Development of a positive genetic selection system for inhibition of protein splicing using mycobacterial inteins in Escherichia coli DNA gyrase subunit A

An intein-based positive genetic selection system was developed to study protein splicing and to provide a selection system with the potential for finding splicing inhibitors. Inteins can be novel antimicrobial targets when present in essential proteins since blocking splicing would kill the organism. For example, pathogenic mycobacteria encode inteins that interrupt DNA gyrase. The gyrase selection system exploits (1) splicing of inteins out of Gyrase A and (2) the dominant lethal effect of quinolone poisoning of DNA gyrase, which in turn blocks replication. The system was adapted for whole-cell high-throughput screening using green fluorescent protein as an automatable readout of viability. To demonstrate the efficacy of this system, mutations that blocked splicing of the Mycobacterium xenopi Gyrase A intein were isolated. Splicing was then assayed at a second temperature to identify inteins with a temperature-sensitive splicing phenotype. Mutations were mapped onto a structure-based sequence alignment, which led to the rational prediction of a temperature-sensitive splicing mutation. GyrA intein subdomain relationships also provided insight into intein evolution.     

InBase: the Intein Database

Inteins are self-catalytic protein splicing elements. InBase (http://www.neb.com/neb/inteins.html), the Intein Database and Registry, is a curated compilation of published and unpublished information about protein splicing. It presents general information as well as detailed data for each intein, including tabulated comparisons and a comprehensive bibliography. An intein-specific BLAST server is now available to assist in identifying new inteins.     

InBase, the Intein Database

InBase, the Intein Database (http://www.neb.com/neb/inteins.html ), is a comprehensive on-line resource that includes the Intein Registry. Inteins are protein splicing elements that mediate a self-catalytic protein splicing reaction. InBase presents general information as well as detailed data for each intein, including tabu-lated comparisons and a comprehensive bibliography.     

Sequential formation of two branched intermediates during protein splicing of class three inteins

Extremophiles - Inteins are the protein equivalent of introns. They are seamlessly removed during post-translational maturation of their host protein (extein). Inteins from extremophiles played a...