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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23802 43474 2011 7 PDF Available
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 IC50 of 8 μg. 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.

Keywords
Tumor necrosis factor-α (TNF-α); Single chain variable fragment (ScFv); Soluble protein; Immobilized metal affinity chromatography (IMAC); Cytotoxicity
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Cloning, expression, purification and characterization of a single chain variable fragment specific to tumor necrosis factor alpha in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 156, Issue 4, 20 December 2011, Pages 238–244
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us