CAJ | 학술논문

目的:通过对大肠杆菌表达重组人骨形成蛋白-2的复性研究,得到高活性的重组人骨形成蛋白-2.方法:在大肠杆菌中通过温度诱导表达重组人骨形成蛋白-2经过Triton X-100清洗之后,又通过DEAE离子交换层析纯化包涵体,包涵体在8 mol/L尿素变性溶解,在氧化-还原(还原型和氧化型谷胱甘肽)复性系统中,通过简单的稀释复性,通过肝素亲和层析一步纯化法纯化重组人骨形成蛋白-2,最后通过诱导C2C12细胞产生碱性磷酸酶检测重组人骨形成蛋白-2活性.结果:温度诱导表达重组人骨形成蛋白-2是以包涵体单体的形式存在,经过几步纯化后,得到高纯度的包涵体.重组人骨形成蛋白-2在不同氧化-还原剂比例,和不同小分子化学辅助剂浓度中复性,得到复性的效率也不同.亲和层析纯化后,本实验得到重组人骨形成蛋白-2的生物学活性比商业化的重组人骨形成蛋白-2更高.结论:重组人骨形成蛋白-2属于转化因子-β家族,此复性方法可能应用于此家族的其他成员同时得到成本低、产量高的重组人骨形成蛋白-2,可能为临床应用创造了良好的条件.
목적:통과대대장간균표체중조인골형성단백-2적복성연구,득도고활성적중조인골형성단백-2.방법:재대장간균중통과온도유도표체중조인골형성단백-2경과Triton X-100청세지후,우통과DEAE리자교환층석순화포함체,포함체재8 mol/L뇨소변성용해,재양화-환원(환원형화양화형곡광감태)복성계통중,통과간단적희석복성,통과간소친화층석일보순화법순화중조인골형성단백-2,최후통과유도C2C12세포산생감성린산매검측중조인골형성단백-2활성.결과:온도유도표체중조인골형성단백-2시이포함체단체적형식존재,경과궤보순화후,득도고순도적포함체.중조인골형성단백-2재불동양화-환원제비례,화불동소분자화학보조제농도중복성,득도복성적효솔야불동.친화층석순화후,본실험득도중조인골형성단백-2적생물학활성비상업화적중조인골형성단백-2경고.결론:중조인골형성단백-2속우전화인자-β가족,차복성방법가능응용우차가족적기타성원동시득도성본저、산량고적중조인골형성단백-2,가능위림상응용창조료량호적조건.
AIM: To get high biological activity of recombinant human bone morphogenetic protein -2 (rhBMP-2) expressed in Escherichia coli by the methods of refolding. METHODS: The rhBMP-2,expressed in Escherichia coli, was washed by Triton X- 100 and further purified by DEAE chromatography.The inclusion bodies were resolved in 8 mol/L urea, and were refolded and dimerized in the redox systems (reduced and oxidized glutathione). Finally, a one - step purification procedure based on the heparin affinity chromatography was implemented. The biological activity of purified rhBMP - 2 were tested by induction of the alkaline phosphatase activity in C2C12 cells. RESULTS: The rhBMP - 2 was expressed in Escherichia coli in a non - active aggregated form of inclusion bodies using a temperature - inducible expression system. The high - purified rhBMP - 2 was obtained in the form of inclusion bodies by several purification courses. The rhBMP - 2 was refolded and dimerized in the redox systems (reduced and oxidized glutathione) and a one - step purification procedure based on the heparin affinity chromatography was implemented to isolate the rhBMP - 2 dimers and monomers. The purified rhBMP - 2 dimers showed the higher biological activity than the commercial rhBMP - 2. CONCLUSIONS: The method achieved the refolding of rhBMP - 2 would be applied to the whole TGF - β superfamily because the BMP - 2 belongs to the superfamily. Meanwhile, the inexpensive,high yield rhBMP - 2 is suitable for clinic application.