The Escherichia Coli‐Derived Fab Fragment of the IgM/κ Antibody IN‐1 Recognizes and Neutralizes Myelin‐Associated Inhibitors of Neurite Growth

Abstract

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.

Identification and Characterization of a Bovine Neurite Growth Inhibitor (bNI-220)

“…5) bNI-220 is present in spinal cord myelin, in line with the previous finding that mAb IN-1 immunostains CNS myelin (32). 6) Immunoprecipitation of bovine CNS myelin by mAb IN-1 or an IN-1 Fab depleted about two thirds of the inhibitory activity and precipitated a high molecular mass complex comprising several bands including a prominent band at 220 kDa (25,31). 7) Analytical analysis of the active gel-eluted 220 kDa band by two-dimensional PAGE revealed one major spot, indicating that bNI-220 was purified to homogeneity.…”

“…A monoclonal antibody (mAb IN-1), which has been raised against rat , neutralizes the neurite growth inhibitory property of differentiated oligodendrocytes and [24][25][26][27][28][29][30]. Immunoprecipitation of CNS myelin proteins by the mAb IN-1 removed more than 50% of inhibitory substrate properties (25,31). Immunohistochemistry revealed that mAb IN-1 stains white matter and myelin in the whole brain and spinal cord from adult rats (32).…”

Regeneration of Lesioned Corticospinal Tract Fibers in the Adult Rat Induced by a Recombinant, Humanized IN-1 Antibody Fragment

“…Recently, it became possible to clone the DNA sequences coding for antibody chains and to express tailor-made recombinant antibodies in large quantities in bacteria (Skerra and Plückthun, 1988;Orlandi et al, 1989). We have cloned the cDNAs for the variable regions of the Fab light and heavy chain, including the antigen binding site of mAb IN-1, and expressed a monovalent, recombinant fragment, rIN-1 Fab (ϳ45 kDa molecular mass), that was able to neutralize Nogo-A in vitro (Bandtlow et al, 1996). rIN-1 Fab can be produced in large quantities, concentrated, stored, and infused as a pure reagent to the CNS in a way potentially also applicable to human patients.…”

Structural insights into the antigenicity of myelin oligodendrocyte glycoprotein

“…Selenomethionine (SeMet)-labeled protein was produced in the methionine-auxotrophic E. coli strain B834(DE3) and grown in minimal medium with SeMet (0.3 mM) substituted for methionine. The cDNA of the variable domains of the mouse monoclonal antibody 8-18C5 was subcloned into the expression vector pASK107, yielding the chimeric (8-18C5)-Fab composed of the 8-18C5 variable domains, the human IgG constant domains, and the Strep tag II fused to the C terminus of the heavy chain (9,10). (8-18C5)-Fab was produced by periplasmic secretion in E. coli and purified by streptavidin affinity chromatography and gel filtration (11).…”