Zhang Yi, Guo Ying-Jun, Sun Shu-han, Yan Hong-li, He Yan
Department of Medical Genetics, The Second Military Medical University, Xiang Yin Road 800, Shanghai 200433, PR China.
Protein Expr Purif. 2004 Mar;34(1):68-74. doi: 10.1016/j.pep.2003.11.025.
Annexin B1 is a novel member of the annexin family of Ca2+- and phospholipid-binding proteins from Cysticercus cellulosae. To obtain high quality annexin B1 for biochemical and biophysical analyses, its cDNA was cloned into the prokaryotic expression vector pJLA503 and the translation initiation codon was immediately under the control of the inducible bacteriophage lambda promoters P(R) and P(L). After induction by shifting temperature, large amounts of non-fusion protein were produced in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 25 mg/L bacterial culture. Western blot analysis showed that recombinant annexin B1 was specifically recognized by serum of pigs infected with cysticercosis. Secondary structure predictions from circular dichroism spectroscopy indicated that alpha-helix is the main secondary structure of the protein. In anticoagulant assays, the recombinant non-fusion protein exhibited dose-dependent effects in modified kaolin partial thromboplastin time (KPTT) prolongation and doubled the clotting time of control human plasma at 60 microg/ml. The expression, purification, and initial characterization of annexin B1 set an important stage for further characterization of the protein.
膜联蛋白B1是来自猪囊尾蚴的Ca2+和磷脂结合蛋白膜联蛋白家族的一个新成员。为了获得用于生化和生物物理分析的高质量膜联蛋白B1，将其cDNA克隆到原核表达载体pJLA503中，翻译起始密码子直接置于可诱导的噬菌体λ启动子P(R)和P(L)的控制之下。通过改变温度进行诱导后，在大肠杆菌中以可溶形式产生了大量非融合蛋白。通过两个连续的离子交换色谱步骤将重组蛋白纯化至同质。最终产量约为每升细菌培养物25毫克。蛋白质印迹分析表明，重组膜联蛋白B1能被感染囊尾蚴病的猪血清特异性识别。圆二色光谱的二级结构预测表明，α-螺旋是该蛋白的主要二级结构。在抗凝试验中，重组非融合蛋白在改良高岭土部分凝血活酶时间（KPTT）延长方面表现出剂量依赖性效应，在60微克/毫升时使对照人血浆的凝血时间加倍。膜联蛋白B1的表达、纯化及初步表征为该蛋白的进一步表征奠定了重要基础。
