A novel phagemid (pTat8) was constructed in this study to improve the quality of a molecule displayed on filamentous phage. The twin-arginine translocation (Tat) pathway was chosen for transporting and integrating a CD147 molecule into a phage particle via gpVIII. The parent vector pComb8-CD147Ex was modified by substituting a Sec signal sequence (PelB) with a twin-arginine signal sequence from trimethylamine N-oxide reductase (TorA). The characteristics of the CD147 displayed on the phage particle were evaluated by Sandwich ELISA and Western immunoblotting. A Tat-dependent leader was found to be superior to the Sec leader for the phage display of CD147. Our findings further support the involvement of an Escherichia coli Tat translocase in mediating the integration of a hydrophobic transmembrane protein into the inner membrane. This modified phagemid will be useful in phage display technique when the correctly folded structure is required (i.e., antibody libraries and ligand–receptor tracing).
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We are grateful to Dr. Nicole Ngo (The Perinatal HIV Prevention Trial Group, Chiang Mai, Thailand) for cooperation in determining the inserted fragment in pTat8-CD147Ex and to Mr. Phawin Charoensook for technical assistance. We also acknowledge Dr. Ian Thomas and Mr. Brian William Habbard for critical reading of this manuscript.
This work was supported by the Thailand Research Fund and the National Center for Genetic Engineering and Biotechnology. R.J.T. thanks the Canadian Institutes of Health Research for funding support.
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Thammawong, P., Kasinrerk, W., Turner, R.J. et al. Twin-arginine signal peptide attributes effective display of CD147 to filamentous phage. Appl Microbiol Biotechnol 69, 697–703 (2006). https://doi.org/10.1007/s00253-005-0242-0
- Phage Display
- Sandwich ELISA
- Phage Particle
- Filamentous Phage
- Phage Display Technique