RESUMO
A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.
Assuntos
Vetores Genéticos , Processamento de Proteína Pós-Traducional , Processamento de Proteína , Proteínas Recombinantes/isolamento & purificação , ATPases Vacuolares Próton-Translocadoras , Marcadores de Afinidade , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Quitina , Concentração de Íons de Hidrogênio , Proteínas Ligantes de Maltose , Métodos , Dados de Sequência Molecular , ATPases Translocadoras de Prótons/metabolismo , TemperaturaRESUMO
Protein splicing is a posttranslational processing event in which an internal polypeptide is excised from a protein precursor and the terminal polypeptides are then ligated together, resulting in the production of two proteins. This report presents direct evidence for protein splicing by demonstrating in vitro splicing of purified precursor that accumulated when the protein splicing element from Pyrococcus DNA polymerase was cloned into a foreign gene. In vitro splicing was temperature and pH dependent. A slowly migrating species exhibited kinetic properties of a splicing intermediate and was shown to be a branched molecule by N-terminal sequencing. The precursor and slowly migrating species were interconvertible in response to pH shifts.
