Plant Methods (Jul 2019)
Heterologous overexpression, purification and functional analysis of plant cellulose synthase from green bamboo
Abstract
Abstract Background The cellulose synthase complex (CSC), composed of cellulose synthase (CesA) proteins, is a catalytic enzyme complex involved in cellulose synthesis in the plant cell. CesA proteins synthesize cellulose microfibrils corresponding to the microtubule direction and export linear products across the plasma membrane. However, the CSC arrangement and the mechanism of cellulose synthesis in plant cells remain unclear. Purified CesA proteins are required to determine biochemical and biophysical characteristics. Results In this study, we constructed, expressed, and purified six heterologously expressed cellulose synthases from Bambusa oldhamii (BoCesA) and analyzed the associated enzyme activity. The conjugating sequences of the maltose-binding protein (MBP) gene and the BoCesA genes were constructed into the expression vector pYES2/CT and were further transformed into yeast cells (BCY123) for fermentation culturing. Purified BoCesA recombinant proteins were obtained by a two-step purification procedure, consisting of immobilized metal affinity chromatography to purify MBP-BoCesAs and size-exclusion chromatography (Superdex-200) to isolate BoCesAs in oligomeric form. The enzymatic activity of oligomeric BoCesAs with 80% purity was determined by partially methylated alditol acetate (PMAA)-coupled gas chromatography–mass spectrometry (GC–MS) analysis. Furthermore, the long fiber-like products synthesized by oligomeric BoCesAs were observed under a transmission electron microscope (TEM) and were further confirmed as cellulose microfibril products. Conclusions In this study, we successfully established a heterologous expression and purification system for BoCesAs. The purified recombinant BoCesA proteins display enzyme activity and can produce protein in milligram quantities for further studies on molecular composition and structure.
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