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A recombinant Fab fragment was prepared from the monoclonal I g M k antibody IN-1, which neutralizes central nervous system myelin-associated neurite growth inhibitors both in vitro and in vivo. The variable domain gene sequences were amplified and cloned after cDNA synthesis from the hybridoma RNA. After insertion into the tet promoter vector pASK85, which provided the constant domains of class IgGl/K, equipped with a Hish tag, large amounts of the Fab fragment were produced in Escherichiu coli by medium cell density fermentation. The Fab fragment was purified to homogeneity by immobilized metal-affinity chromatography and its biochemical activity was compared with the original IN-I antibody. In an assay for neurite outgrowth and fibroblast spreading, the Fab fragment showed a similar neutralizing effect on inhibitory substrate properties of central nervous system myelin as the unpurified IgM, although an approximately tenfold higher concentration was necessary. Immunoprecipitation experiments revealed a more selective antigen-binding behaviour for the Fab fragment. The Fab fragment was also successfully applied for antigen detection in immunohistochemical analyses. Therefore, the recombinant Fab fragment of IN-1 shows full functionality in vitro and appears to be well suited for replacing the monoclonal IgM in investigations on fiber tract regeneration in vivo.Keywords: antibody fragment ; axonal regeneration; bacterial expression ; neurite growth inhibitor; tetracycline promoter.The development of methods for the production of functional antibody fragments in E. coli [l] has enabled the rapid preparation of large quantities of these proteins and facilitated their modification by protein engineering for a variety of applications. When a hybridoma cell line is available that synthesizes a monoclonal antibody of interest, its variable domain genes can thus be rescued by PCR cloning [2] and directly utilized for recombinant protein production. In addition, novel properties or effector functions of the bacterially produced, engineered antibody fragment can be exploited and the modified antibody can be used as a reagent in biochemical research, medical diagnosis, and possible therapy.Several advantages of this strategy, for example the small size of the recombinant Ig fragment compared with a whole antibody or its possible production as a fusion protein with a reporter enzyme or a cellular toxin, have been widely recognized 131.

The synthetic antibody model "M41" was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine C kappa and CH1 gamma 1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P2(1)2(1)2(1) with unit cell dimensions a = 104.7 A, b = 113.9 A, c = 98.8 A and diffracted X-rays to a nominal resolution of 2.5 A.

The cDNAs for the two variable domains of the antibody 9E10 were cloned from the hybridoma cell line. A chimeric 9E10 F a b fragment was produced in E. coli under control of the tightly controlled tetracycline promoter. The functional F ab fragment was isolated in a single step via a His 6 -tag, which also served for its recognition by a nickel chelatealkaline phosphatase conjugate. Thus, the recombinant F.,i, fragment permitted the immunochemical detection of the myc tag in a sandwich ELISA. The dissociation constant for the interaction with the myc tag peptide was determined as 80 ± 5 nM by fluorescence titration. In an attempt to produce the smaller 9E10 F v fragment it was found that its VH domain alone can be readily isolated from E. coli as a soluble protein. This unusual behaviour may be explained by the 18 amino acid-long CDR-H3 and could be of value in the design of 'single domain' antibodies.

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