Heterologous expression and characterization of xyloglucan endotransglycosylases from Populus tremula x tremuloides

Sammanfattning: Xyloglucan endotransglycosylases (XETs) are enzymes involvedin the reorganization of the primary cell wall. This thesisdescribes several aspects of XET16A from hybrid aspen (Populustremula x tremuloides). The gene was isolated from the hybridaspen cDNA library and the protein was expressed heterologouslyin the methylotrophic yeast Pichia pastoris. The yield ofpurified, active PttXET16A from shake flask cultivations wasaround 10 - 20 mg/litre. Different characteristics, such asstability, temperature- and pH optimum were studied. We useddeglycosylated PttXET16A wild type enzyme as well as a mutatedenzyme to elucidate the role and the importance of theglycosylation of the protein. The three dimensional structureof PttXET16A was determined by X-ray crystallography. This wasthe first XET structure to be published and it contributed to abetter understanding of the mechanism and substrate specificityof the enzyme. Keywords:xyloglucan endotransglycosylase,transglycosylation, hybrid aspen, cell expansion, heterologousexpression, Pichia pastoris, N-glycosylation, X-raycrystallography