Efficient phage display of intracellularly folded proteins mediated by the TAT pathway

• Published in: Protein engineering, design and selection Vol. 24; no. 6; pp. 473 - 484
• Main Authors: Speck, Janina, Arndt, Katja M., Müller, Kristian M.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.06.2011
• Subjects: Bacteriophages - genetics; Cell Survival; Cloning, Molecular; Cysteine; Cytosol; Endoribonucleases - genetics; Endoribonucleases - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fluorescence; Green fluorescent protein; Intracellular Space - metabolism; Luminescent Proteins; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Oligomerization; Peptide Library; Phage display; Plasmids - genetics; Protein Engineering - methods; Protein Folding; Protein transport; red fluorescent protein; Secretory Pathway; Signal recognition particle; Signal Recognition Particle - genetics; Signal Recognition Particle - metabolism; Temperature; Translocation; yellow fluorescent protein; phagemid display; protein engineering; selection; g3p; protein design
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzr001

Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function.