2022
DOI: 10.3390/ijms231911537
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Enzymatic Construction of DARPin-Based Targeted Delivery Systems Using Protein Farnesyltransferase and a Capture and Release Strategy

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), whi… Show more

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Cited by 3 publications
(3 citation statements)
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“…Using an isoprenoid analogue incorporating bioorthogonal functionality, this strategy can be employed to create a plethora of modified proteins for a variety of applications. To investigate whether C10NorOPP could be used to construct site-specific protein–fluorophore conjugates for targeted cancer cell imaging, an anti-EpCAM Ac2-KCVIA DARPin (D1, 17 ) engineered to contain a C-terminal CVIA sequence was selected as the targeting moiety (Figure A); this approach has been previously explored with other FPP analogues. , The D1 DARPin was prenylated with C10NorOPP (D1-Nor, 18 ), followed by conjugation with TAMRA-tetrazine, 13 , to generate the D1-TAMRA conjugate ( 19 ), whose identity was confirmed using LC-MS (Figure S8). D1-TAMRA was shown to specifically label EpCAM + MCF-7 cells in flow cytometry experiments, and that labeling could be completely competed away using excess unmodified D1 protein ( 17 ) (Figure B,C).…”
Section: Resultsmentioning
confidence: 99%
See 1 more Smart Citation
“…Using an isoprenoid analogue incorporating bioorthogonal functionality, this strategy can be employed to create a plethora of modified proteins for a variety of applications. To investigate whether C10NorOPP could be used to construct site-specific protein–fluorophore conjugates for targeted cancer cell imaging, an anti-EpCAM Ac2-KCVIA DARPin (D1, 17 ) engineered to contain a C-terminal CVIA sequence was selected as the targeting moiety (Figure A); this approach has been previously explored with other FPP analogues. , The D1 DARPin was prenylated with C10NorOPP (D1-Nor, 18 ), followed by conjugation with TAMRA-tetrazine, 13 , to generate the D1-TAMRA conjugate ( 19 ), whose identity was confirmed using LC-MS (Figure S8). D1-TAMRA was shown to specifically label EpCAM + MCF-7 cells in flow cytometry experiments, and that labeling could be completely competed away using excess unmodified D1 protein ( 17 ) (Figure B,C).…”
Section: Resultsmentioning
confidence: 99%
“…Furthermore, the development of prenylation as a tool for selective protein modification is a burgeoning field. In the past, analogues of FPP capable of SPAAC or CuAAC have been employed for the creation of protein conjugates that can be used for selective visualization of cancer cells. The assembly of protein–drug conjugates that link functional proteins with small molecules, including fluorescent dyes and drugs, has proven to be valuable for diagnostic and therapeutic purposes. Bioorthogonal chemistry is particularly useful for generating protein conjugates because it allows modification to occur at a specific position on the protein, which leads to the production of homogeneous conjugates with a uniform structural composition. In earlier work, site-specifically labeled protein conjugates obtained by prenylating Designed Ankyrin Repeat Proteins (DARPins) , with different orthogonal probes allowed selective labeling and killing of cancer cells.…”
Section: Introductionmentioning
confidence: 99%
“…The modification process does not adversely affect the properties of the antibody, and the modification site is located far from the antigen binding site, ensuring that there is no reduction in affinity. In addition, a different geranyl pyrophosphate analog bearing an aromatic aldehyde group was used to introduce a single modification into a DARPin (designed ankyrin repeat protein) for further conjugation with a fluorophore or a drug [222].…”
Section: Introduction Of a Protein Tag For Subsequent Carbonyl Group ...mentioning
confidence: 99%