ABSTRACT
A cDNA combinatorial antibody library of mouse variable immunoglobulin fragments has been constructed from mice immunized with rhIFN-beta1b. For this purpose, cDNAS of immunoglobulin variable heavy (V(H)) and variable light (V(L)) chains genes amplified from splenocytes were joined with linker DNA to form ScFv's (single-chain Fv-antibodies). The obtained ScFv-DNA pool was cloned into a phagemid vector and used for Esherichia coli transformation. Using the phage display technique, bacterial clones producing single-chain antibodies specific to rhIFN-beta1b were selected. The following characteristics of the combinatorial library were determined in this work: abundance, functional size, and the initial ScFv-DNA diversity in the library constructed. High specificity of interaction between phage displayed ScFv's and rhIFN-beta1b has been demonstrated.
Subject(s)
Gene Library , Immunoglobulin Fragments/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , Interferon-beta/immunology , Animals , Cloning, Molecular , Combinatorial Chemistry Techniques , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Female , Humans , Immunoblotting , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Fragments/immunology , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/immunology , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/immunology , Interferon beta-1b , Mice , Mice, Inbred BALB CABSTRACT
The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Escherichia coli/genetics , Immunoglobulin Fragments/biosynthesis , Interferon-alpha/immunology , Animals , Antibodies, Monoclonal/genetics , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Humans , Immunoglobulin Fragments/genetics , Inclusion Bodies , Interferon alpha-2 , Mice , Plasmids , Protein Renaturation , Recombinant ProteinsABSTRACT
The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-alpha2b is shown.
