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J Biotechnol. 2011 Dec 20;156(4):238-44. doi: 10.1016/j.jbiotec.2011.06.039. Epub 2011 Jul 6.

Cloning, expression, purification and characterization of a single chain variable fragment specific to tumor necrosis factor alpha in Escherichia coli.

Author information

1
Centre for Bioseparation Technology, VIT University, Vellore, Tamil Nadu, India.

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.

PMID:
21763363
DOI:
10.1016/j.jbiotec.2011.06.039
[Indexed for MEDLINE]

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