IP Library › Granted Patent US 12,703,722
Granted Patent B2
US 12,703,722 · App. 17/782,366 · Granted Aug 11, 2026

Poptag peptide and uses thereof

Inventors: Keren Lasker (Palo Alto, CA); Steven Boeynaems (Stanford, CA); Aaron David Gitler (Foster City, CA); Lucy Shapiro (Palo Alto, CA)
Assignees: CZ Biohub SF, LLC; The Board of Trustees of the Leland Stanford Junior University
C07K14/195C12N15/62C07K2317/622C07K2319/735
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Quick Facts
Patent No.
US 12,703,722
App. No.
17/782,366
Filed
Jun 3, 2022
Granted
Aug 11, 2026
Kind
B2
Art Unit
1672
USPC
424/185.1
Abstract

Proteins and fusion proteins for forming membraneless 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).

Claims (26)

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 is a single domain antibody or a single-chain variable fragment (scfv) or comprises an immunoglobulin heavy chain variable region.

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

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

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

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

9 . The polynucleotide of claim 8 , comprising a promoter operably linked to the nucleic acid sequence.

10 . An in vitro or ex vivo cell comprising a polynucleotide encoding the fusion protein of claim 1 , wherein the cell expresses the fusion protein.

11 . The cell of claim 10 , wherein the cell is a eukaryotic cell.

12 . The cell of claim 11 , wherein the eukaryotic cell is a mammalian cell.

13 . The cell of claim 10 , wherein the cell comprises:

a. a first polynucleotide encoding a first fusion protein comprising SEQ ID NO:1 and a first heterologous amino acid sequence and;

b. a second polypeptide encoding a second fusion protein comprising SEQ ID NO:1 and a second heterologous amino acid sequence, wherein the first heterologous amino acid sequence and first heterologous amino acid sequence are different heterologous amino acid sequences.

14 . The cell of claim 13 , wherein the different heterologous amino acid sequences are different enzymes.

15 . 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, wherein the compartments are membraneless synthetic organelles;

lysing the cell; and

isolating the compartments from cell lysate material, wherein the compartments comprise the product, thereby purifying the product from the cell.

16 . The method of claim 15 , wherein the product is formed by performing a reaction in the compartments.

17 . The method of claim 16 , wherein the amino acid sequence comprises an enzyme and the enzyme catalyzes production of the product.

18 . The method of claim 15 , 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.

19 . The method of claim 15 , comprising a step of performing a reaction in the compartments to form the product after the expressing and before the lysing.

20 . The cell of claim 10 , wherein the polypeptide sequence comprises SEQ ID NO:1.

21 . The fusion protein of claim 1 , wherein the polypeptide sequence comprises SEQ ID NO:1.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 30, 2026
From: CHAN ZUCKERBERG BIOHUB, INC.
To: CZ BIOHUB SF, LLC
Reel/Frame 075133/0789 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 22, 2022
From: THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
To: CHAN ZUCKERBERG BIOHUB, INC.; THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
Reel/Frame 060271/0499 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 3, 2022
From: LASKER, KEREN; BOEYNAEMS, STEVEN; GITLER, AARON DAVID; SHAPIRO, LUCY
To: THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
Reel/Frame 060097/0079 →
Continuity (2)
Provisional Application 62944936 · Dec 6, 2019
Related Publication 20230044825A1 · Feb 9, 2023
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