Cell surface display in biomedical applications and biofuels production (2010)

Cell surface display allows peptides or proteins to be expressed on the cell exterior as\nfusions to natural host anchoring motifs. It is a powerful technique with a myriad of applications\nin protein engineering, environmental bioremediation, biocatalysis, as well as vaccine and therapeutics development. Compared to intracellular expression, the main advantages of this technology include direct access to large target molecules that cannot diffuse into the cell,\nstabilization of enzymes or proteins by attaching them to the cell surface, and elimination of\ntime-consuming protein purification steps. This thesis describes our efforts of applying the cell surface display technology to address some of the challenges in biomedical research and biofuels production.\nIdentification of T cell epitopes is a critical, but often difficult step in developing peptidebased\nvaccines and T cell immunotherapies. Unlike antibody that recognizes free soluble\nantigens, T cell receptor (TCR) recognizes its epitope bound to major histocompatibility\ncomplex (MHC) expressed on antigen presenting cells (APCs). In addition, the examination of T\ncell epitope activity requires the use of professional APCs, which are difficult to isolate, expand,\nand maintain. To address these issues, we have developed a facile, accurate, and high-throughput method for T cell epitope mapping by displaying pathogen-derived peptide libraries in complex with MHC on yeast cell surface. Using human MHC class II protein DR1 and influenza A virus\nas a model system, this method was successfully used to pinpoint a 17-amino-acid-long T cell epitope from the entire influenza A virus genome. The production of peptide-MHC (pMHC) tetramer, especially class II pMHC tetramer, is very time-consuming and labor-intensive and often show low avidity, thus represents another\nchallenge in the biomedical research area. To address these limitations, we sought to engineer\nMHC monomers with high TCR-binding affinity. The wild-type DR2-MBP85-99 complex, which\nis associated with multiple sclerosis, was successfully displayed on insect cell surface and bound specific TCR tetramers in an epitope-dependent manner, providing the basis of a high throughput screening method to identify DR2 variants with improved affinity by directed evolution. A library of DR2 variants in complex with MBP was created and screened using specific TCR tetramers. After one round of cell sorting, potential variants with improved TCR-binding affinity have been enriched. Further rounds of enrichment are in process.\nLignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. The central technological impediment to a\nmore widespread utilization of lignocellulose is the absence of low-cost technology to break\ndown its major component ??? cellulose. Consolidated bioprocessing (CBP), which combines\nenzyme production, cellulose hydrolysis, and fermentation in a single step, has been proposed to\nsignificantly lower the cellulosic ethanol production cost. However, the great potential of CBP cannot be realized using microorganisms available today. In an effort to develop a CBP-enabling microorganism, we developed an engineering strategy to enable yeast cells to hydrolyze and\nferment cellulose simultaneously by displaying trifunctional minicellulosomes on the surface.\nThe system developed here solved the technical difficulties of displaying multiple proteins and\nrepresents a useful platform for elucidating principles of cellulosome construction and mode of action. Continuing efforts are being directed to improving the hydrolytic efficiency of the surface engineered yeast with a focus of increasing the enzyme display levels.