title: Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support.
abstract: Anti TNF-α molecules are important as therapeutic agents for many of the autoimmune diseases in chronic stage. Here we report the expression and purification of a recombinant single chain variable fragment (ScFv) specific to TNF-α from inclusion bodies. In contrast to the conventional on column refolding using the soft gel supports, an efficient methodology using monolithic matrix has been employed. Nickel (II) coupled to convective interaction media (CIM) support was utilized for this purpose with 6M guanidine hydrochloride (GuHCl) as the chaotropic agent. The protein purified after solubilization and refolding proved to be biologically active with an IC₅₀ value of 15 μg. To the best of our knowledge, this is the first report showing the application of methacrylate based chromatographic supports for matrix-assisted refolding and purification of Escherichia coli inclusion bodies. The results are promising to elaborate the methodology further to exploit the potential positive features of monoliths in protein refolding science.
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore 632014, Tamil Nadu, India.
authors.forename: Krishnan
authors.initials: K
authors.lastname: Sushma
authors.forename: Chuvappumkal Joseph
authors.initials: CJ
authors.lastname: Bilgimol
authors.forename: Mookambeswaran A
authors.initials: MA
authors.lastname: Vijayalakshmi
authors.forename: Padikara Kutty
authors.initials: PK
authors.lastname: Satheeshkumar
mesh_terms: D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D006801:Humans; D017510:Protein Folding; D057127:Single-Chain Antibodies / Q000737:chemistry*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry*
chemical_list: D057127:Single-Chain Antibodies; D014409:Tumor Necrosis Factor-alpha