RESUMO
Centella asiatica (L.) Urban (Apiaceae), a small annual plant that grows in India, Sri Lanka, Malaysia, and other parts of Asia, is well-known as a medicinal herb with a long history of therapeutic uses. The bioactive compounds present in C. asiatica leaves include ursane-type triterpene sapogenins and saponins-asiatic acid, madecassic acid, asiaticoside, and madecassoside. Various bioactivities have been shown for these compounds, although most of the steps in the biosynthesis of triterpene saponins, including glycosylation, remain uncharacterized at the molecular level. This chapter describes an approach that integrates partial enzyme purification, proteomics methods, and transcriptomics, with the aim of reducing the number of cDNA candidates encoding for a glucosyltransferase involved in saponin biosynthesis and facilitating the elucidation of the pathway in this medicinal plant.
Assuntos
Centella/genética , Centella/metabolismo , DNA Complementar , Perfilação da Expressão Gênica/métodos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Proteômica/métodos , Centella/química , Biologia Computacional , Ativação Enzimática , Glucosiltransferases/química , Extratos Vegetais , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Medicinais/química , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Saponinas/biossínteseRESUMO
Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography.