Kopitar-Jerala N, Gubensek F, Turk V
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Biol Chem. 2000 Dec;381(12):1245-9. doi: 10.1515/BC.2000.152.
Human stefin A is an inhibitor of lysosomal cysteine proteinases cathepsin B, H, L and S. In the present report we describe the cloning and expression of anti-stefin A Fab fragment A22 in E. coli. We have determined the nucleotide sequences of the antibody heavy and light chain and compared them to the murine immunoglobulin germ line sequences. Expression of the two antibody chains was achieved using a single vector with a PhoA promoter and coding regions placed after the signal sequences, directing them to the periplasmic space. The A22 Fab fragment was extracted from the periplasmic space and expression was confirmed by Western blot analysis. The recombinant A22 Fab fragment had an affinity for stefin A comparable to the original monoclonal antibody, as determined by ELISA.
人微小抑制素A是溶酶体半胱氨酸蛋白酶组织蛋白酶B、H、L和S的抑制剂。在本报告中，我们描述了抗微小抑制素A Fab片段A22在大肠杆菌中的克隆和表达。我们测定了抗体重链和轻链的核苷酸序列，并将它们与小鼠免疫球蛋白种系序列进行了比较。使用具有PhoA启动子的单一载体实现了两条抗体链的表达，编码区位于信号序列之后，将它们导向周质空间。从周质空间提取A22 Fab片段，并通过蛋白质免疫印迹分析证实了表达。通过酶联免疫吸附测定法确定，重组A22 Fab片段对微小抑制素A的亲和力与原始单克隆抗体相当。
