设计了两段不同序列长度透明颤菌血红蛋白(VGB)的天然低氧启动子基因(Pvgb-L,Pvgb-S),分别与绿色荧光蛋白(GFP)或加强型绿色荧光蛋白(TRIGFP)基因克隆到pBluescript II SK-质粒中。根据重组菌在不同溶氧条件下荧光蛋白基因转录水平和表达量,经RT-PCR和荧光光谱分析证明,在低氧条件下,短链低氧启动子(Pvgb-S)能更有效地诱导下游基因GFP的表达,并有利于蛋白的正确折叠表达,其诱导效果受溶氧浓度影响而差异显著。在低氧培养条件下,低氧启动子Pvgb-S可有效诱导重组五碳糖代谢基因在大肠杆菌W3110(ΔpflB,ΔadhE)中的表达,提高重组菌木糖代谢,从而增加乳酸产量。
Aim To purify and characterize flammulin, a basic protein with anti-tumoractivities. Methods Ammonium sulfate, ethanol fractionation and column chromatography were used forseparation and purification. Electrophoretic analysis, amino acid analysis, and MS of flammulin werecarried out. Results Flammulin was purified to electrophoretic homogeneity and crystallized. With amolecular mass of 19891.13 Da, pI 8.9, λ_(max) = 276 - 278 nm, λ_(min) = 250 nm, flammulin wascharacterized by its lack of methionine. Fingerprint mapping of flammulin was determined by MALDI-MSfollowing in-gel protease digestion; no close matches were identified. Conclusion Flammulin waspurified to electrophoretic homogeneity, and its characteristics are discussed for the first time.
