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Bioconjug Chem. 2017 Oct 18;28(10):2560-2568. doi: 10.1021/acs.bioconjchem.7b00398. Epub 2017 Sep 15.

Affinity-Based Purification of Polyisocyanopeptide Bioconjugates.

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Department of Molecular Materials, Institute for Molecules and Materials, Radboud University , Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Geert Grooteplein 26, 6525 GA Nijmegen, The Netherlands.
Department of Biomedical Engineering and Institute for Complex Molecular Systems, Laboratory of Immunoengineering, Eindhoven University of Technology , De Zaale 15, 5612 AP Eindhoven, The Netherlands.
Mechano(bio)chemistry, Max Planck Institute of Colloids and Interfaces , Potsdam-Golm Science Park, 14424 Potsdam, Germany.


Water-soluble polyisocyanopeptides (PICs) are a new class of synthetic polymers that mimic natural protein-based filaments. Their unique semiflexible properties combined with a length of several hundred nanometers have recently enabled a number of biomedical applications ranging from tissue engineering to cancer immunotherapy. One crucial step toward the further development of PICs for these applications is the efficient and controlled synthesis and purification of PIC-biomolecule conjugates. Considering the large size of PICs and the biomolecules to be conjugated, conjugation reactions do usually not proceed to completion due to steric effects. As a consequence, purification of the reaction mixture is necessary to separate the obtained bioconjugates from unreacted biomolecules. As a direct result of the semiflexible nature of PICs, standard polymer and protein purification methods based on molecular weight have not been successful. Here, we introduce a new affinity-based purification method utilizing biotin as an affinity tag. PICs decorated with a controlled and tunable density of biotin molecules (biotinPICs) were efficiently bound to and eluted from a monoavidin resin in buffered aqueous solution. Using these biotinPICs, two different protein conjugates were synthesized, one carrying the enzyme alkaline phosphatase (PhoA) and the other T-cell activating anti-CD3 antibodies. The resulting biotinPIC-protein conjugates were successfully obtained in high purity (>90%) and without any loss of protein activity. The high purity greatly simplifies the analysis of biotinPIC bioconjugates, such as the determination of the average number of biomolecules conjugated per biotinPIC chain. Most importantly, it allows for the direct and straightforward application of the obtained bioconjugates in the desired applications. The new method developed may further be adapted for the purification of other advanced bioconjugates that are difficult to obtain in high purity with the available standard methods.

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