The Ohio State University

Roland Saldanha

Alan M Lambowitz

Georg Mohr

Manabu Matsuura

Steven Zimmerly

Huatao Guo

Clifford James Beall

Jiam Yang

Methods for preparing nucleotide integrases are provided. The nucleotide integrases are prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as 'exogenous RNA', with a group II intron-encoded protein. The exogenous RNA is prepared by in vitro transcription of a DNA molecule which comprises a group II intron sequence. In one embodiment, the group II intron-encoded protein is made by introducing into a host cell a DNA molecule that comprises at least the open reading frame sequence of a group II intron and then expressing the open reading frame sequence in the host cell. The DNA molecule may comprise the open reading frame sequence operably linked to a promoter, preferably an inducible promoter. Thereafter, the cell is fractionated and the protein is recovered and combined in vitro with the exogenous RNA to provide RNP particles having nucleotide integrase activity. In another embodiment, the DNA molecule comprise a group II intron sequence that encodes both a group II intron RNA as well as a group II intron encoded protein. The DNA molecule is then expressed in the host cell to provide RNP particles that comprise the group II intron-encoded protein bound to the group II intron RNA. Thereafter, the RNP particles comprising the group II intron-encoded protein and the group II intron RNA are isolated from the cell and treated with a nuclease to remove the RNA and to provide the group II-intron encoded protein. The group II intron-encoded protein is then combined in vitro with the exogenous RNA to provide RNP particles having nucleotide integrase activity.

Methods of making an RNP particle having nucleotide integrase activity

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.

Methods for preparing nucleotide integrases

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Columbus, OH, United States of America

Roland Saldanha