{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,2,21]],"date-time":"2025-02-21T12:49:24Z","timestamp":1740142164132,"version":"3.37.3"},"reference-count":27,"publisher":"Bentham Science Publishers Ltd.","issue":"3","funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["2018R1D1A1B07040941"],"award-info":[{"award-number":["2018R1D1A1B07040941"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["eurekaselect.com"],"crossmark-restriction":true},"short-container-title":["PPL"],"published-print":{"date-parts":[[2021,5,7]]},"abstract":"<jats:sec>\n<jats:title>Background:<\/jats:title>\n<jats:p>Orientia tsutsugamushi (Ot) is an obligate, intracellular, gram-negative\nbacterium causing scrub typhus. Some of its encoded proteins play key roles in the adhesion and internalization\nof the Ot strain into host cells and are suitable resources for vaccine development and\ntools for scrub typhus diagnosis. Surface cell antigen (Sca) proteins, classified as autotransporter\n(AT) proteins, are one of the largest protein families involved in bacterial pathogenesis and can be\npromising candidates for vaccine development. These proteins are typically large and contain inhibitory\ndomains; therefore, recombinant proteins without such domains have been evaluated for\nthis purpose. However, the expression for recombinant Sca proteins containing the AT domain,\nwhich might largely affect their protective role against scrub typhus, has not been analyzed and optimized.<\/jats:p>\n<\/jats:sec>\n<jats:sec>\n<jats:title>Objective:<\/jats:title>\n<jats:p>In this study, we optimized expression and purification conditions for individual Ot Sca\nprotein fragments [ScaA (27\u20131461), ScaC (257\u2013526), ScaD (26\u2013998), and ScaE (35\u2013760)] harboring\nthe AT domain.<\/jats:p>\n<\/jats:sec>\n<jats:sec>\n<jats:title>Methods:<\/jats:title>\n<jats:p>To this end, we subcloned sequences of codon-optimized DNA encoding Sca protein\nfragments into the Escherichia coli expression vector. In addition, the expression condition for individual\nSca fragments was optimized, and the proteins were purified using one-step Ni-NTA column\nmethod. The purified fractions were re-folded by serial dilution method, followed by BCA\nquantification and densitometric analysis to estimate the yield and purity of proteins.<\/jats:p>\n<\/jats:sec>\n<jats:sec>\n<jats:title>Results:<\/jats:title>\n<jats:p>We prepared platforms for expression of recombinant Sca protein fragments [ScaA\n(27\u20131461), ScaC (257\u2013526), ScaD (26\u2013998), and ScaE (35\u2013760)] containing an AT domain without\nthe signal peptide and transmembrane (TM) domain. The protein yield per liter of culture with\n&gt;70% of purity was ScaC (257\u2013576), ScaE (35\u2013760), ScaD (26-998), and ScaA (27-1461) in order.<\/jats:p>\n<\/jats:sec>\n<jats:sec>\n<jats:title>Conclusion:<\/jats:title>\n<jats:p>Our results could be used to develop Sca AT-domain based vaccines and tools for\nscrub typhus diagnosis with rapid and cost-effective ways.<\/jats:p>\n<\/jats:sec>","DOI":"10.2174\/0929866527666200924144908","type":"journal-article","created":{"date-parts":[[2020,9,25]],"date-time":"2020-09-25T04:59:29Z","timestamp":1601009969000},"page":"241-248","update-policy":"https:\/\/doi.org\/10.2174\/bsp_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["An Inducible Expression System for Recombinant Sca Proteins with an Autotransporter Domain from Orientia Tsutsugamushi in Escherichia coli"],"prefix":"10.2174","volume":"28","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3550-1633","authenticated-orcid":true,"given":"Hyejin","family":"Cho","sequence":"first","affiliation":[{"name":"Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 46241, South Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3703-5461","authenticated-orcid":true,"given":"Kwang-Sun","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 46241, South Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"965","reference":[{"key":"ref=1","doi-asserted-by":"publisher","first-page":"14371","DOI":"10.1073\/pnas.0804090105","volume":"105","author":"Wagner S.","year":"2008","unstructured":"Wagner S.; Klepsch M.M.; Schlegel S.; Appel A.; Draheim R.; Tarry M.; H\u00f6gbom M.; van Wijk K.J.; Slotboom D.J.; Persson J.O.; de Gier J.W.; Tuning Escherichia coli for membrane protein overexpression. 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