{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T08:09:36Z","timestamp":1780387776645,"version":"3.54.1"},"reference-count":67,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T00:00:00Z","timestamp":1763683200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T00:00:00Z","timestamp":1763683200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Proteins"],"published-print":{"date-parts":[[2026,4]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>The generation of antibodies against integral membrane proteins (IMPs) presents unique challenges due to IMPs' low natural abundance, reduced biosynthesis rate, limited level of extraction, and low purification yield. That stems from their intricate structure defining the conformal epitope location. This study introduces a novel approach to identify crucial amino acid residues involved in the interaction between antibodies and tumor\u2010associated transmembrane antigens based on utilizing a prokaryotic cell\u2010free expression (CFPE) system. We considered human transmembrane prostate androgen\u2010induced protein 1 (hTMEPA1) as the target antigen. It is a single\u2010pass \u03b1\u2010helical protein possessing hallmarks of a promising cancer biomarker. A several\u2010step procedure was implemented to determine key residues of hTMEPA1. We created mimotope mAbs based on in silico analysis of immunogenic regions followed by target protein antigen production in the CFPE system and further antibody characterization. The results demonstrated an exceptional way for robust and high\u2010throughput target protein production combined with in\u2010depth biophysical and immunochemical characterization of therapeutic and diagnostic antibody\u2010based molecules.<\/jats:p>","DOI":"10.1002\/prot.70088","type":"journal-article","created":{"date-parts":[[2025,11,22]],"date-time":"2025-11-22T05:40:37Z","timestamp":1763790037000},"page":"961-972","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Elucidation of Critical Amino Acid Residues for Antibody and Transmembrane Antigen Interactions via Cell\u2010Free Protein Expression\u2010Based Approach"],"prefix":"10.1002","volume":"94","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-1866-4283","authenticated-orcid":false,"given":"Mikhail S.","family":"Karbyshev","sequence":"first","affiliation":[{"name":"Department of Biotechnology Moscow Polytechnic University (Moscow Polytech)  Moscow Russian Federation"},{"name":"Department of Biochemistry and Molecular Biology Pirogov Russian National Research Medical University  Moscow Russian Federation"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristina O.","family":"Baskakova","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology Pirogov Russian National Research Medical University  Moscow Russian Federation"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pavel K.","family":"Kuzmichev","sequence":"additional","affiliation":[{"name":"Research \u0421enter for Molecular Mechanisms of Aging and Age\u2010Related Diseases  Moscow Russian Federation"},{"name":"Research Center for Molecular Mechanisms of Aging and Age\u2010Related Diseases Moscow Institute of Physics and Technology  Dolgoprudniy Russian Federation"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivan S.","family":"Okhrimenko","sequence":"additional","affiliation":[{"name":"Research \u0421enter for Molecular Mechanisms of Aging and Age\u2010Related Diseases  Moscow Russian Federation"},{"name":"Research Center for Molecular Mechanisms of Aging and Age\u2010Related Diseases Moscow Institute of Physics and Technology  Dolgoprudniy Russian Federation"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eduard V.","family":"Bocharov","sequence":"additional","affiliation":[{"name":"Research \u0421enter for Molecular Mechanisms of Aging and Age\u2010Related Diseases  Moscow Russian Federation"},{"name":"Research Center for Molecular Mechanisms of Aging and Age\u2010Related Diseases Moscow Institute of Physics and Technology  Dolgoprudniy Russian Federation"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Irina M.","family":"Kolesnikova","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology Pirogov Russian National Research Medical University  Moscow Russian Federation"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olga P.","family":"Shatova","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology Pirogov Russian National Research Medical University  Moscow Russian Federation"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aleksandr V.","family":"Shestopalov","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology Pirogov Russian National Research Medical University  Moscow Russian Federation"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergei A.","family":"Rumyantsev","sequence":"additional","affiliation":[{"name":"Department of Oncology, Hematology and Radiation Therapy Pirogov Russian National Research Medical University  Moscow Russian Federation"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,11,21]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0117955"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-bioeng-092115-025322"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-323-95072-5.00008-0"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1134\/S0006297923090031"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.eng.2020.11.013"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.trecan.2024.07.002"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1007\/s40259-024-00682-1"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.2174\/0929866528666210415120234"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/cplu.202300678"},{"key":"e_1_2_11_11_1","doi-asserted-by":"crossref","unstructured":"H. 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