Cloning and expression of cellulase XF-818 of Xylella fastidiosa in Escherichia Coli

• Autor(a) principal: Wulff, Nelson Arno
• Data de Publicação: 2003
• Outros Autores: Carrer, Helaine, Pascholati, Sérgio Florentino
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Scientia Agrícola (Online)
• Texto Completo: https://www.revistas.usp.br/sa/article/view/21909
• topic: clorose variegada dos citros; celulases; clonagem e expressão; citrus variegated chlorosis; cellulases; cloning and expresssion

Xylella fastidiosa's genome was the first of a plant pathogen to be completely sequenced. Through comparative sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. However, the biological role of each gene should be assigned experimentally. On this regard, heterologous protein expression is a powerful tool to produce proteins from such genes, allowing their characterization. X. fastidiosa lives inside xylem vessels and eventually would degrade pit membranes from xylem cells to move radialy into the host. The identification of several putative plant cell wall degrading enzymes on X. fastidiosa genome prompted the assession of the function of such proteins. The open reading frame (ORF) Xf-818 was cloned into expression vector pET20b and E. coli cells harboring such plasmid exhibited cellulase activity. Using IPTG at 0.4 mmol L-1 with a 12 h incubation at 32°C are the best conditions to produce higher amounts of heterologous protein. The enzyme degrades cellulose confirming the endoglucanase activity of Xf-818.