Patent ID: 8846577
Filing Date: 2014-09-30
Classification: C07K,C12N,C40B

Abstract:
1. A filamentous phage library display method for polypeptides of interest comprising the steps of (a) constructing a filamentous phage or phagemid vector containing an expression cassette for a fusion polypeptide comprising a polypeptide of interest and an N-terminal signal sequence promoting cotranslational translocation of the fusion polypeptide across a cytoplasmic membrane of a Gram-negative bacteria, wherein the signal sequence promoting cotranslational translocation is based on the signal recognition particle pathway is selected from the group consisting of signal sequences of TorT, SfmC, FocC, CcmH, Yral, TolB, NikA, FlgI, and DsbA; (b) constructing a combinatorial library of phage or phagemid vectors by cloning of a DNA library encoding polypeptides of interest into the expression cassette of a plurality of vectors of step (a), wherein said DNA library is a cDNA library or a combinatorial DNA library that encodes a library based on alternative scaffolds; (c) transforming suitable Gram-negative bacteria with the library of vectors of step (b) wherein the polypeptides of interest encoded by the DNA library are cotranslationally translocated across the cytoplasmic membrane of Gram-negative bacteria, and wherein the polypeptides of interest are displayed on phage particles; and (d) performing phage display selection cycles to separate the phage particles based on properties of the displayed polypeptides of interest.