Internalization
AssigneeMorpho Sys AG
InventorMarkus Enzelberger
Life of this patent
Estimating the term…
Abstract
A target internalized within a cell (and a binding member that specifically binds thereto) can be identified in an efficient manner by segregating (or substantially segregating) genetic material encoding the binding member from genetic material encoding a binding member that binds to a target that is not internalized. This can be achieved by employing a display library of binding members having a genotype/phenotype linkage via a non-fusion protein format, whereby genetic material encoding non-in-ternalized targets can be segregated (or substantially segregated) without lysing the cells. Internalized genetic material subsequently can be isolated and amplified.
Claims7
A method for recovering a nucleic acid molecule encoding a binding member of a complex internalized in a target cell, comprising the following steps of:
- (a) contacting a target cell with a diverse collection of bacteriophage particles, wherein each or substantially all of said bacteriophage particles display a binding member on its surface, wherein said binding member is displayed as a non-fusion (poly)peptide with a phage coat protein of said bacteriophage particle and wherein each or substantially all of said bacteriophage particles comprise a nucleic acid molecule encoding the displayed binding member, wherein said display as a non-fusion (poly)peptide is characterized by a disulfide bond between the phage coat protein and the binding member,
- (b) allowing for binding of the binding member displayed on the bacteriophage particle to its target, thereby allowing for the formation of at least one complex, each of said complexes comprising a bacteriophage particle with its displayed binding member and its target,
- (c) culturing the target cell under conditions that allow internalization of at least one of said complexes into the cell,
- (d) eluting the bacteriophage particles displaying a binding member that are not internalized under conditions that substantially no cell lysis occurs, wherein the eluting is carried out under reducing conditions such that said disulfide bond is cleaved,
- (e) lysing the target cell comprising the internalized complexes, and
- (f) recovering from the lysed cell the nucleic acid molecule encoding a binding member from at least one of the internalized complexes.
Dependencies are inferred from the claim text.