Deimmunisation oF Enhanced Green Fluorescent Protein

  • Timothy Eyes

    Student thesis: Phd


    The discovery and application of GFP (Green Fluorescent Protein) has made a very significant contribution to biological research. Detection of a genetically encoded fluorescent signal has proven to be an extremely powerful tool for monitoring biological events both in vitro and in vivo. Whole organism applications for EGFP (enhanced GFP, the widely used variant of GFP) span a broad range of key disciplines which encompass cell biology, immunology and developmental biology. Furthermore, EGFP has specific applications in monitoring tumour development, as an indicator of genetic modification and cell tracking in vivo. However, there is strong evidence that EGFP elicits an immune response when used in animal models. Stimulation of a host immune response can have very serious consequences for the integrity and reliability of experimental studies, including the rejection of cells expressing EGFP. The aim of this study was to develop an EGFP construct that has reduced immunogenicity in the BALB/c model strain but maintains the fluorescent functionality to the original EGFP. The major histocompatability complex (MHC) class I immunodominant epitopes of EGFP in both BALB/c and C57BL/6 strain mice have been described previously. EGFP was recombinantly expressed in E.coli and saturation mutagenesis was used to generate a library of EGFP variants where the MHC class I anchor site in the dominant epitope is mutated to reduce immunogenic potential, termed 'deimmunisation.' The library was screened for fluorescent EGFP variants which possessed anchor site mutations that reduced immunogenicity based on computational prediction. The EGFP Y200T variant was isolated with near identical fluorescent properties to the parent EGFP.BALB/c mice were immunised with a tumour cell line expressing EGFP or EGFP Y200T. The Y200T mutation completely abrogated a humoral antibody response to EGFP in vivo. The data suggests that through a single mutation the immunodominant epitope in EGFP can be disrupted and tolerance enhanced in mice. This presents a novel strategy for improving tolerance to heterologously expressed proteins without the requirement for immunosuppressive intervention.
    Date of Award1 Aug 2014
    Original languageEnglish
    Awarding Institution
    • The University of Manchester

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