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US 2021/0355175 A1Published application
Orthogonal protein heterodimers
AssigneeUniversity of Washington
InventorsZibo CHEN, Scott BOYKEN, Sherry BERMEO, Robert A. LANGAN, David BAKER
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Abstract
Disclosed herein are designed heterodimer proteins, monomeric polypeptides capable of forming heterodimer proteins, protein scaffolds including such polypeptides, and methods for using the heterodimer proteins and subunit polypeptides for designing logic gates.
Claims118
A designed heterodimer protein, comprising:
- (a) a monomer A polypeptide, wherein the monomer A polypeptide is a non-naturally occurring polypeptide comprising 1-5 alpha helices connected by amino acid linkers;
- and
- (b) a monomer B polypeptide, wherein the monomer B polypeptide is a non-naturally occurring polypeptide comprising 1-5 alpha helices connected by amino acid linkers,
- wherein monomer A and monomer B non-covalently interact to form the designed heterodimer protein.
1 more element
A non-naturally occurring polypeptide comprising a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along the length of the amino acid sequence selected from the group consisting of SEQ ID NOS:
- 1-290.
The non-naturally occurring polypeptide of claim 8, wherein amino acid changes from the reference amino acid sequence are conservative amino acid substitutions.
A protein comprising 2, 3, 4, or more non-naturally occurring polypeptides having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity along the length of the amino acid sequence selected from the group consisting of SEQ ID NOS:
- 1-290, wherein the 2, 3, 4, or more naturally occurring polypeptides are covalently linked.
A protein scaffold, comprising
- a) a first designed component comprised of any number of monomer A polypeptides and/or monomer B polypeptides, each from different heterodimers, connected into a single component by amino acid linkers.
- b) a second designed component, comprising corresponding monomers for each monomer A and/or monomer B in the first designed component one;
- wherein the first and second designed components interact to form the protein scaffold, and wherein each monomer A only interacts in the scaffold with its monomer B mate.
A designed heterodimer protein comprising:
- a) asymmetric buried hydrogen bond networks incorporated into regularly repeating backbone structures;
- and
- b) helix hairpin helix monomers wherein the supercoil phases of the helices are fixed at 0, 90, 180, or 270 degrees and the supercoil twist (ω0) and helical twist (ω1) are held constant for either a two layer left handed super coil (ω0=−2.85 and ω1=102.85), or a 5 layer untwisted bundle (ω0=0 and ω1=100)
Use of the polypeptide, protein, heterodimer protein, protein scaffold, nucleic acid, expression vector, and/or cell of any preceding claim for any suitable purposed, including but not limited to those disclosed herein such as designing protein logic gates.
A fusion protein comprising a polypeptide of the formula X-B-Z, wherein:
- (a) the X domain is a non-naturally occurring polypeptide comprising 1, 2, or 3 alpha helices, wherein the X domain is capable of non-covalently binding to a first target;
- (b) the Z domain is a non-naturally occurring polypeptide comprising 1, 2, or 3 alpha helices, wherein the Z domain is capable of non-covalently binding to either (i) a second target that differs from the first target, or (ii) a different non-naturally occurring polypeptide comprising 1, 2, or 3 alpha helices;
- and
- (c) the B domain is an amino acid linker;
- wherein a combined number of alpha helices from the X domain and the Z domain is 4, 5, or 6;
- and
- wherein the X domain and the Z domain interact at a binding interface, wherein the binding interface comprises a hydrogen bond network in which at least one side chain in each alpha helix hydrogen of the X domain bonds with a side chain in an alpha helix in the Z domain, and wherein the binding interface comprises a plurality of hydrophobic residues.
4 more elements
A kit or composition, comprising at least two fusion proteins comprising the formula X-B-Z, wherein
- the B domain in each fusion protein is independently a polypeptide linker;
- the X domain in each fusion protein comprises a first non-naturally occurring polypeptide comprising 1, 2, or 3 alpha helices;
- the Z domain in each fusion protein comprises a second non-naturally occurring polypeptide comprising 1, 2, or 3 alpha helices, wherein a combined number of alpha helices from the X domain and the Z domain in each individual fusion protein is 4, 5, or 6;
- wherein the X domain and the Z domain interact at a binding interface, wherein the binding interface comprises a hydrogen bond network in which at least one side chain in each X domain alpha helix bonds with a side chain in an alpha helix in the Z domain;
- wherein the X domain in a first fusion protein is capable of non-covalently binding to a first target;
- the Z domain in a second fusion protein is capable of non-covalently binding to a second target;
- and
- the X domains and Z domains in each individual fusion protein that are not capable of non-covalently binding to the first target or the second target are capable of non-covalently binding to an X or a Z domain of a different fusion protein in the plurality of fusion proteins.
5 more elements
A method for target detection, comprising
- (a) contacting a biological sample with at least two fusion proteins, wherein each of the at least two fusion proteins comprises the formula X-B-Z, wherein
- each B is independently a polypeptide linker;
- each X domain comprises a first non-naturally occurring polypeptide comprising 1, 2, or 3 alpha helices;
- each Z domain comprises a second non-naturally occurring polypeptide comprising 1, 2, or 3 alpha helices, wherein a combined number of alpha helices from the X domain and the Z domain in each individual fusion protein is 4, 5, or 6;
- wherein the X domain and the Z domain interact at a binding interface, wherein the binding interface comprises a hydrogen bond network in which at least one side chain in each X domain alpha helix bonds with a side chain in an alpha helix in the Z domain;
- wherein
- the X domain in a first fusion protein is capable of non-covalently binding to a first target;
- the Z domain in a second fusion protein is capable of non-covalently binding to a second target;
- and
- the X domains and Z domains in each individual fusion protein that are not capable of non-covalently binding to the first target or the second target are capable of non-covalently binding to an X or a Z domain of a different fusion protein in the plurality of fusion proteins;
- (b) detecting non-covalent binding of the two or more fusion proteins to the first target and/or the second target in the biological sample.
8 more elements
A composition comprising
- (a) a first polypeptide comprising 2 alpha helices, wherein the first polypeptide is capable of non-covalently binding a first target;
- and
- (b) a second polypeptide comprising 2 alpha helices, wherein the first polypeptide is capable of non-covalently binding to the second polypeptide, and wherein the second polypeptide is capable of non-covalently binding a second target that differs from the first target;
- wherein:
- (i) a binding affinity of the first polypeptide for the first target is approximately equal to a binding affinity of the second polypeptide for the second target;
- and
- (ii) the binding affinity of the first polypeptide for the first target and the binding affinity of the second polypeptide for the second target are greater than the binding affinity of the first target and the second target for each other.
4 more elements
A composition comprising
- (a) a first polypeptide comprising 2 alpha helices, wherein the first polypeptide is capable of non-covalently binding a first target;
- and
- (b) a second polypeptide comprising 2 alpha helices, wherein the first polypeptide is capable of non-covalently binding to the second polypeptide, and wherein the second polypeptide is capable of non-covalently binding a second target that differs from the first target;
- wherein:
- (i) a binding affinity of the first polypeptide for the second polypeptide is greater than a binding affinity of the second polypeptide for the second target;
- (ii) a binding affinity of the first polypeptide for the first target is approximately equal to a binding affinity of the second polypeptide for the second target;
- and
- (iii) the binding affinity of the first polypeptide for the first target and the binding affinity of the second polypeptide for the second target are greater than the binding affinity of the first target and the second target for each other.
5 more elements
A composition comprising
- (a) a first polypeptide comprising 4 alpha helices, wherein the first polypeptide is capable of non-covalently binding a first target;
- and
- (b) a second polypeptide comprising 4 alpha helices, wherein the second polypeptide is capable of non-covalently binding a second target that differs from the first target;
- wherein:
- (i) a binding affinity of the first target for the second target is greater than a binding affinity of the first polypeptide for the first target;
- (ii) a binding affinity of the first polypeptide for the first target is approximately equal to a binding affinity of the second polypeptide for the second target;
- and
- (iii) the sum of the binding affinity of (A) the first polypeptide for the first target and (B) the binding affinity of the second polypeptide for the second target, is greater than the binding affinity of the first target and the second target.
5 more elements
A heterodimer, comprising:
- (a) a monomer A polypeptide, wherein the monomer A polypeptide is a non-naturally occurring polypeptide comprising 1-5 alpha helices, wherein adjacent alpha helices may optionally be connected by an amino acid linker;
- and
- (b) a monomer B polypeptide, wherein the monomer B polypeptide is a non-naturally occurring polypeptide comprising 1-5 alpha helices, wherein adjacent alpha helices may optionally be connected by an amino acid linker;
- wherein the monomer A polypeptide and the monomer B polypeptide non-covalently interact to form the designed heterodimer protein.
1 more element
A protein scaffold, comprising
- (a) a fusion protein comprising of 2, 3, 4, or more polypeptides, wherein each polypeptide present in the fusion protein is a non-naturally occurring polypeptide comprises 1-5 alpha helices, wherein adjacent alpha helices may optionally be connected by an amino acid linker;
- wherein each polypeptide in the fusion protein is capable of non-covalently interacting with a binding partner, and wherein the fusion protein does not comprise a binding partner for any polypeptide present in the fusion protein;
- and
- (b) a binding partner for at least one of the polypeptides present in the fusion protein;
- wherein the fusion protein and the binding partner non-covalently interact to form the protein scaffold, wherein an interaction specificity between the binding partner and the at least polypeptide in the fusion protein are determined by at least one hydrogen bond network at the interface between the binding partner and the at least one polypeptide.
2 more elements
The protein scaffold any one of claims 102-111, wherein the binding interface comprises at least 25% hydrophobic residues.
Dependencies are inferred from the claim text.