Opsin polypeptides and methods of use thereof
The present disclosure provides opsins, including variant opsins with increased activity and/or increased trafficking to the plasma membrane. The opsins are useful in therapeutic and screening applications, which are also provided.
1. An isolated fusion polypeptide comprising:
a) an opsin polypeptide comprising an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31;
b) an endoplasmic reticulum (ER) export signal; and
c) a membrane trafficking signal.
2. The fusion polypeptide of claim 1 , wherein the ER export signal comprises the amino acid sequence VXXSL, where X is any amino acid; or FXYENE (SEQ ID NO: 4), where X is any amino acid.
3. The fusion polypeptide of claim 1 , wherein the polypeptide further comprises a fluorescent protein disposed between (a) and (b).
4. The fusion polypeptide of claim 1 , wherein the membrane trafficking signal comprises an amino acid sequence having at least 90% amino acid sequence identity to an amino acid sequence selected from:
MDYGGALSAVGRELLFVTNPVVVNGS;
(SEQ ID NO: 6)
MAGHSNSMALFSFSLLWLCSGVLGTEF;
(SEQ ID NO: 7)
MGLRALMLWLLAAAGLVRESLQG;
(SEQ ID NO: 8)
MRGTPLLLVVSLFSLLQD;
(SEQ ID NO: 9)
and
KSRITSEGEYIPLDQIDINV.
(SEQ ID NO: 10)
5. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) an opsin polypeptide comprising an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31;
b) a membrane trafficking signal comprising the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 10); and
c) an ER export signal comprising the amino acid sequence FCYENEV (SEQ ID NO:5).
6. An isolated variant opsin polypeptide comprising, in order from amino terminus to carboxyl terminus: a) an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31; and b) an endoplasmic reticulum (ER) export signal.
7. An isolated polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide of claim 1 .
8. The polynucleotide of claim 7 , wherein the fusion polypeptide-encoding nucleotide sequence is operably linked to a promoter that provides for neuron-selective expression.
9. A recombinant expression vector comprising the isolated polynucleotide of claim 7 .
10. A method of modulating the voltage potential of a cell in response to a light stimulus, the method comprising exposing a cell that is genetically modified with the recombinant expression vector of claim 9 to a light stimulus, wherein the encoded variant opsin is expressed in the cell, and wherein in response to exposure to a light stimulus, the voltage potential of the cell is modulated.
11. The fusion polypeptide of claim 1 , wherein the opsin polypeptide comprises an amino acid sequence having at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31.
12. The fusion polypeptide of claim 1 , wherein the opsin polypeptide comprises the amino acid sequence set forth in SEQ ID NO:31.
13. The fusion polypeptide of claim 1 , wherein the ER export signal comprises the amino acid sequence VKESL (SEQ ID NO: 1), VLGSL (SEQ ID NO: 2), or FCYENE (SEQ ID NO: 5).
14. The fusion polypeptide of claim 1 , wherein the membrane trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:10).
15. The polynucleotide of claim 7 , wherein the nucleotide sequence is codon optimized for expression in a mammalian cell.
16. The polynucleotide of claim 7 , wherein the nucleotide sequence is operably linked to a promoter.
17. The recombinant expression vector of claim 9 , wherein the recombinant vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
18. A genetically modified cell comprising the polynucleotide of claim 7 .
19. A genetically modified cell comprising the recombinant vector of claim 9 .
20. The genetically modified cell of claim 19 , wherein the cell is a mammalian cell.
21. The genetically modified cell of claim 20 , wherein the cell is a neuron.