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Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via imprinting of the widely used Strep-tag II affinity peptide (Strep-MIP). Electrochemistry, surface-sensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tagged enzymes: an O 2 -tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface‐exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high‐resolution structural data of flexible surface‐exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin‐based MIP that recognizes recombinant proteins via imprinting of the widely used Strep‐tag II affinity peptide (Strep‐MIP). Electrochemistry, surface‐sensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep‐MIP electrosynthesis. The functionality of this novel platform was verified with two Strep‐tagged enzymes: an O2‐tolerant [NiFe]‐hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep‐MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology. [ABSTRACT FROM AUTHOR]