Park, Heesun Oh, Jeong-Seok Lee, Jonghwan Bang, Jinho Park, Keunwan Jeong, Suhyeon Park, Seho Woo, Jae-Sung Kim, Sunghyun 2024-08-08T01:00:07Z 2024-08-08T01:00:07Z 2024-08-08 2024-08 0961-8368 https://pubs.kist.re.kr/handle/201004/150371 Affinity precipitation is an attractive method for protein purification due to its many advantages, including the rapid capture of target proteins, simple processing, high specificity, and ease of scale-up. We previously reported a robust antibody purification method using Ca2+-dependent precipitation of ZZ-hCSQ2, a fusion protein of human calsequestrin 2, and the antibody-binding protein ZZ. However, the stability of this fusion protein was not sufficiently high for industrial use because the antibody recovery yield decreased to 60% after being reused 10 times. To identify a more stable calsequestrin (CSQ), we calculated Rosetta energy values for the folding stabilities of various CSQ homologs and selected human CSQ1 (hCSQ1) with lowest energy value (-992.6) as the new CSQ platform. We also identified that the linker sequence between ZZ and CSQ was vulnerable to proteases and alkaline pH by N-terminal protein sequencing. Therefore, we changed the linker to four asparagine (4N) sequences, which were shorter and less flexible than the previous glycine-rich linker. The new version of ZZ-CSQ, ZZ-4N-hCSQ1, was stable in a protease-containing conditioned medium obtained from the cultured Chinese hamster ovary cell or high pH condition (0.1M sodium hydroxide) for more than 5 days and could be reused at least 25 times for antibody purification without loss of recovery yield. The antibodies purified by ZZ-4N-hCSQ1 precipitation also showed greater purity (similar to 33.6-fold lower host cell DNA and similar to 6.4-fold lower host cell protein) than those purified by protein A chromatography. These data suggest that ZZ-4N-hCSQ1 precipitation is more efficient and can achieve cost-effectiveness of up to 12.5-fold cheaper than previous antibody purification methods and can lower the production costs of therapeutic antibodies. English Cold Spring Harbor Laboratory Press Stable and reusable calcium-responsive biopolymer for affinity precipitation of therapeutic antibodies Article 10.1002/pro.5066 1 Protein Science, v.33, no.8 Protein Science 33 8 N scie scopus 001280094500001 2-s2.0-85199979637 Biochemistry & Molecular Biology Biochemistry & Molecular Biology Article LIGAND MONOCLONAL-ANTIBODY PROTEIN-A PURIFICATION CALSEQUESTRIN antibody purification calcium-dependent calsequestrin cost-effectiveness protein engineering reuse Z-domain