Recombinant anti-stefin A Fab fragment: sequence analysis of the variable region 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 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.
Kopitar-Jerala
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Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Gubensek
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Turk
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D000911:Antibodies, Monoclonal / Q000378:metabolism; D000915:Antibody Affinity; D001483:Base Sequence; D003001:Cloning, Molecular; D055312:Cystatin A; D015891:Cystatins / Q000276:immunology*; D015853:Cysteine Proteinase Inhibitors / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D055312:Cystatin A; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C026407:CSTA protein, human