Abstract
Protein-based conjugates have been extensively utilized in various biotechnological and therapeutic applications. In order to prepare homogeneous conjugates, site-specific modification methods and efficient purification strategies are both critical factors to be considered. The development of general and facile conjugation and purification strategies is therefore highly desirable. Here, we apply a capture and release strategy to create protein conjugates based on Designed Ankyrin Repeat Proteins (DARPins), which are engineered antigen-binding proteins with prominent affinity and selectivity. In this case, DARPins that target the epithelial cell adhesion molecule (EpCAM), a diagnostic cell surface marker for many types of cancer, were employed. The DARPins were first genetically modified with a C-terminal CVIA sequence to install an enzyme recognition site and then labeled with an aldehyde functional group employing protein farnesyltransferase. Using a capture and release strategy, conjugation of the labeled DARPins to a TAMRA fluorophore was achieved with either purified proteins or directly from crude E. coli lysate and used in subsequent flow cytometry and confocal imaging analysis. DARPin-MMAE conjugates were also prepared yielding a construct manifesting an IC50 of 1.3 nM for cell killing of EpCAM positive MCF-7 cells. The method described here is broadly applicable to enable the streamlined one-step preparation of protein-based conjugates.
Original language | English (US) |
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Article number | 11537 |
Journal | International journal of molecular sciences |
Volume | 23 |
Issue number | 19 |
DOIs | |
State | Published - Oct 2022 |
Bibliographical note
Funding Information:This work was supported by NIH grants R01 GM084152 and R35 GM141853 (M.D.D.); Chweizerische Nationalfonds Grant 310030_192689 (A.P.); M.R. was supported by Innovation Research Fund Basic Research Award from the Dana-Farber Cancer Institute.
Publisher Copyright:
© 2022 by the authors.
Keywords
- biorthogonal conjugation
- protein farnesyltransferase
- site-specific protein conjugation
- targeted drug delivery
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University Imaging Centers
Sanders, M. A. (Program Director)
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