A twin-arginine translocation (Tat)-mediated phage display system.
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.
Paschke
Matthias
M
Institut für Biochemie, Charité-Universitätsmedizin Berlin, Monbijoustrasse 2A, Berlin D-10117, Germany. matthias.paschke@charite.de
Höhne
Wolfgang
W
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C408585:twin-arginine translocase complex, E coli; D049452:Green Fluorescent Proteins