Traceless protein splicing utilizing evolved split inteins.
Proc Natl Acad Sci U S A
; 106(27): 10999-1004, 2009 Jul 07.
Article
em En
| MEDLINE
| ID: mdl-19541616
ABSTRACT
Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins. Ideally, inteins would seamlessly splice polypeptides together with no remnant sequences and at high efficiency. Here, we describe experiments that identify the branched intermediate, a transient step in the overall splicing reaction, as a key determinant of the splicing efficiency at different splice-site junctions. To alter intein specificity, we developed a cell-based selection scheme to evolve split inteins that splice with high efficiency at different splice junctions and at higher temperatures. Mutations within these evolved inteins occur at sites distant from the active site. We present a hypothesis that a network of conserved coevolving amino acids in inteins mediates these long-range effects.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Evolução Molecular
/
Processamento de Proteína
/
Inteínas
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article