence crystal growth but whose influence is largely ignored by current methods.  My research focuses on the protein as a variable in crystallography. Using recombinant antibody molecules, protein evolution and crystallography techniques, we intend to create a hyper-crystallizable peptide-binding single-chain antibody (scFv), that readily grows crystals using multiple space groups and binding partners.

Analysis of this antibody will itself provide scientific insight in terms of the biophysical parameters and amino acid contact residues which correlate with an increased propensity to crystallize. It has greater potential as a general tool: the cognate peptide can be readily introduced into any second protein of interest (POI); the two proteins combined to form a complex. By virtue of the scFv’s propensity to crystallize, the POI will be also incorporated into a lattice network and structural data obtained for both molecules (see Figure below). This work fits into our larger goals of understanding and manipulating immunological proteins for therapeutic benefit and would be broadly useful as a scaffold to crystallize otherwise recalcitrant proteins.