Xi Hualong, Yuan Ruosen, Chen Xiaoxu, Gu Tiejun, Cheng Yue, Li Zhuang, Jiang Chunlai, Kong Wei, Wu Yongge
National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, China.
BCHT Biotechnology Company, Changchun 130012, China.
Protein Expr Purif. 2016 Oct;126:26-32. doi: 10.1016/j.pep.2016.05.004. Epub 2016 May 5.
An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.
本研究成功且简便地制备了一种抗狂犬病病毒糖蛋白的单链抗体片段，其方向为VL-连接子-VH，命名为scFv57RN。scFv57RN蛋白主要在大肠杆菌的包涵体中表达。经过洗涤和纯化，最终通过柱上复性程序获得包涵体。通过凝胶排阻色谱进一步纯化以去除无活性的多聚体。从1升细菌培养物中回收了约360毫克最终产物。使用快速荧光灶抑制试验测定，最终产物对CVS-11毒株显示出950 IU/mg的高中和效价。我们的研究证明了一种从包涵体中大量生产具有活性的scFV57RN的高效方法，该方法可能适用于其他不溶性蛋白质的纯化。
