Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display.
Even proteins that fold well in bacteria are frequently displayed poorly on filamentous phages. Low protein presentation on phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in phage assembly, we tested the display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved phage display from tenfold enrichment to >1,000-fold enrichment per phage display selection round. We named this method 'SRP phage display' and envision broad applicability, especially when displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.
Steiner
Daniel
D
Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Forrer
Patrik
P
Stumpp
Michael T
MT
Plückthun
Andreas
A
D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods; D046188:Protein Modification, Translational; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000235:genetics; D018271:Signal Recognition Particle / Q000235:genetics* / Q000378:metabolism
D019151:Peptide Library; D021382:Protein Sorting Signals; D018271:Signal Recognition Particle