US 11,453,883 B2Granted

Display systems for proteins of interest

AssigneeBio-Rad Abd Serotec GmbH

InventorFrancisco Ylera

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Abstract

Described herein is a protein display selection method which uncouples a protein of interest (POI) library from the display selection system. Display of the POI can be achieved by forming a covalent bond between the POI and the anchor protein post expression either by enzymatic protein ligation (e.g. SpyLigase, SnoopLigase, sortase, butelase, peptiligase etc.) or by spontaneous covalent bond formation (e.g. SpyTag/SpyCatcher, SnoopTag/SnoopCatcher, etc.). The POI library is fused to a tethering sequence, for example SpyTag, at the C-terminus of the POI which then forms a covalent bond to a capture sequence found on an anchor protein, for example, the SpyCatcher-fused anchor protein, e.g., a SpyCatcher-geneIII protein (SpyCatcher-pIII) fusion, for the most common form of phage display. Nucleic acid constructs, host cell systems and methods of producing the protein display systems are also provided.

Claims21

Dependent claims

A combination of vectors comprising:

a) a first vector comprising a genetic construct (GC1) comprising polynucleotide encoding a protein of interest (POI) fused, in frame, to a tether sequence;
and
b) a second vector comprising a genetic construct (GC2) comprising polynucleotide encoding an anchor protein fused, in frame, to a capture sequence,
the tether sequence and capture sequence forming a covalent bond when brought into contact with one another either spontaneously or with the help of an enzyme;
or
a combination of vectors comprising:
a) a first vector comprising a genetic construct (GC1) comprising polynucleotide encoding a protein of interest (POI) fused, in frame, to a capture sequence;
and
b) a second vector comprising a genetic construct (GC2) comprising polynucleotide encoding an anchor protein fused, in frame, to a tether sequence,
the tether sequence and capture sequence forming a covalent bond when brought into contact with one another either spontaneously or with the help of an enzyme;
the POI being selected from antibodies, antibody fragments, single chain antibodies, scFv, scFab, or single domain antibodies and the anchor protein being selected from pIII, pVI, pVII, pVIII, or pIX.

8 more elements

Dependencies are inferred from the claim text.

Facts

Application: 16/375,894Filed 5 Apr 2019 · us provisional application
Priority: 5 Apr 2018
Published: US 2019/0309312 A110 Oct 2019
Granted: 27 Sep 2022
Term ends: Not estimatedThe source records don’t support an estimate.

Classification13 codes

C12N 15/70
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: vectors or expression systems…
C07K 16/241
Against cytokines, lymphokines or interferons: tumor Necrosis Factors
C07K 16/32
Against material from animals or humans: against translation products of oncogenes
C07K 19/00
Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently