(5cx) Protein Engineering for Biomedical Applications and Biofuels Production

The overall theme of my PhD thesis research is to apply cell surface display and directed evolution strategies to address challenges in biomedical research and biofuels production. Throughout my thesis study, I have been working with various types of proteins (human membrane proteins including T-cell receptors (TCRs) and major histocompatibility complexes (MHC), and enzymes from bacteria, fungi, and yeast) and a wide variety of expression systems (E. coli, yeast, insect cells, and mammalian cells) as described below.

1. Development of a new tool for T-cell epitope mapping. Identification of T-cell epitopes (antigenic peptides recognized by T cells) is a critical step in studying and modulating the immune responses to tumors, infectious agents, and autoantigens. We developed a facile, accurate, and high-throughput method for T-cell epitope identification using yeast displaying pathogen-derived peptide library. For the first time, we demonstrated that yeast cells displaying the peptide-MHC (pMHC) complexes could be used as artificial antigen presenting cells. Using human MHC class II molecule HLA-DR1 and influenza A virus (X31/A/Aichi/68) as a model system, we showed that this method can be used to rapidly pinpoint a 17-amino-acid-long epitope from the entire influenza A virus genome. Although the method was demonstrated by identifying a viral epitope, it should be generally applicable to the identification of T-cell epitopes from other systems such as cancer and autoimmune diseases.

2. Directed evolution of human T-cell receptor (TCR) ligands with improved affinity. The development of pMHC (TCR ligand) tetramer staining has revolutionized the field of T-cell research. However, pMHC tetramers are very time-consuming and labor-intensive to prepare and often show low avidity. To address these limitations, we sought to engineer MHC monomers with high TCR-binding affinity. The human MHC class II molecule DR2, which is associated with multiple sclerosis, was chosen to be our engineering target. By fusing the leucine zipper dimerization motifs, the wild-type DR2 was successfully displayed on insect cell surface and was capable of binding a candidate epitope involved in multiple sclerosis (MBP85-99) and activating specific T-cell hybridomas. More importantly, the surface displayed DR2 bound specific TCR tetramers in an epitope-dependent manner. Coupled with directed evolution and fluorescence-activated cell sorting (FACS), DR2 variants with improved affinity could be readily identified from a mutagenesis library in a high throughput manner. Currently, the validation of the described screening method is in process.

3. Lignocellulosic ethanol production using yeast displaying an engineered mini-cellulosome. In an effort to develop a consolidated bioprocessing-enabling microorganism, we created a recombinant cellulolytic yeast strain by displaying an engineered mini-cellulosome on the surface. The mini-cellulosome consists of a mini-scaffoldin derived from C. thermocellum and three types of cellulases - an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. The surface expression and functionality of the mini-cellulosome was confirmed by various detection methods. The engineered mini-cellulosome showed both enzyme-enzyme synergy and enzyme proximity synergy. In addition, the engineered mini-cellulosome was stable at 4 C for at least four months. More importantly, yeast cells displaying the engineered mini-cellulosome could directly convert amorphous cellulose to ethanol; and they also showed activity towards crystalline cellulose. The evaluation of ethanol production from crystalline cellulose is in progress.