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

• Published in: Separation and purification technology Vol. 307; p. 122758
• Main Authors: Wang, Lei, Lan, Huiling, Guan, Weimin, Han, Juan, Liu, Yihan, Wang, Yu, Mao, Yanli, Wang, Yun
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 15.02.2023
• Subjects: Affinity ligand; Enzyme immobilization; High specificity; Purification; Thermo-responsive polymers; Affinity ligand; Thermo-responsive polymers; Enzyme immobilization; Purification; High specificity
• ISSN: 1383-5866; 1873-3794
• DOI: 10.1016/j.seppur.2022.122758

Aptamer-derived UCST-type thermoresponsive polymers was constructed to enhance the effective contact frequency and specific capture efficiency, and achieved the efficient and selective immobilization of Cyt C from low concentration and complexity of pig heart extract. [Display omitted] •An aptamer-derived UCST-type thermoresponsive polymer was synthesized.•PEAAG-apt allowed the specific and high-affinity binding of aptamer to Cyt C.•Spontaneously cooling induced the easy separation of the PEAAG-apt-immobilized Cyt C.•Immobilized Cyt C displayed high activity via maintaining its original conformation. 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.