Poptag peptide and uses thereof

Patent metadata

Field	Value
Publication number	US-2023044825-A1
Application number	US-202017782366-A
Country	US
Kind code	A1
Filing date	Dec 4, 2020
Priority date	Dec 6, 2019
Publication date	Feb 9, 2023
Grant date	—

Abstract

Proteins and fusion proteins for forming merbraneless droplets in cells are provided. Described herein, is the development of a protein, named PopTag, that drives phase separation when it is part of a chimeric fusion protein. PopTag is engineered from the PopZ protein, found in a-proteobacteria (including Caulobacter crescentus ). Despite PopZ being exclusively found in this clade of bacteria, the PopTag can drive protein phase separation in other prokaryotes (e.g., E. coli ) and eukaryotes (e.g., human cells).

First claim

1 . A fusion protein comprising an amino acid sequence linked to a polypeptide sequence comprising SEQ ID NO: 1, or a variant thereof as set forth in Table 1, wherein the amino acid sequence is heterologous to the polypeptide sequence.

2 . The fusion protein of claim 1 , wherein the polypeptide sequence is at least 95% identical to SEQ ID NO: 1.

3 . The fusion protein of claim 1 , wherein the amino acid sequence is an epitope-binding polypeptide.

4 . The fusion protein of claim 3 , wherein the epitope-binding polypeptide comprises an immunoglobulin heavy chain variable region.

5 . The fusion protein of claim 4 , wherein the epitope-binding polypeptide is a single domain antibody or a single-chain variable fragment (scfv).

6 . The fusion protein of claim 1 , wherein the amino acid sequence is a target-binding polypeptide.

7 . The fusion protein of claim 1 , wherein the amino acid sequence comprises a fluorescent protein.

8 . The fusion protein of claim 1 , wherein the amino acid sequence comprises an enzyme.

9 . A polynucleotide comprising a nucleic acid sequence that encodes the fusion protein of claim 1 .

10 . The polynucleotide of claim 9 , comprising a promoter operably linked to the nucleic acid sequence.

11 . (canceled)

12 . A cell comprising a polynucleotide encoding the fusion protein of claim 1 , wherein the cell expresses the fusion protein.

13 . The cell of claim 12 , wherein the cell is a eukaryotic cell.

14 . The cell of claim 13 , wherein the eukaryotic cell is a mammalian cell.

15 . The cell of claim 13 , wherein the eukaryotic cell is a plant or yeast cell.

16 . The cell of claim 12 , wherein the cell comprises:
a. a first polynucleotide encoding a first fusion protein and; b. a second polypeptide encoding a second fusion protein, wherein the first fusion protein and the second fusion protein comprise a polypeptide sequence comprising SEQ ID NO: 1 or a variant thereof as set forth in Table 1 and comprise different heterologous amino acid sequences.

17 . The cell of claim 16 , wherein the different heterologous amino acid sequences are different enzymes.

18 . A method of purifying a product from a cell, the method comprising,
expressing in the cell the fusion protein of claim 1 , wherein the fusion protein forms compartments in the cell; optionally performing a reaction in the compartments to form the product; lysing the cell; and isolating the compartments from cell lysate material, wherein the compartments comprise the product, thereby purifying the product from the cell.

19 . The method of claim 18 , wherein the product is formed by performing a product in the compartments.

20 . The method of claim 19 , wherein the amino acid sequence comprises an enzyme and the enzyme catalyzes production of the product.

21 . The method of claim 18 , wherein the cell produces the product and the amino acid sequence comprises a binding polypeptide that binds the product, thereby binding the product to the compartment.

22 - 23 . (canceled)

Assignees

• Chan Zuckerberg Biohub Inc
• The Board Of Trustees Of The Leland Stanford Junior Univesity

Inventors

• Lasker Keren
• Boeynaems Steven
• Gitler Aaron David
• Shapiro Lucy

Classifications

• C07K2319/735

  containing a domain for self-assembly, e.g. a viral coat protein (includes phage display) · CPC title

• C07K14/195 Primary

  from bacteria · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title