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Fig. 1 | BMC Biotechnology

Fig. 1

From: A novel synthetic trivalent single chain variable fragment (tri-scFv) construction platform based on the SpyTag/SpyCatcher protein ligase system

Fig. 1

Construction and Purification of SpyTag/SpyCatcher based anti-HER3 Tri-scFv. a Schematic of the SpyTag/SpyCatcher based anti-HER3 Tri-scFv ligation reaction. SpyCatcher was fused to the C-terminus of anti-HER3 scFv (VL-(G4S)3-VH-GGS-SpyCatcher). Anti-HER3 Tri-scFv was generated by mixing monomeric scFv-SpyCatcher and a synthetic Tri-SpyTag peptide together at 6:1 M ratio. VL: variable light domain, VH: variable heavy domain. b Reducing SDS-PAGE analysis of TriSpyTag peptide and scFv-SpyCatcher ligation products. scFv-SpyCatcher and Tri-SpyTag peptide have molecular weights of 41 kDa and 4.88 kDa, respectively. After successful ligation, trivalent scFv (Tri-scFv) and divalent scFv (Di-scFv) had a molecular weight of 128 kDa and 87 kDa, respectively. c Size-exclusion chromatogram (SEC) of Tri-scFv ligation reaction. Inset shows SDS-PAGE analysis of two fractions eluted from 30 to 40 mins. d Further separation of Tri-scFv using SEC-HPLC. Inset shows SDS-PAGE analysis of fraction eluted from 15 to 17 mins. e Bioanalyzer electropherograms of SEC-HPLC separated Tri-scFv under non-reducing conditions. The observed molecular weight (MW) of Tri-scFv is 135 kDa

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BMC Biotechnology

ISSN: 1472-6750

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