Recombinant anti-stefin A F-ab fragment: Sequence analysis of the variableregion and expression in Escherichia coli

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 antistefin A F-ab fragment A22 in E. coli. We have determined the nucle otide sequences of the antibody heavy and light chain and compared them to the murine immunoglobulin germ line sequences. Expression of the two antibo dy chains was achieved using a single vector with a PhoA promoter and codin g regions placed after the signal sequences, directing them to the periplas mic space. The A22 F-ab fragment was extracted from the periplasmic space a nd expression was confirmed by Western blot analysis. The recombinant A22 F -ab fragment had an affinity for stefin A comparable to the original monocl onal antibody, as determined by ELISA.