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. 2022 Dec 9;20:578. doi: 10.1186/s12967-022-03794-w

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — Formation of the Stx1B-scFv OKT3 conjugate. a Schematic representation of the conjugation approach. AzK was site-specifically incorporated at position E129 in scFv OKT3 and at K9 in Stx1B. Then, the azido groups in purified Stx1B K9AzK and scFv OKT3 E129AzK selectively reacted with the DBCO group on the methyltetrazine-DBCO and trans-cyclooctene-PEG4-DBCO linkers by SPAAC. When mixed, the methyltetrazine-functionalized Stx1B K9Tz and trans-cyclooctene-functionalized scFv OKT3 E129TCO coupled with each other via an IEDDA reaction to form the Stx1B-scFv OKT3 conjugate. b Qualitative SDS-PAGE analysis of the IEDDA reaction mixture (lane 4). Lane 1, Stx1B K9AzK; lane 2, scFv OKT3E129AzK; lane 3, SEC purified Stx1B-scFv OKT3 conjugate. Calculated molecular weights (MW_calc) of the individual proteins are indicated in the legend to Fig. 2. M, molecular weight marker, the band sizes are indicated on the left margin of the gel. The gel was stained with Coomassie protein stain