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Conditional protein splicing via disulfide bond formation
Author(s) -
Nicastri Michael C.,
Xega Kristina,
Reitter Julie N.,
Mills Kenneth V.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.789.3
Subject(s) - intein , protein splicing , rna splicing , dithiothreitol , mutagenesis , chemistry , biochemistry , disulfide bond , in vitro , cleavage (geology) , microbiology and biotechnology , genetics , biology , gene , mutation , enzyme , rna , paleontology , fracture (geology)
Inteins are intervening polypeptides that self‐catalyze their own excision from the flanking polypeptides, or exteins, in a process called protein splicing. An intein interrupting the DNA PolII in Methanoculleus marisnigri (Mma) can facilitate protein splicing when over‐expressed in E. coli. However, when overexpressed in gor– E. coli , we isolate mostly unspliced precursor. This precursor can be induced to undergo N‐terminal cleavage upon in vitro incubation with 1,4‐dithiothreitol. Unusual migration on SDS‐PAGE, resolvable by reduction, suggests the presence of a disulfide bond in the unspliced precursor. Mutagenesis analysis indicates that the Cys residues involved in the bond are within the intein, suggesting that this inducible intein may be able to regulate the expression of foreign flanking exteins. This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.