Abstract
BackgroundThe 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.ResultsIn 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.ConclusionsIn 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.
Highlights
The cellulose synthase complex (CSC), composed of cellulose synthase (CesA) proteins, is a catalytic enzyme complex involved in cellulose synthesis in the plant cell
The full-length BoCesA5 protein (120 kDa) under E. coli expression resulted in a protein fragment (~ 60 kDa) or invalid expression (Fig. 3a), indicating the BoCesA5 would be unstable in prokaryotic expression
We attempted to use the eukaryotic system for BoCesA5 expression
Summary
The cellulose synthase complex (CSC), composed of cellulose synthase (CesA) proteins, is a catalytic enzyme complex involved in cellulose synthesis in the plant cell. A major renewable substance derived from plant cell walls, is ubiquitous in woody plants. With respect to fast-growing woody monocots, 3–5-year-old bamboo could be a potential polysaccharide resource for renewable material based on biomass yield [3] and composition analysis [4]. Several studies on fast-growing monocots have referenced the growth and development of moso bamboo, Phyllostachys edulis, demonstrating the mechanism of rapid shoot growth, cell wall biosynthesis and cell metabolism using draft genome sequencing [5], transcriptome sequencing [6] and signal pathway analysis [7].
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