A twin-arginine translocation (Tat)-mediated phage display system |
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| Authors: | Paschke Matthias Höhne Wolfgang |
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| Affiliation: | 1. Division of Hematology and Center for Cellular and Molecular Therapeutics, Children''s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA;1. Serviço de Urologia. Hospital Santo Antonio, Porto, Portugal;2. Servicio de Histología. Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain;3. Laboratorio de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain;4. Molecular Discovery Research, GlaxoSmithKline R&D, Stevenage, Herts SG1 2NY, United Kingdom;1. Human Retrovirus Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA;2. Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA;3. Laboratory of Immunoregulation, Division of Viral Products, Office of Vaccines, Center for Biologics, FDA, Silver Spring, MD, USA;4. Inovio Pharmaceuticals, Plymouth Meeting, PA, USA;1. Department of Medicine, Brigham and Women''s Hospital and Harvard Medical School, Boston, Mass;2. Department of Chemistry, University of Gdansk, Gdansk, Poland;3. Wihuri Research Institute, Biomedicum Helsinki 1, Helsinki, Finland;1. Department of Biotechnology, Hoseo University, Asan City, Chungnam 336-795, South Korea;2. Department of Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA;3. National Research Laboratory of Defense Proteins, College of Pharmacy, Pusan National University, Busan 609-735, South Korea;4. Department of Pharmacy, Korea University, Sejong 339-700, South Korea;5. Research Institute for Basic Science, Hoseo University, Asan City, Chungnam 336-795, South Korea;1. Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia;2. Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands;3. Laboratory of Bioluminescence Biotechnology, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk 660041, Russia |
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| Abstract: | The major limitation of conventional phage display is caused by its dependence on the Sec translocation pathway. All proteins displayed on filamentous phages must first be transported into the bacterial periplasm in an unfolded state via the Sec translocation machinery. Proteins that require a cytoplasmic environment and/or cytoplasmic components for folding, or that contain "stop transfer" signals, or reach their native state before they interact with the Sec proteins are not compatible with the Sec pathway. They can never be presented using conventional phage display. We have developed an alternative phage display system, termed the TPD system, which overcomes these limitations of conventional phage display by exploiting the properties of the twin-arginine translocation (Tat) pathway. The Tat pathway only exports folded proteins that have already attained their native conformation in the cytoplasm. We investigated the functional efficiency of the TPD system by displaying and panning for a mutant of the green fluorescent protein. |
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