Prokaryotic Expression Analysis of A PpCAD Gene from Elephant Grass

Cinnamyl Alcohol Dehydrogenase (CAD), a multifunctional enzyme that catalyzes the last step in lignin monolignol biosynthesis, has significant meanings to plant lignification improvement. In the present study, a prokaryotic expression vector of pGEX-PpCAD was constructed based on the cDNA sequence of PpCAD gene, aiming to investigate the in vitro expression property of PpCAD protein. The E. coli strain BL21 was used as expression host. The results showed that soluble fusion protein GST-PpCAD could be induced under the condition of 0.02 mmol/L IPTG, 28℃ and 60 min, but the expression level was low. The induced fusion protein was about 69 kDa and the PpCAD was about 40 kDa, which was consistent with the prediction. Meanwhile, fusion protein would convert to inclusion body with increase of induction time, and it was mostly existed in inclusion body no matter how to optimize induction conditions. Ethyl alcohol and low temperature treatment would slow down the induction of fusion protein, but had no effect on the inclusion body. Frozen treatment was conductive to the extraction of inclusion bodies. Refer to this link:http://www.cusabio.com/Clone/dnaT-1089563.html