Poptag peptide and uses thereof
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).
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.