Claims for Patent: 5,804,418

Summary for Patent: 5,804,418

Title:	Methods for preparing nucleotide integrases
Abstract:	The present invention provides new, improved, and easily manipulable methods for making nucleotide integrases. In one embodiment, the nucleotide integrase is prepared by introducing a DNA molecule which comprises a group II intron DNA sequence into a host cell. The group II intron DNA sequence is then expressed in the host cell such that RNP particles having nucleotide integrase activity are formed in the cell. Such RNP particles comprise an exiced group II intron RNA encoded by the introduced DNA molecule and a group II intron-encoded protein encoded by the introduced DNA molecule. Thereafter, the nucleotide integrase is isolated from the cell. In another embodiment, the nucleotide integrase is prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as \"exogenous RNA\", with a group II intron-encoded protein. In another embodiment, the nucleotide integrase is prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as \"exogenous RNA\", with an RNA-protein complex which comprises a group II intron-encoded protein. Preferably, the exogenous RNA is prepared by in vitro transcription of a DNA molecule which comprises the group II intron sequence. Preferably, the group II intron-encoded protein is made by introducing into a host cell a DNA molecule which comprises the open reading frame sequence of a group II intron, and then expressing the the open reading sequence in the host cell such that the group II intron-encoded protein encoded by the open reading frame sequence is formed in the cell. Preferably, the RNA-protein complex is made by introducing into a host cell a DNA molecule comprising a group II intron DNA sequence which encodes a splicing-defective group II intron RNA. The present invention also relates to a nucleotide integrase and an improved method for making RNA-protein complexes for use in preparing nucleotide integrases in vitro.
Inventor(s):	Lambowitz; Alan Marc (Columbus, OH), Mohr; Georg (Columbus, OH), Saldanha; Roland (Columbus, OH), Matsuura; Manabu (Columbus, OH)
Assignee:	The Ohio State University Research Foundation (Columbus, OH)
Application Number:	08/752,238
Patent Claims:	1. A method for preparing a nucleotide integrase which cleaves a double-stranded DNA substrate, said method comprising the following steps: (a) providing a DNA molecule comprising a group II intron DNA sequence, wherein the group II intron DNA sequence encodes a group II intron RNA and comprises an open reading frame sequence which encodes a group II intron-encoded protein; (b) introducing the DNA molecule into a host cell; (c) expressing the group II intron DNA sequence in the host cell, to provide an excised group II intron RNA and a group II intron-encoded protein molecule, wherein the protein and the RNA combine in the host cell to form the nucleotide integrase; (d) obtaining the nucleotide integrase of step (c) from the host cell. 2. The method of claim 1 wherein the DNA molecule further comprises a promoter operably linked to the group II intron DNA sequence. 3. The method of claim 2 wherein the promoter is an inducible promoter. 4. The method of claim 2 wherein the DNA molecule further comprises a nucleotide sequence which encodes a tag for facilitating isolation of the nucleotide integrase from the host cell; and wherein the method further comprises expressing the nucleotide sequence which encodes the tag in the host cell to provide a tagged group II intron-encoded protein; and wherein step (d) involves employing the tag to recover the nucleotide integrase. 5. The method of claim 4 wherein the sequence which encodes the tag is located at the 5' end or the 3' end of the open reading frame sequence of the group II intron DNA sequence. 6. The method of claim 2 further comprising the steps of: introducing a DNA sequence encoding at tRNA which corresponds to the genetic code of the group II intron DNA sequence into the host cell before step (b) and expressing the tRNA-encoding DNA sequence in the host cell. 7. The method of claim 1 wherein the DNA molecule is prepared by the following steps of: preparing a synthetic group II intron DNA sequence; wherein the group II intron DNA sequence comprises a sequence of nucleotides that bind to the recognition site of the substrate DNA; and incorporating the synthetic group II intron DNA sequence into the DNA molecule. 8. The method of claim 1 wherein the group II intron DNA sequence comprises the DNA sequence of the Ll.ltrB intron and the RNP particles comprise an excised Ll.ltrB intron RNA and an ltra protein. 9. The method of claim 1 wherein the group II intron DNA sequence comprises a modified DNA sequence of the Ll.ltrB intron and the RNP particles comprise a modified excised Ll.ltrB intron RNA and an ltra protein molecule. 10. The method of claim 1 wherein the group II inton DNA sequence comprises a modified DNA sequence of the Ll.ltrB intron and the RNP particles comprise a modified excised Ll.ltrB intron RNA and a modified ltra protein molecule. 11. The method of claim 1 wherein the host cell is E. coli. 12. The method of claim 8 wherein the host cell is E. coli. 13. A method of preparing a nucleotide integrase in vitro comprising the steps of: (a) providing an isolated, excised, group II intron RNA; (b) providing an isolated group II intron-encoded protein; and (c) incubating the excised, group II intron RNA with the group II intron-encoded protein for a sufficient time to form a nucleotide integrase comprising the excised, group II intron RNA bound to the group II intron-encoded protein. 14. The method of claim 13 wherein the group II intron-encoded protein is produced by a process comprising the steps of: (a) providing a DNA molecule comprising an open reading frame sequence of a group II intron, said open reading frame sequence being operably linked to a promoter; (b) introducing the DNA molecule of step (a) into a host cell; (c) expressing the open reading frame sequence in the host cell to provide the group II intron-encoded protein; and (d) isolating the group II intron-encoded protein from the host cell. 15. The method of claim 13 wherein the DNA molecule further comprises a sequence which encodes a tag that facilitates isolation of the group II intron-encoded protein from the host cell; and wherein the method further comprises expressing the nucleotide sequence which encodes the tag in the host cell to provide a tagged group II intron-encoded protein; and wherein step (d) involves obtaining a tagged nucleotide integrase from the host cell. 16. The method of claim 15 wherein the sequence which encodes the tag is located at a position selected from the 5' end and the 3' end of the open reading frame sequence. 17. The method of claim 13 wherein the open reading frame sequence encodes the ltrA protein and wherein the excised, group II RNA is selected from the group consisting of an unmodified, excised Ll.ltrB intron RNA and a modified, excised Ll.ltrB intron RNA. 18. A method of preparing a nucleotide integrase in vitro comprising the steps of: (a) providing an exogenous RNA which comprises an excised group II intron RNA; (b) providing an RNA-protein complex, wherein the RNA-protein complex comprises a protein having an amino acid sequence encoded by a group II intron and RNA that is free of excised, group II intron RNA molecules having a sequence that encodes said protein; said RNA-protein complex being prepared by the following steps: (i) providing an isolated DNA molecule comprising a group II intron DNA sequence, wherein said group II intron DNA sequence encodes a group II intron-encoded protein and a splicing defective group II RNA (ii) introducing the DNA molecule into a host cell; (iii) expressing the mutated group II intron DNA sequence in the host cell, wherein an RNA-protein complex comprising the group II intron-encoded protein and the splicing-defective group II RNA are formed in the cell (iv) obtaining the RNA-protein complex of step (iii) from the host cell; and (c) incubating the exogenous RNA of step (a) with the RNP particle preparation for a sufficent time to form a nucleotide integrase comprising the excised group II RNA and the protein having an amino acid sequence encoded by a group II intron. 19. An isolated nucleotide integrase comprising an excised Ll.ltrB intron RNA and an ltra protein molecule.

Details for Patent 5,804,418

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26