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Periplasmic secretion, extraction and purification of recombinant tagless anti-tumor necrosis factor (TNF) alpha single chain variable fragment (scFv) in E. coli

Author: 
Balaji Parthasarathy, Satheeshkumar, P. K., Krishnan Venkataraman and Vijayalakshmi, M. A.
Subject Area: 
Life Sciences
Abstract: 

Present day therapeutics against TNF have some disadvantages in the form of cost, immunogenicity, etc., resulting in a need for an approach using small antigen binding antibody fragments called scFv (single chain variable fragment). In this study, construct with anti-TNFscFv was developed and expressed in E.coli under optimal conditions of 30oC and 37 M IPTG. Periplasmic secretion was achieved using 2% glycine and 1% Triton X-100in the culture medium. Addition of 0.5 M sorbitol ensured that the protein was expressed as a soluble form. Secreted protein was purified by immobilized metal affinity chromatography (IMAC) and confirmed by immunoblot.

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