Method of producing dicer
View Patent ↗The present disclosure provides a method for producing a Dicer polypeptide in a prokaryotic host cell. The present disclosure further provides a purified Dicer complex. The present disclosure further provides kits for producing a Dicer polypeptide in a prokaryotic host cell.
1. A method of producing a Dicer polypeptide in a prokaryotic host cell, the method comprising:
a) expressing a first polypeptide in a first prokaryotic host cell, wherein the first polypeptide comprises an amino acid sequence having at least 85% sequence identity to:
(i) amino acids 1-1008 of the amino acid sequence of SEQ ID NO: 1 comprising an ATPase/helicase domain, a DUF domain, and a PAZ domain,
(ii) amino acids 1-1068 of the amino acid sequence of SEQ ID NO: 1 comprising an ATPase/helicase domain, a DUF domain, and a PAZ domain,
(iii) amino acids 605-1008 of the amino acid sequence of SEQ ID NO: 1 comprising a DUF and a PAZ domain,
(iv) amino acids 605-1068 of the amino acid sequence of SEQ ID NO: 1 comprising a DUF and a PAZ domain,
(v) amino acids 886-1008 of the amino acid sequence of SEQ ID NO: 1 comprising a PAZ domain, or
(vi) amino acids 886-1068 of the amino acid sequence of SEQ ID NO: 1 comprising a PAZ domain, and wherein said first polypeptide lacks an RNase IIIa domain and an RNase IIIb domain; and
b) expressing a second polypeptide in the first prokaryotic host cell or in a second prokaryotic host cell, wherein the second polypeptide comprises an amino acid sequence having at least 85% sequence identity to:
(i) amino acids 1235 to 1922 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa domain, and RNaseIIIb domain, and a dsRBD domain),
(ii) amino acids 1296 to 1922 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa domain, an RNaseIIIb domain, and dsRBD domain),
(iii) amino acids 1235 to 1772 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa and RNaseIIIb domain, or
(iv) amino acids 1296 to 1772 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa and an RNaseIIIb domain, and wherein said second polypeptide lacks at least one of: a DUF domain and a PAZ domain, wherein said first and second polypeptides spontaneously associate to form an enzymatically active Dicer complex that has endoribonuclease activity.
2. The method of claim 1 , wherein the second polypeptide is expressed in the first prokaryotic host cell.
3. The method of claim 1 , wherein the second polypeptide is expressed in the second prokaryotic host cell.
4. The method of claim 1 , further comprising purifying the Dicer complex from the first and/or the second prokaryotic host cell.
5. The method of claim 1 , wherein the prokaryotic host cell is Escherichia coli.
6. The method of claim 1 , wherein the first polypeptide lacks amino acids 1-604 of the amino acid sequence of SEQ ID NO: 1 (a DExD/H-box domain).
7. A genetically modified prokaryotic host cell, wherein the prokaryotic host cell is genetically modified with a first recombinant expression vector comprising a nucleotide sequence encoding a first polypeptide, wherein said first polypeptide comprises an amino acid sequence having at least 85% sequence identity to:
(i) amino acids 1-1008 of the amino acid sequence of SEQ ID NO: 1 comprising an ATPase/helicase domain, a DUF domain, and a PAZ domain,
(ii) amino acids 1-1068 of the amino acid sequence of SEQ ID NO: 1 comprising an ATPase/helicase domain, a DUF domain, and a PAZ domain,
(iii) amino acids 605-1008 of the amino acid sequence of SEQ ID NO: 1 comprising a DUF and a PAZ domain,
(iv) amino acids 605-1068 of the amino acid sequence of SEQ ID NO: 1 comprising a DUF and a PAZ domain,
(v) amino acids 886-1008 of the amino acid sequence of SEQ ID NO: 1 comprising a PAZ domain, or
(vi) amino acids 886-1068 of the amino acid sequence of SEQ ID NO: 1 comprising a PAZ domain, and wherein said first polypeptide lacks an RNase IIIa domain and an RNase IIIb domain.
8. The genetically modified prokaryotic host cell of claim 7 , wherein the prokaryotic host cell is further genetically modified with a second recombinant expression vector comprising a nucleotide sequence encoding a second polypeptide, wherein said second polypeptide comprises an amino acid sequence having at least 85% sequence identity to:
(i) amino acids 1235 to 1922 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa domain, and RNaseIIIb domain, and a dsRBD domain,
(ii) amino acids 1296 to 1922 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa domain, an RNaseIIIb domain, and dsRBD domain,
(iii) amino acids 1235 to 1772 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa and RNaseIIIb domain, or
(iv) amino acids 1296 to 1772 of the amino acid sequence of SEQ ID NO: 1 an RNaseIIIa and an RNaseIIIb domain, and wherein said second polypeptide lacks at least one of: a DUF domain and a PAZ domain, wherein said first and second polypeptides spontaneously associate to form an enzymatically active Dicer complex.
9. A genetically modified prokaryotic host cell, wherein the prokaryotic host cell is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding a second polypeptide, wherein said second polypeptide comprises an amino acid sequence having at least 85% sequence identity to:
(i) amino acids 1235 to 1922 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa domain, and RNaseIIIb domain, and a dsRBD domain,
(ii) amino acids 1296 to 1922 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa domain, an RNaseIIIb domain, and dsRBD domain,
(iii) amino acids 1235 to 1772 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa and RNaseIIIb domain, or
(iv) amino acids 1296 to 1772 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa and an RNaseIIIb domain, and wherein said second polypeptide lacks at least one of: a DUF domain and a PAZ domain.
10. A kit comprising:
a) a first recombinant expression vector comprising a nucleotide sequence encoding a first polypeptide, wherein said first polypeptide comprises an amino acid sequence having at least 85% sequence identity to:
(i) amino acids 1-1008 of the amino acid sequence of SEQ ID NO: 1 comprising an ATPase/helicase domain, a DUF domain, and a PAZ domain,
(ii) amino acids 1-1068 of the amino acid sequence of SEQ ID NO: 1 comprising an ATPase/helicase domain, a DUF domain, and a PAZ domain,
(iii) amino acids 605-1008 of the amino acid sequence of SEQ ID NO: 1 comprising a DUF and a PAZ domain,
(iv) amino acids 605-1068 of the amino acid sequence of SEQ ID NO: 1 comprising a DUF and a PAZ domain,
(v) amino acids 886-1008 of the amino acid sequence of SEQ ID NO: 1 comprising a PAZ domain, or
(vi) amino acids 886-1068 of the amino acid sequence of SEQ ID NO: 1 comprising a PAZ domain, and wherein said first polypeptide lacks an RNase IIIa domain and an RNase IIIb domain; and
b) a second recombinant expression vector comprising a nucleotide sequence encoding a second polypeptide, wherein said second polypeptide comprises amino acid sequence having at least 85% sequence identity to:
(i) amino acids 1235 to 1922 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa domain, and RNaseIIIb domain, and a dsRBD domain,
(ii) amino acids 1296 to 1922 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa domain, an RNaseIIIb domain, and dsRBD domain,
(iii) amino acids 1235 to 1772 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa and RNaseIIIb domain, or
(iv) amino acids 1296 to 1772 of the amino acid sequence of SEQ ID NO: 1 comprising an RNaseIIIa and an RNaseIIIb domain, and wherein said second polypeptide lacks at least one of: a DUF domain and a PAZ domain.
11. The kit of claim 10 , wherein the first polypeptide lacks amino acids 1-604 of the amino acid sequence of SEQ ID NO: 1 (a DExD/H-box domain).
12. The method of claim 1 , wherein the second polypeptide lacks a double-stranded RNA binding domain.
13. The method of claim 1 , wherein the second polypeptide lacks a DUF domain and a PAZ domain.
14. The genetically modified prokaryotic host cell of claim 8 , wherein the second polypeptide lacks a DUF domain and a PAZ domain.
15. The genetically modified prokaryotic host cell of claim 9 , wherein the second polypeptide lacks a DUF domain and a PAZ domain.
16. The kit of claim 10 , wherein the second polypeptide lacks a DUF domain and a PAZ domain.
17. The method of claim 1 , wherein at least one of the first and second polypeptides further comprises a heterologous polypeptide that provides for a detectable signal and/or facilitates protein purification or isolation.
18. The genetically modified prokaryotic host cell of claim 7 , wherein the first polypeptide further comprises a heterologous polypeptide that provides for a detectable signal and/or facilitates protein purification or isolation.
19. The genetically modified prokaryotic host cell of claim 8 , wherein at least one of the first and second polypeptides further comprises a heterologous polypeptide that provides for a detectable signal and/or facilitates protein purification or isolation.
20. The genetically modified prokaryotic host cell of claim 9 , wherein the second polypeptide further comprises a heterologous polypeptide that provides for a detectable signal and/or facilitates protein purification or isolation.
21. The kit of claim 10 , wherein at least one of the first and second polypeptides further comprises a heterologous polypeptide that provides for a detectable signal and/or facilitates protein purification or isolation.