Screening and use of reagents which block or activate intein splicing utilizing natural or homologous exteins

Abstract

In accordance with the present invention, there are provided selection systems and methods for screening for agents that control splicing of inteins in their native host protein (extein) or in homologous exteins. Specifically, there are provided positive genetic selection systems for the screening of agents which inhibit or activate protein splicing which comprise: a host cell containing a chromosomal gene encoding either a drug-resistant form of a target enzyme or a wild-type target enzyme, and a plasmid-borne gene encoding either a drug-sensitive form of the target enzyme, which is dominantly cytotoxic upon interaction with the drug, or a dominantly cytotoxic form of the target enzyme. In these systems the plasmid-borne gene contains an intein, and the inhibition or activation of splicing of the dominant cytotoxic form of the target enzyme by a given reagent results in the survival or death of the host cell. More specifically, positive genetic selection systems that utilize the M. xenopi GyrA intein or M. tuberculosis DnaB helicase intein are provided. Similar reporter systems utilizing native or homologous exteins and systems utilizing controllable inteins are provided, as are methods of controlling in vivo expression of proteins by modulating protein splicing with inhibiting or activating agents, and methods of controlling the delivery of proteinaceous drugs in vivo by modulating protein splicing.

Description

RELATED APPLICATIONS [0001] This application is a Continuation of application Ser. No. 10 / 324,023 filed Dec. 18, 2002, which is a Divisional of application Ser. No. 09 / 430,221 filed Oct. 29, 1999, now U.S. Pat. No. 6,521,425, issued Feb. 18, 2003, all of which are herein incorporated by reference.BACKGROUND OF THE INVENTION [0002] The present invention relates to the screening for and use of Agents that either inhibit or activate protein splicing of inteins (IVPS). Specifically, disclosed herein is the development of two specific reporter systems for Gyrase A and DnaB inteins. Agents screened for in accordance with the present invention can be used to control protein splicing for any purpose, in vivo or in vitro, including antimicrobial activity of organisms containing inteins in essential genes. More specifically, the present invention relates to the use of inteins expressed in modified or unmodified native protein splicing precursors or homologous extein systems to screen for muta...

AI Technical Summary

Benefits of technology

The present invention provides selection systems and methods for selecting or screening for mutations or agents that control the splicing of inteins. These systems use the native host protein or a homologous extein in any host organism to identify inhibitors or activators of protein splicing. The invention also provides methods for controlling in vivo expression of proteins and delivering proteinaceous drugs in vivo by modulating protein splicing. The "agent" includes peptides, peptide derivatives, natural products, or synthetic molecules.

Problems solved by technology

While a general method of screening for antimicrobial agents using the M. tuberculosis RecA intein in a thymidylate synthetase (TS) reporter system has been described (Belfort, U.S. Pat. No. 5,795,731, issued Aug. 18, 1998), this system suffers from several limitations.

While not wishing to be bound by theory, it is believed that such inefficient protein splicing in the foreign extein context occurs because the flanking extein is, in effect, the substrate of the intein.

The substrate specificity of the intein limits acceptable extein sequences, hence the native extein sequence is the optimal substrate, whereas foreign extein sequences may not be acceptable substrates at all.

Additionally, exteins may affect the packing at the intein active site, or global folding of the intein and / or precursor, hence the use of a foreign extein may result in improper folding of the intein or precursor and inefficient or no splicing.

Moreover, expression of an extein gene that naturally contains an intein in a foreign host, for example E. coli or yeast, may not be efficient (Perler et al.

Images