人微管结合蛋白4微管结合区的体外表达

Expression of Microtubule Binding Domain of Human Microtubule-associated Protein 4

目的 :构建表达人微管结合蛋白 4微管结合区 (Microtubule -bindingdomain ,MTB)的大肠杆菌工程菌。方法 :将 1.2 7Kb编码MTB的DNA片段按正确方向亚克隆到原核生物表达载体pET - 3a。得到表达质粒JS3,并转化大肠杆菌BL2 1(DE3)。结果 :IPTG诱导下 ,表达产物的SDS -PAGE分子量大约是 4 0kD ,并能被抗MAP4抗体特异地识别。此外 ,重组MTB蛋白能在体外结合微管。结论 :人微管结合蛋白 4微管结合区具有微管结合活性。

Objective:To construct the engineered E.coli .strain which expresses microtubule-binding domain (MTB) of human microtubule-associated protein 4 (MAP4).Methods:A 1.27Kb DNA fragment encoded MTB was subcloned into expression vector pET-3a.One expression plasmid JS3 was constructed and transferred into BL21(DE3).Results:The molecular weight of expression products was about 40kD on SDS-PAGE.A specific antibody against MAP4 recognized them on Western Blot.In addition,recombinant MTB proteins bound microtubules in vitro.Conclusion:recombinant MTB proteins of human MAP4 retained the MT-binding activity.