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Title:Cell surface display in biomedical applications and biofuels production
Author(s):Wen, Fei
Director of Research:Zhao, Huimin
Doctoral Committee Chair(s):Zhao, Huimin
Doctoral Committee Member(s):Kenis, Paul J.A.; Rao, Christopher V.; Kranz, David M.
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):T cell
Major histocompatibility complex (MHC)
directed evolution
Consolidated bioprocessing (CBP)
yeast surface display
baculovirus display
affinity engineering
directed evolution
pMHC tetramer
Abstract:Cell surface display allows peptides or proteins to be expressed on the cell exterior as fusions to natural host anchoring motifs. It is a powerful technique with a myriad of applications in 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, stabilization of enzymes or proteins by attaching them to the cell surface, and elimination of time-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. Identification of T cell epitopes is a critical, but often difficult step in developing peptidebased vaccines and T cell immunotherapies. Unlike antibody that recognizes free soluble antigens, T cell receptor (TCR) recognizes its epitope bound to major histocompatibility complex (MHC) expressed on antigen presenting cells (APCs). In addition, the examination of T cell epitope activity requires the use of professional APCs, which are difficult to isolate, expand, and 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 as 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 challenge in the biomedical research area. To address these limitations, we sought to engineer MHC monomers with high TCR-binding affinity. The wild-type DR2-MBP85-99 complex, which is 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. Lignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. The central technological impediment to a more widespread utilization of lignocellulose is the absence of low-cost technology to break down its major component – cellulose. Consolidated bioprocessing (CBP), which combines enzyme production, cellulose hydrolysis, and fermentation in a single step, has been proposed to significantly 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 ferment cellulose simultaneously by displaying trifunctional minicellulosomes on the surface. The system developed here solved the technical difficulties of displaying multiple proteins and represents 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.
Issue Date:2010-05-14
Rights Information:Copyright 2010 Fei Wen
Date Available in IDEALS:2010-05-14
Date Deposited:May 2010

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