title: Cloning, expression, purification and characterization of a single chain variable fragment specific to tumor necrosis factor alpha in Escherichia coli.
abstract: Anti TNF-α molecules have been used as therapeutic agents in a variety of human diseases such as Rheumatoid arthritis, Ankylosing spondylitis, Chron's diseases, Psoriasis, etc., where high levels of TNF-α plays a destructive role. The limitations of the present TNF-α inhibitors in terms of size, tissue penetration and immunogenicity, etc., provoked the search for small anti TNF-α molecules. In the present study, a single chain variable fragment (ScFv) construct was made from a monoclonal antibody of the class IgG raised against TNF-α was used. The anti TNF-α ScFv was well expressed as soluble form in Escherichia coli BL21 (DE3), which was purified to homogeneity by commercial methacrylate monolith-convective interaction media (CIM) supports using two different chemistries, immobilized metal affinity chromatography (IMAC) with copper ions followed by anion exchange chromatography. The anti TNF-α ScFv found to be inhibiting the TNF-α mediated cytotoxicity in MCF-7 cells with an IC(50) of 8μg. Data presented here are promising and encouraging to further optimize anti TNF-α ScFv production in larger scale with higher recovery at a cheaper price for therapeutic purposes.
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore, Tamil Nadu, India.
authors.forename: Krishnan
authors.initials: K
authors.lastname: Sushma
authors.forename: Mookambeswaran A
authors.initials: MA
authors.lastname: Vijayalakshmi
authors.forename: Venkataraman
authors.initials: V
authors.lastname: Krishnan
authors.forename: Padikara Kutty
authors.initials: PK
authors.lastname: Satheeshkumar
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D012965:Sodium Chloride; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D014409:Tumor Necrosis Factor-alpha; D012965:Sodium Chloride