{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T16:01:26Z","timestamp":1790438486799,"version":"4.1.0"},"reference-count":11,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"vor","delay-in-days":10,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Recombinant protein purification is a critical step prior to their use in therapeutics and diagnostics. Affinity and ion\u2010exchange chromatography are among the most widely used purification strategies; however, both rely on extensive trial\u2010and\u2010error optimization and require chemical additives or high salt concentrations for protein elution. These conditions necessitate additional, time\u2010consuming downstream dialysis steps to remove residual chemicals. To address these limitations, we propose a light\u2010triggered protein elution strategy that eliminates the need for chemical elution agents and simplifies downstream processing. This approach exploits PhoCl1, a photocleavable protein that undergoes light\u2010induced cleavage into an N\u2010terminal empty barrel and a short C\u2010terminal peptide fragment (Zhang et\u00a0al., 2017; Lu et\u00a0al., 2021). We engineered a fusion construct in which a 6\u00d7His tag is positioned at the N\u2010terminus of PhoCl1, while the protein of interest (POI) is fused to its C\u2010terminus (6\u00d7His\u2013PhoCl1\u2013POI). Following immobilization of the whole\u2010cell lysate on Ni\u2010NTA resin and removal of nonspecifically bound proteins, light exposure induces PhoCl1 cleavage. The His\u2010tagged N\u2010terminal fragment remains bound to the resin, while the POI is selectively released into the supernatant. Using this method, we have achieved protein purities exceeding 90%, demonstrating a rapid, reagent\u2010free, and highly efficient alternative to conventional protein purification workflows. Here, for demonstration purposes, we have included a POI, riboflavin kinase (\n                    <jats:italic>Mj<\/jats:italic>\n                    RibK) found in\n                    <jats:italic>Methanocaldococcus jannaschii<\/jats:italic>\n                    . In this protocol, we describe the design, expression, and characterization of PhoCl1 fusion proteins using an intracellular expression system in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    , with all steps optimized for cytosolic production. \u00a9 2026 Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Light\u2010Activated Affinity\u2010Based Recombinant Protein Purification\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70440","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T11:41:28Z","timestamp":1789126888000},"source":"Crossref","is-referenced-by-count":0,"title":["Light\u2010Activated Molecular Purification (LAMP): A Photo\u2010Responsive Affinity\u2010Chromatography Protocol for Recombinant Protein Purification"],"prefix":"10.1002","volume":"6","author":[{"given":"Krishna","family":"Agrawal","sequence":"first","affiliation":[{"name":"Biological Sciences and Engineering IIT Gandhinagar  Gujarat India"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aishwarya","family":"Satyanarayana","sequence":"additional","affiliation":[{"name":"Biological Sciences and Engineering IIT Gandhinagar  Gujarat India"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yashwant","family":"Kumar","sequence":"additional","affiliation":[{"name":"Chemical Engineering IIT Gandhinagar  Gujarat India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karthik","family":"Pushpavanam","sequence":"additional","affiliation":[{"name":"Chemical Engineering IIT Gandhinagar  Gujarat India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,11]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF02779018"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpba.2015.02.037"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471140864.ps1001s68"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471140864.ps0611s61"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.bioconjchem.4c00284"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1039\/d1sc01059j"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010024\u201055212\u2010y"},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.drudis.2013.11.008"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb1006s85"},{"key":"e_1_2_6_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/cben.201600002"},{"key":"e_1_2_6_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4222"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70440","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70440","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70440","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T15:54:55Z","timestamp":1790438095000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70440"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9]]},"references-count":11,"journal-issue":{"issue":"9","published-print":{"date-parts":[[2026,9]]}},"alternative-id":["10.1002\/cpz1.70440"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70440","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9]]},"article-number":"e70440"}}