Capillary zone electrophoresis (CZE) was used to monitor the whole unfolding process of bovine insulin resulted from the dithiothreitol (DTT) reduction. An off-line matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was employed to determine the molecular weight of the reaction products at the same time to confirm the observation results obtained by CZE. The structural change during the process of bovine insulin unfolding could be observed directly from the electropherogram and information of protein unfolding could be obtained simultaneously. The results indicated that as an effective tool of monitoring the conformational change of protein,the method of CZE was simple,quick,sensitive and lower sample consumption.

Diabetes mellitus is a typical metabolic disease.Its complications cause the main damage and lead to high mortality and disability eventually.The exact mechanism of diabetes is still unknown at present,and no radical cure of it is available.Therefore,the prevention of diabetes has become a priority.Metabolomics as a new technology can identify and measure the entire metabolic changes in the organism,and therefore has been widely applied to diabetes related studies with its enormous potential.

Artemisinin-based combination therapies (ACTs) are recommended to be the most effective therapies for the first-line treatment of uncomplicated falciparum malaria. However, artemisinin is often in short supply and unaffordable to most malaria patients, which limits the wide use of ACTs. Production of amorpha-4,11-diene, an artemisinin precursor, was investigated by engineering a heterologous isoprenoid biosynthetic pathway in Escherichia coli. The production of amorpha-4,11-diene was achieved by expression of a synthetic amorpha-4,11-diene synthase gene in Escherichia coli DHGT7 and further improved by about 13.3 fold through introducing the mevalonate pathway from Enterococcus faecalis. After eliminating three pathway bottlenecks including amorpha-4,11-diene synthase, HMG-CoA reducase and mevalonate kinase by optimizing the metabolic flux, the yield of amorpha-4,11-diene was increased by nearly 7.2 fold and reached at 235 mg/L in shaking flask culture. In conclusion, an engineered Escherichia coli was constructed for high-level production of amorpha-4,11-diene.
Alkyl and Aryl Transferases ; genetics ; Antimalarials ; metabolism ; Artemisinins ; metabolism ; Enterococcus faecalis ; genetics ; Escherichia coli ; genetics ; metabolism ; Metabolic Engineering ; methods ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Sesquiterpenes ; metabolism ; Transformation, Bacterial