One-step purification of target enzymes using interaction- and structure-based design of aptamer-affinity responsive polymers: Selective immobilization and enhanced stability

Abstract

One-step purification enzymes directly from crude extract has emerged as an attractive option by reducing the purification steps and cost. Despite extensive research, a viable one-step purification technique is far from being realized. The major obstacles are the low concentration and complexity of crude extract. To address these problems, a reversibly dissolved and highly specific affinity material, aptamer-derived UCST-type thermoresponsive polymer (PEAAG-apt), was engineered for enhancing the effective contact frequency and selective immobilization efficiency. PEAAG-apt allowed the specific and high-affinity binding of aptamer to cytochrome C (Cyt C) in soluble state above room temperature, where the selective immobilization efficiency was improved to 92 % in 30 min. Moreover, spontaneously cooling induced the easy separation of the PEAAG-apt-immobilized Cyt C directly from pig heart extract, compatible with the recycling process with up to 76 % catalytic activity in the sixth cycle. Compared with covalently immobilized Cyt C, PEAAG-apt-immobilized Cyt C displayed excellent activity via maintaining its original conformation and behaving as homogeneous catalysis. Such high enzymatic activity was further ensured even in aggressive pH and long-term storage. Our developed material opens a new avenue to highly efficient one-step purification of enzymes directly from crude extract.