Patent ID: 8852911

Claim:
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.