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.

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Figure 1: Scheme of the expression cassette of the pDST phagemid vector series.
Figure 2: Display yields of the various POIs on phage particles.
Figure 3: Increased display yields using SRP-dependent signal sequences.
Figure 4: Protein translation and translocation of the various unfused POIs to the periplasm.
Figure 5: Display yields and periplasmic translocation of DARPin E3_5 and TrxA using various Sec- and SRP-dependent signal sequences.


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We thank Patrick Amstutz and H. Kaspar Binz for valuable discussions and J. Beckwith and D. Huber for advice on experimental procedures. This work was supported by the Swiss National Center of Competence in Research (NCCR) in Structural Biology and by KTI Discovery.

Author information

A.P. conceived the project; D.S., P.F., M.T.S. and A.P. designed experiments; D.S. performed the experiments; D.S., P.F., M.T.S. and A.P. analyzed the data; D.S., P.F., M.T.S. and A.P. wrote the manuscript.

Note: Supplementary information is available on the Nature Biotechnology website.

Correspondence to Andreas Plückthun.

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The technology in this paper has been patented by D.S., M.T.S., P.T. and A.P.

Supplementary information

Supplementary Figure 1

Schematic representation of the phagemid pDST22. (PDF 306 kb)

Supplementary Table 1

E. coli strains. (PDF 109 kb)

Supplementary Table 2

Signal sequences. (PDF 84 kb)

Supplementary Methods

Phagemid cloning. (PDF 111 kb)

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