Protein complementation assay as a display system for screening protein libraries in the intracellular environment
Pow, Andrew James (2008) Protein complementation assay as a display system for screening protein libraries in the intracellular environment. PhD thesis, Queensland University of Technology.
A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display.
This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) Escherichia coli proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display.
In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.
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|Item Type:||QUT Thesis (PhD)|
|Supervisor:||Morris, Charles & Harris, Jonathan|
|Keywords:||protein complementation assay (PCA), â-lactamase, two-hybrid, molecular display, protein libraries, intracellular proteins, library screening, high-throughput screening, mammalian expression system, flow cytometry, single cell sorting, CCF2/AM, nitrocefin, HEK 293, HEK 293T, biopharmaceuticals|
|Divisions:||Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
|Institution:||Queensland University of Technology|
|Deposited On:||11 Feb 2010 04:22|
|Last Modified:||21 Sep 2016 04:49|
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