Heterologous expression and purification of Nicotiana benthamiana Cellulose synthase-like B (NbCslB)
Abstract: Hemicelluloses are synthesized by proteins encoded by genes from the cellulose synthasegene superfamily. One subgroup of this gene family is the cellulose synthase-like B, which islargely uncharacterized and unexplored. The common model organism Nicotianabenthamiana has one such gene in its genome, NbCslB, encoding a membrane protein. Theexpression of this gene has previously been studied in vivo, but in order to study the protein invitro a viable solubilization and purification protocol is required. This study evaluated the useof the detergent n-Dodecyl β-D-maltoside (DDM) for solubilization, followed by purificationusing immobilized metal ion affinity chromatography (IMAC), and thereafter reconstitutionof the protein into proteoliposomes. SDS-PAGE as well as Western blot analyses showed thatthe purification was successful and provided a pure sample of protein. Throughout theanalyses performed, an anti-FLAG antibody was discovered to bind well to the protein, andthereby be especially useful for analysis. An activity assay was performed on the purifiedprotein, to characterize its function and evaluate whether the protein had maintained itsactivity and conformation after the steps of purification and reconstitution. No activity couldbe detected in the enzymatic assay, which indicated that the purification protocol may havebeen too rough on the protein, that the reconstitution was not successful, or that the assayconditions were not optimal. These results can be used as a base for future research, where theprotocols for solubilization, purification, and reconstitution should be further refined in orderto obtain an end result where the purified protein is active. When an active and pure proteinsample is achieved, it will be possible to perform further attempts at characterizing thefunction of the protein using enzymatic activity assays. Additionally, the results showed thatthe choice of antibody can be crucial for proper analysis of this protein.
AT THIS PAGE YOU CAN DOWNLOAD THE WHOLE ESSAY. (follow the link to the next page)