Methods and compositions for assessing protein function

Patent metadata

Field	Value
Publication number	US-2020407708-A1
Application number	US-202016910507-A
Country	US
Kind code	A1
Filing date	Jun 24, 2020
Priority date	Jun 28, 2019
Publication date	Dec 31, 2020
Grant date	—

Abstract

Compositions and methods for assessing protein function are provided. A modified SpyCatcher protein that can include a tag is provided. Modified SpyCatcher proteins linked to a protein of interest, such as a nuclease are also provided. The methods include contacting a SpyCatcher protein and a SpyTagged protein to form a complex that may further include a protein of interest, one or more nucleic acids, and/or a nuclease. The methods can be used to purify a protein of interest or identify or target a protein binding site in a nucleic acid.

First claim

We claim:

1 . A modified SpyCatcher polypeptide comprising at least one heterologous cysteine residue.

2 . The modified SpyCatcher polypeptide of claim 1 , comprising a cysteine at an amino acid residue corresponding to amino acid position 50 of SEQ ID NO: 6.

3 . The modified SpyCatcher polypeptide of claim 2 , comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 7.

4 . The modified SpyCatcher polypeptide of claim 3 , comprising the amino acid sequence of SEQ ID NO: 7.

5 . The modified SpyCatcher polypeptide of claim 2 , further comprising a tag covalently linked to the cysteine residue.

6 . The modified SpyCatcher polypeptide of claim 5 , wherein the tag comprises biotin.

7 . The modified SpyCatcher polypeptide of claim 1 , wherein the SpyCatcher polypeptide is covalently linked to a protein of interest.

8 . The modified SpyCatcher polypeptide of claim 7 , wherein the protein of interest comprises a nuclease.

9 . The modified SpyCatcher polypeptide of claim 8 , wherein the nuclease is a micrococcal nuclease.

10 . The modified SpyCatcher polypeptide of claim 8 , wherein the nuclease is a catalytically inactive Cas9 nuclease.

11 . The modified SpyCatcher polypeptide of claim 7 , wherein the protein of interest is a Tn5 transposase.

12 . A nucleic acid encoding the SpyCatcher polypeptide of claim 2 .

13 . A vector comprising the nucleic acid of claim 12 .

14 . A kit comprising the nucleic acid of claim 12 .

15 . The kit of claim 14 , further comprising a nucleic acid encoding a SpyTag or SpyTag-SNAC tag.

16 . A method of purifying a protein of interest, comprising:
(a) contacting the SpyCatcher polypeptide of claim 5 with a protein of interest covalently linked to a SpyTag polypeptide or a SpyTag-SNAC tag polypeptide under conditions sufficient for the SpyCatcher polypeptide and the SpyTag polypeptide or SpyTag-SNAC tag polypeptide to form a covalently bound SpyCatcher-SpyTag-protein of interest complex or a SpyCatcher-SpyTag-SNAC tag-protein of interest complex; (b) contacting the SpyCatcher-SpyTag-protein of interest complex or SpyCatcher-SpyTag-SNAC tag-protein of interest complex with a binding partner for the tag covalently linked to the SpyCatcher polypeptide under conditions sufficient for the binding partner to bind to the tag; (c) performing one or more washes of the complex; (d) cleaving the SpyTag or the SpyTag-SNAC tag from the complex; and (e) isolating the protein of interest.

17 . The method of claim 16 , wherein the contacting the SpyCatcher polypeptide with the protein of interest covalently linked to the SpyTag polypeptide or the SpyTag-SNAC tag polypeptide is performed in a lysate of a cell expressing the protein of interest covalently linked to the SpyTag polypeptide or the SpyTag-SNAC tag polypeptide.

18 . The method of claim 16 , wherein the tag is biotin and the binding partner for the tag is streptavidin.

19 . A method of mapping protein binding to a nucleic acid, comprising:
(a) contacting the modified SpyCatcher polypeptide of claim 5 with a protein of interest covalently linked to a SpyTag polypeptide or a SpyTag-SNAC tag polypeptide and a population of nucleic acids under conditions sufficient for the protein of interest to bind to the nucleic acids and the SpyCatcher polypeptide and the SpyTag polypeptide or SpyTag-SNAC tag polypeptide to bind to form a SpyCatcher-SpyTag-protein-nucleic acid complex or a SpyCatcher-SpyTag-SNAC tag-protein-nucleic acid complex; (b) contacting the SpyCatcher-SpyTag-protein-nucleic acid complex or SpyCatcher-SpyTag-SNAC tag-protein-nucleic acid complex with a binding partner for the tag covalently linked to the SpyCatcher polypeptide under conditions sufficient for the binding partner to bind to the tag; (c) contacting the complex with a nuclease under conditions sufficient for the nuclease to cleave the nucleic acid flanking the complex; (d) performing one or more washes of the complex; (e) disassociating the SpyCatcher-SpyTag-protein or SpyCatcher-SpyTag-SNAC tag-protein from the nucleic acid; and (f) isolating the nucleic acid.

20 . The method of claim 19 , wherein the contacting the modified SpyCatcher polypeptide with the protein of interest covalently linked to the SpyTag polypeptide or the SpyTag-SNAC tag polypeptide and the population of nucleic acids is in a permeabilized cell expressing the protein of interest covalently linked to the SpyTag polypeptide or the SpyTag-SNAC tag polypeptide.

21 . The method of claim 20 , further comprising identifying the isolated nucleic acid.

22 . A method of mapping protein binding to a nucleic acid, comprising:
(a) contacting a SpyCatcher polypeptide covalently linked to a nuclease with a protein of interest covalently linked to a SpyTag polypeptide or a SpyTag-SNAC tag polypeptide and a population of nucleic acids under conditions sufficient for the protein of interest to bind to the nucleic acids and the SpyCatcher polypeptide and the SpyTag polypeptide or SpyTag-SNAC tag polypeptide to bind to form a SpyCatcher-SpyTag-protein-nucleic acid complex or a SpyCatcher-SpyTag-SNAC tag-protein-nucleic acid complex; (b) incubating the SpyCatcher-SpyTag-protein-nucleic acid complex or the SpyCatcher-SpyTag-SNAC tag-protein-nucleic acid complex under conditions sufficient to activate the nuclease; (c) performing one or more washes of the complex; (d) dissociating the SpyCatcher-SpyTag-protein or the SpyCatcher-SpyTag-SNAC tag-protein from the nucleic acid; and (e) isolating the nucleic acid.

23 . The method of claim 22 , wherein the contacting the SpyCatcher polypeptide with the protein of interest covalently linked to the SpyTag polypeptide or the SpyTag-SNAC tag polypeptide is performed in:
(i) a permeabilized cell expressing the protein of interest covalently linked to the SpyTag polypeptide or the SpyTag-SNAC tag polypeptide; or (ii) a permeabilized cell co-expressing the SpyCatcher polypeptide covalently linked to the nuclease and the protein of interest covalently linked to the SpyTag polypeptide or the SpyTag-SNAC tag polypeptide.

24 . The method of claim 22 , further comprising identifying the nucleic acid.

25 . A method of mapping protein binding to a nucleic acid, comprising:
(a) contacting the SpyCatcher polypeptide of claim 11 with a protein of interest covalently linked to a SpyTag polypeptide or a SpyTag-SNAC tag polypeptide and a population of nucleic acids under conditions sufficient for the protein of interest to bind to the nucleic acids and the SpyCatcher polypeptide and the SpyTag polypeptide or SpyTag-SNAC tag polypeptide to bind to form a SpyCatcher-SpyTag-protein-nucleic acid complex or a SpyCatcher-SpyTag-SNAC tag-protein-nucleic acid complex; (b) incubating the SpyCatcher-SpyTag-protein-nucleic acid complex or the SpyCatcher-SpyTag-SNAC tag-protein-nucleic acid complex under conditions sufficient to activate the Tn5 transposase; (c) performing one or more washes of the complex; (d) dissociating the SpyCatcher-SpyTag-protein or the SpyCatcher-SpyTag-SNAC tag-protein from the nucleic acid; and (e) isolating the nucleic acid.

26 . The method of claim 25 , the contacting the SpyCatcher polypeptide with the protein of interest covalently linked to the SpyTag polypeptide or the SpyTag-SNAC tag polypeptide is performed in:
(i) a permeabilized cell expressing the protein of

Assignees

• Univ Oregon

Inventors

• Mcknight Jeffrey

Classifications

• C07K1/13

  Labelling of peptides · CPC title

• C07K1/107

  by chemical modification of precursor peptides · CPC title

• C07K14/315

  from Streptococcus (G), e.g. Enterococci · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title