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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Letter Article

An Inducible Expression System for Recombinant Sca Proteins with an Autotransporter Domain from Orientia Tsutsugamushi in Escherichia coli

Author(s): Hyejin Cho and Kwang-Sun Kim*

Volume 28, Issue 3, 2021

Published on: 24 September, 2020

Page: [241 - 248] Pages: 8

DOI: 10.2174/0929866527666200924144908

Price: $65

Abstract

Background: Orientia tsutsugamushi (Ot) is an obligate, intracellular, gram-negative bacterium causing scrub typhus. Some of its encoded proteins play key roles in the adhesion and internalization of the Ot strain into host cells and are suitable resources for vaccine development and tools for scrub typhus diagnosis. Surface cell antigen (Sca) proteins, classified as autotransporter (AT) proteins, are one of the largest protein families involved in bacterial pathogenesis and can be promising candidates for vaccine development. These proteins are typically large and contain inhibitory domains; therefore, recombinant proteins without such domains have been evaluated for this purpose. However, the expression for recombinant Sca proteins containing the AT domain, which might largely affect their protective role against scrub typhus, has not been analyzed and optimized.

Objective: In this study, we optimized expression and purification conditions for individual Ot Sca protein fragments [ScaA (27–1461), ScaC (257–526), ScaD (26–998), and ScaE (35–760)] harboring the AT domain.

Methods: To this end, we subcloned sequences of codon-optimized DNA encoding Sca protein fragments into the Escherichia coli expression vector. In addition, the expression condition for individual Sca fragments was optimized, and the proteins were purified using one-step Ni-NTA column method. The purified fractions were re-folded by serial dilution method, followed by BCA quantification and densitometric analysis to estimate the yield and purity of proteins.

Results: We prepared platforms for expression of recombinant Sca protein fragments [ScaA (27–1461), ScaC (257–526), ScaD (26–998), and ScaE (35–760)] containing an AT domain without the signal peptide and transmembrane (TM) domain. The protein yield per liter of culture with >70% of purity was ScaC (257–576), ScaE (35–760), ScaD (26-998), and ScaA (27-1461) in order.

Conclusion: Our results could be used to develop Sca AT-domain based vaccines and tools for scrub typhus diagnosis with rapid and cost-effective ways.

Keywords: Orientia tsutsugamushi, scrub typhus, Sca proteins, autotransporter domain, Escherichia coli, heterologous, expression.

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Graphical Abstract

[1]
Wagner, S.; Klepsch, M.M.; Schlegel, S.; Appel, A.; Draheim, R.; Tarry, M.; Högbom, M.; van Wijk, K.J.; Slotboom, D.J.; Persson, J.O.; de Gier, J.W. Tuning Escherichia coli for membrane protein overexpression. Proc. Natl. Acad. Sci. USA, 2008, 105(38), 14371-14376.
[http://dx.doi.org/10.1073/pnas.0804090105] [PMID: 18796603]
[2]
Rosano, G.L.; Ceccarelli, E.A. Recombinant protein expression in Escherichia coli: Advances and challenges. Front. Microbiol., 2014, 5, 172.
[http://dx.doi.org/10.3389/fmicb.2014.00172] [PMID: 24860555]
[3]
Gopal, G.J.; Kumar, A. Strategies for the production of recombinant protein in Escherichia coli. Protein J., 2013, 32(6), 419-425.
[http://dx.doi.org/10.1007/s10930-013-9502-5] [PMID: 23897421]
[4]
Schlegel, S.; Rujas, E.; Ytterberg, A.J.; Zubarev, R.A.; Luirink, J.; de Gier, J.W. Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels. Microb. Cell Fact., 2013, 12, 24.
[http://dx.doi.org/10.1186/1475-2859-12-24] [PMID: 23497240]
[5]
Hjelm, A.; Karyolaimos, A.; Zhang, Z.; Rujas, E.; Vikström, D.; Slotboom, D.J.; de Gier, J.W. Tailoring Escherichia coli for the l-rhamnose PBAD promoter-based production of membrane and secretory proteins. ACS Synth. Biol., 2017, 6(6), 985-994.
[http://dx.doi.org/10.1021/acssynbio.6b00321] [PMID: 28226208]
[6]
Baumgarten, T.; Ytterberg, A.J.; Zubarev, R.A.; de Gier, J.W. Optimizing recombinant protein production in the Escherichia coli periplasm alleviates stress. Appl. Environ. Microbiol., 2018, 84(12), e00270-e18.
[http://dx.doi.org/10.1128/AEM.00270-18] [PMID: 29654183]
[7]
Seong, S.Y.; Choi, M.S.; Kim, I.S. Orientia tsutsugamushi infection: Overview and immune responses. Microbes Infect., 2001, 3(1), 11-21.
[http://dx.doi.org/10.1016/S1286-4579(00)01352-6] [PMID: 11226850]
[8]
Kweon, S.S.; Choi, J.S.; Lim, H.S.; Kim, J.R.; Kim, K.Y.; Ryu, S.Y.; Yoo, H.S.; Park, O. Rapid increase of scrub typhus, South Korea, 2001-2006. Emerg. Infect. Dis., 2009, 15(7), 1127-1129.
[http://dx.doi.org/10.3201/eid1507.080399] [PMID: 19624938]
[9]
Paris, D.H.; Shelite, T.R.; Day, N.P.; Walker, D.H. Unresolved problems related to scrub typhus: A seriously neglected life-threatening disease. Am. J. Trop. Med. Hyg., 2013, 89(2), 301-307.
[http://dx.doi.org/10.4269/ajtmh.13-0064] [PMID: 23926142]
[10]
Seong, S.Y.; Huh, M.S.; Jang, W.J.; Park, S.G.; Kim, J.G.; Woo, S.G.; Choi, M.S.; Kim, I.S.; Chang, W.H. Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56. Infect. Immun., 1997, 65(4), 1541-1545.
[http://dx.doi.org/10.1128/IAI.65.4.1541-1545.1997] [PMID: 9119501]
[11]
Seong, S.Y.; Kim, H.R.; Huh, M.S.; Park, S.G.; Kang, J.S.; Han, T.H.; Choi, M.S.; Chang, W.H.; Kim, I.S. Induction of neutralizing antibody in mice by immunization with recombinant 56 kDa protein of Orientia tsutsugamushi. Vaccine, 1997, 15(16), 1741-1747.
[http://dx.doi.org/10.1016/S0264-410X(97)00112-6] [PMID: 9364677]
[12]
Seong, S.Y.; Kim, M.K.; Lee, S.M.; Odgerel, Z.; Choi, M.S.; Han, T.H.; Kim, I.S.; Kang, J.S.; Lim, B.U. Neutralization epitopes on the antigenic domain II of the Orientia tsutsugamushi 56-kDa protein revealed by monoclonal antibodies. Vaccine, 2000, 19(1), 2-9.
[http://dx.doi.org/10.1016/S0264-410X(00)00167-5] [PMID: 10924780]
[13]
Henderson, I.R.; Navarro-Garcia, F.; Desvaux, M.; Fernandez, R.C.; Ala’Aldeen, D. Type V protein secretion pathway: The autotransporter story. Microbiol. Mol. Biol. Rev., 2004, 68(4), 692-744.
[http://dx.doi.org/10.1128/MMBR.68.4.692-744.2004] [PMID: 15590781]
[14]
Paxman, J.J.; Lo, A.W.; Sullivan, M.J.; Panjikar, S.; Kuiper, M.; Whitten, A.E.; Wang, G.; Luan, C.H.; Moriel, D.G.; Tan, L.; Peters, K.M.; Phan, M.D.; Gee, C.L.; Ulett, G.C.; Schembri, M.A.; Heras, B. Unique structural features of a bacterial autotransporter adhesin suggest mechanisms for interaction with host macromolecules. Nat. Commun., 2019, 10(1), 1967.
[http://dx.doi.org/10.1038/s41467-019-09814-6] [PMID: 31036849]
[15]
Wells, T.J.; Tree, J.J.; Ulett, G.C.; Schembri, M.A. Autotransporter proteins: Novel targets at the bacterial cell surface. FEMS Microbiol. Lett., 2007, 274(2), 163-172.
[http://dx.doi.org/10.1111/j.1574-6968.2007.00833.x] [PMID: 17610513]
[16]
Henderson, I.R.; Nataro, J.P. Virulence functions of autotransporter proteins. Infect. Immun., 2001, 69(3), 1231-1243.
[http://dx.doi.org/10.1128/IAI.69.3.1231-1243.2001] [PMID: 11179284]
[17]
Ha, N.Y.; Cho, N.H.; Kim, Y.S.; Choi, M.S.; Kim, I.S. An autotransporter protein from Orientia tsutsugamushi mediates adherence to nonphagocytic host cells. Infect. Immun., 2011, 79(4), 1718-1727.
[http://dx.doi.org/10.1128/IAI.01239-10] [PMID: 21282412]
[18]
Cherry, J.D.; Gornbein, J.; Heininger, U.; Stehr, K. A search for serologic correlates of immunity to Bordetella pertussis cough illnesses. Vaccine, 1998, 16(20), 1901-1906.
[http://dx.doi.org/10.1016/S0264-410X(98)00226-6] [PMID: 9796041]
[19]
Hewlett, E.L.; Halperin, S.A. Serological correlates of immunity to Bordetella pertussis. Vaccine, 1998, 16(20), 1899-1900.
[http://dx.doi.org/10.1016/S0264-410X(98)00228-X] [PMID: 9796040]
[20]
Cutter, D.; Mason, K.W.; Howell, A.P.; Fink, D.L.; Green, B.A.; St Geme, J.W.S., III. Immunization with Haemophilus influenzae Hap adhesin protects against nasopharyngeal colonization in experimental mice. J. Infect. Dis., 2002, 186(8), 1115-1121.
[http://dx.doi.org/10.1086/344233] [PMID: 12355362]
[21]
Ha, N.Y.; Kim, Y.; Choi, J.H.; Choi, M.S.; Kim, I.S.; Kim, Y.S.; Cho, N.H. Detection of antibodies against Orientia tsutsugamushi Sca proteins in scrub typhus patients and genetic variation of sca genes of different strains. Clin. Vaccine Immunol., 2012, 19(9), 1442-1451.
[http://dx.doi.org/10.1128/CVI.00285-12] [PMID: 22787193]
[22]
Letunic, I.; Doerks, T.; Bork, P. SMART: Recent updates, new developments and status in 2015. Nucleic Acids Res., 2015, 43(Database issue), D257-D260.
[http://dx.doi.org/10.1093/nar/gku949] [PMID: 25300481]
[23]
UniProt Consortium, T. UniProt: The universal protein knowledgebase. Nucleic Acids Res., 2018, 46(5), 2699.
[http://dx.doi.org/10.1093/nar/gky092] [PMID: 29425356]
[24]
Baneyx, F.; Mujacic, M. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol., 2004, 22(11), 1399-1408.
[http://dx.doi.org/10.1038/nbt1029] [PMID: 15529165]
[25]
De Geyter, J.; Tsirigotaki, A.; Orfanoudaki, G.; Zorzini, V.; Economou, A.; Karamanou, S. Protein folding in the cell envelope of Escherichia coli. Nat. Microbiol., 2016, 1(8), 16107.
[http://dx.doi.org/10.1038/nmicrobiol.2016.107] [PMID: 27573113]
[26]
Cho, N.H.; Kim, H.R.; Lee, J.H.; Kim, S.Y.; Kim, J.; Cha, S.; Kim, S.Y.; Darby, A.C.; Fuxelius, H.H.; Yin, J.; Kim, J.H.; Kim, J.; Lee, S.J.; Koh, Y.S.; Jang, W.J.; Park, K.H.; Andersson, S.G.; Choi, M.S.; Kim, I.S. The Orientia tsutsugamushi genome reveals massive proliferation of conjugative type IV secretion system and host-cell interaction genes. Proc. Natl. Acad. Sci. USA, 2007, 104(19), 7981-7986.
[http://dx.doi.org/10.1073/pnas.0611553104] [PMID: 17483455]
[27]
Benz, I.; Schmidt, M.A. Structures and functions of autotransporter proteins in microbial pathogens. Int. J. Med. Microbiol., 2011, 301(6), 461-468.
[http://dx.doi.org/10.1016/j.ijmm.2011.03.003] [PMID: 21616712]

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