Article ID Journal Published Year Pages File Type
1178336 Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2007 7 Pages PDF
Abstract

As the Cne PRP8 intein is active and exists in an essential gene of an important fungal pathogen, inhibitors of splicing and assays for intein activity are of interest. The self-splicing activity of Cne PRP8, the intein from the Prp8 gene of Cryptococcus neoformans, was assessed in different heterologous fusion proteins expressed in Escherichia coli. Placement of a putatively inactive variant of the intein adjacent to the α-complementation peptide abolished the peptide's ability to restore β-galactosidase activity, while an active variant allowed complementation. This α-complementation peptide therefore provides a facile assay of splicing which can be used to test potential inhibitors. When placed between two heterologous protein domains, splicing was impaired by a β-branched amino acid immediately preceding the intein, while splicing occurred only with a hydroxyl or thiol immediately following the intein. Both these assays sensitively report impairment of splicing and provide information on how context constrains the splicing ability of Cne PRP8.

Related Topics
Physical Sciences and Engineering Chemistry Analytical Chemistry
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