福氏志贺菌硫氧还过氧化物酶的表达、纯化、活性检测及晶体生长的初步研究

Sf2523蛋白属于硫氧还蛋白过氧化物酶(Prx)家族,在有氧代谢过程中消除活性氧,起到保护生命大分子的重要作用.通过构建原核表达体系,可溶性表达并纯化了Sf2523酶蛋白,并对蛋白进行了过氧化物酶活性检测,证明其依然具有天然活性,酶的核心结构并未发生变化.由分子排阻色谱结果发现,酶蛋白体内和体外聚集状态不同,离体蛋白聚集状态不稳定,趋于二体化.将纯化的单体蛋白即时进行了结晶实验,初筛长出了针状晶体.通过进一步切除标签蛋白进行优化处理,最终得到了均匀的三维单晶.

Expression,Purification,Activity Detection and Crystal Growth Studies on Sf2523 Protein from Shigella flexneri 2a Strain 301

Sf2523 protein is a member from the Thioredoxin Peroxidase(Prx) family.Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides,thus conferring resistance to oxidative stress.Sf2523 protein can protect the large biological molecules from reactive oxygen specie,playing an important role during aerobic metabolism.After the construction of prokaryotic expression system,the Sf2523 protein was expressed in soluble state,and purified by Ni Affinity Chromatography and Size Exclusive Chromatography.The peroxidase activity experiment proved that it still has a natural enzyme activity and the core structure has not changed.Enzyme protein in vivo and in vitro has different state of aggregation.The purified monomeric protein of crystallization experiments,screening out the needle-like crystals.Through further resection of label protein to optimize the processing,finally,a homogeneous three-dimensional crystal was got.