{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T01:59:13Z","timestamp":1785376753980,"version":"3.55.0"},"reference-count":27,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T00:00:00Z","timestamp":1778803200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T00:00:00Z","timestamp":1778803200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100004735","name":"Natural Science Foundation of Hunan Province","doi-asserted-by":"publisher","award":["2024JJ4053"],"award-info":[{"award-number":["2024JJ4053"]}],"id":[{"id":"10.13039\/501100004735","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2026,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Shark\u2010derived variable new antigen receptors (VNARs) are promising single\u2010domain antibodies due to their compact size, high stability, and cryptic epitope access. While conventional screening via phage display is highly effective, complementary approaches can further expand the methodological toolkit. We developed a phage\u2010free bacterial two\u2010hybrid (BATCH) platform for rapid antigen\u2010specific VNAR discovery from immunized\n                    <jats:italic>Chiloscyllium plagiosum<\/jats:italic>\n                    . A Cre recombinase\u2010immunized spleen cDNA library (~10\n                    <jats:sup>8<\/jats:sup>\n                    diversity) was cloned into pUT18c, screened against pKT25c\u2010Cre in BTH101 cells using dual lacZ and CmR readouts enhanced by triple\n                    <jats:italic>lacO<\/jats:italic>\n                    sites and cAMP feedback, yielding two unique binders (VNAR1 and VNAR2) in 16\u2009h. AlphaFold3 modeling highlighted CDR3 dominance in the Cre paratope, with C84\u2013C90 disulfide stabilizing the loop; mutagenesis halved enzyme\u2010linked immunosorbent assay affinity and disrupted BATCH function but retained transcriptional activation in 293 T cells (~27% EGFP+). This 1\u2010 to 2\u2010day screening phase provides a streamlined alternative to conventional methods, facilitating the rapid identification of stable VNARs. By leveraging a simplified in vivo selection process, this BATCH\u2010based platform offers a practical tool for VNAR discovery in various therapeutics and diagnostics applications.\n                  <\/jats:p>","DOI":"10.1002\/pro.70620","type":"journal-article","created":{"date-parts":[[2026,5,16]],"date-time":"2026-05-16T04:01:27Z","timestamp":1778904087000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A rapid phage\u2010free platform for antigen\u2010specific\n                    <scp>VNAR<\/scp>\n                    screening with\n                    <scp>BATCH<\/scp>\n                    system"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-0554-7853","authenticated-orcid":false,"given":"Wenjie","family":"Tang","sequence":"first","affiliation":[{"name":"MOE Key Laboratory of Rare Pediatric Diseases, Center for Medical Genetics, School of Life Sciences Central South University  Changsha Hunan China"},{"name":"College of Life Sciences and Medicine, Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine Zhejiang Sci\u2010Tech University  Hangzhou Zhejiang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinming","family":"Hu","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Rare Pediatric Diseases, Center for Medical Genetics, School of Life Sciences Central South University  Changsha Hunan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanping","family":"Zhu","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Rare Pediatric Diseases, Center for Medical Genetics, School of Life Sciences Central South University  Changsha Hunan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7419-6232","authenticated-orcid":false,"given":"Yiqian","family":"Zeng","sequence":"additional","affiliation":[{"name":"Department of Trauma Intensive Care Unit, Zhuzhou Hospital Affiliated to Xiangya School of Medicine Central South University  Zhuzhou Hunan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiyuan","family":"Li","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Rare Pediatric Diseases, Center for Medical Genetics, School of Life Sciences Central South University  Changsha Hunan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingfeng","family":"Gu","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Rare Pediatric Diseases, Center for Medical Genetics, School of Life Sciences Central South University  Changsha Hunan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junjie","family":"Yang","sequence":"additional","affiliation":[{"name":"College of Life Sciences and Medicine, Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine Zhejiang Sci\u2010Tech University  Hangzhou Zhejiang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo","family":"Zhong","sequence":"additional","affiliation":[{"name":"College of Life Sciences and Medicine, Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine Zhejiang Sci\u2010Tech University  Hangzhou Zhejiang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yike","family":"Chen","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Rare Pediatric Diseases, Center for Medical Genetics, School of Life Sciences Central South University  Changsha Hunan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-5221-333X","authenticated-orcid":false,"given":"Xianxin","family":"Yan","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Rare Pediatric Diseases, Center for Medical Genetics, School of Life Sciences Central South University  Changsha Hunan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianqing","family":"Chen","sequence":"additional","affiliation":[{"name":"College of Life Sciences and Medicine, Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine Zhejiang Sci\u2010Tech University  Hangzhou Zhejiang China"},{"name":"Zhejiang Sci\u2010Tech University Shaoxing Academy of Biomedicine Co., Ltd  Shaoxing Zhejiang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaofeng","family":"Jiang","sequence":"additional","affiliation":[{"name":"College of Life Sciences and Medicine, Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine Zhejiang Sci\u2010Tech University  Hangzhou Zhejiang China"},{"name":"Zhejiang Sci\u2010Tech University Shaoxing Academy of Biomedicine Co., Ltd  Shaoxing Zhejiang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhengbing","family":"Lyu","sequence":"additional","affiliation":[{"name":"College of Life Sciences and Medicine, Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine Zhejiang Sci\u2010Tech University  Hangzhou Zhejiang China"},{"name":"Zhejiang Sci\u2010Tech University Shaoxing Academy of Biomedicine Co., Ltd  Shaoxing Zhejiang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Faxiang","family":"Li","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Rare Pediatric Diseases, Center for Medical Genetics, School of Life Sciences Central South University  Changsha Hunan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guodong","family":"Chen","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Rare Pediatric Diseases, Center for Medical Genetics, School of Life Sciences Central South University  Changsha Hunan China"},{"name":"Furong Laboratory  Changsha Hunan China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,5,15]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymeth.2012.07.018"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-023-30623-x"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0213394"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/374168a0"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40364-025-00755-5"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2018.1489633"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.fsi.2023.108535"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.3390\/md21040237"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.10.5752"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-2581-1_10"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2025.140290"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1080\/10826068.2019.1566145"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-3469-1_2"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0294874"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.abb.2012.04.023"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.prot108601"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2016.01864"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2006.12.045"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1110\/ps.051709505"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1038\/srep24648"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-27611-y"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1093\/bib\/bbac520"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2022.105736"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.3390\/md20050307"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.fsi.2024.109661"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.fsi.2025.110411"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.4161\/19420862.2015.989032"}],"container-title":["Protein Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pro.70620","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/pro.70620","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pro.70620","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T01:33:42Z","timestamp":1785375222000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/pro.70620"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,15]]},"references-count":27,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2026,6]]}},"alternative-id":["10.1002\/pro.70620"],"URL":"https:\/\/doi.org\/10.1002\/pro.70620","archive":["Portico"],"relation":{},"ISSN":["0961-8368","1469-896X"],"issn-type":[{"value":"0961-8368","type":"print"},{"value":"1469-896X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,15]]},"assertion":[{"value":"2026-01-26","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-05-05","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-05-15","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e70620"}}