An in vivo screening system against protein splicing useful for the isolation of non-splicing mutants or inhibitors of the RecA intein of Mycobacterium tuberculosis.
Protein splicing involves the self-catalyzed excision of an intervening sequence, the intein, from a precursor protein, with the concomitant ligation of the flanking extein sequences to yield a new polypeptide. The ability of inteins to promote protein splicing even when inserted into a foreign context has facilitated the study of the modulation of protein splicing. In this paper, we describe an in vivo screening system for the isolation of mutations or inhibitors that interfere with protein splicing mediated by the RecA intein of Mycobacterium tuberculosis. It involves the activation of the cytotoxic CcdB protein by protein splicing, such that host cells survive in the presence of inducer only when protein splicing is blocked. The coding sequence for the RecA intein was inserted in-frame into the polylinker region of an inducible lacZ alpha-ccdB fusion vector, leading to inactivation of the CcdB toxin unless the intein is excised by protein splicing. Depending on the objective of the screening procedure, its stringency can be modified by altering the level of expression of the intein-CcdB fusion protein. To induce large amounts of CcdB fusion proteins, the fusion protein is expressed from a high-copy-number plasmid. Such a screening system detects even low levels of protein splicing and we have used it to show that protein splicing of the RecA intein is compatible with any amino acid in the extein position adjacent to the N-terminal splice junction. In order to search for protein splicing inhibitors, which may attenuate protein splicing by less than an order of magnitude, we have also constructed a low-copy-number intein-CcdB plasmid so that the host cells can survive when splicing of the expressed CcdB fusion protein is only moderately suppressed. We anticipate that the CcdB-based in vivo screening system will find uses in the analysis of structural and mechanistic aspects of protein splicing.
Lew
Belinda M
BM
Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA.
Paulus
Henry
H
D000596:Amino Acids / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D019154:Protein Splicing / Q000235:genetics*; D011938:Rec A Recombinases / Q000235:genetics*
D000596:Amino Acids; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D007544:Isopropyl Thiogalactoside; D011938:Rec A Recombinases