Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8359323 | Protein Expression and Purification | 2018 | 27 Pages |
Abstract
The development of therapeutic antibodies in their various forms has been a constant challenge since the development of the first monoclonal antibodies in 1975. This is especially true for the development of therapeutic single chain variable (scFv) fragments in Escherichia coli. In a previous study the selection of a tissue factor inhibiting single chain variable fragment (TFI-scFv) isolated from the Thomlinson IÂ +Â J phage libraries was described. Although the initial findings were promising, additional characterization of the antibody fragment and subsequent application was hampered due low protein yield. This study reports on: i) the improved expression of a previously low yielding TFI-scFv in the cytoplasm of E. coli BL21 (DE3) through modifications to the expression systems in conjunction with codon optimization ii) evaluation of two commercial methods of protein recovery: in vitro refolding and the utilization of cold shock expression systems in conjunction with E. coli SHuffle. Results showed that TFI-scFv could be expressed at higher levels in the cytoplasm of E. coli than previously achieved in the periplasm. Both the in vitro refolding and cold shock strategies were capable of producing functional TFI-scFv with varying degrees of success. These procedures could be applied to improve the production of other problematic low yielding scFv isolated from phage display repositories in order to facilitate their characterization.
Keywords
GSHMRCGSSGIMACTBSGdnHClCAIscFvIPTGDMSOE.coliEscherichia coliisopropyl β-D-1-thiogalactopyranosideTris buffered salineDimethyl sulfoxideprothrombin timeCodon adaptation indexFusion partnerMedical Research CouncilCold shockTissue factorSingle chain variable fragmentInclusion bodiesDisulfide bridgereduced glutathioneimmobilized metal affinity chromatographyoxidized glutathione
Related Topics
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Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Jan-G. Vermeulen, Felicity Burt, Esta van Heerden, Errol Cason, Muriel Meiring,