{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T01:00:07Z","timestamp":1774314007773,"version":"3.50.1"},"reference-count":35,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Microb Cell Fact"],"published-print":{"date-parts":[[2010,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>Leptospirosis, a zoonosis caused by <jats:italic>Leptospira<\/jats:italic> spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in <jats:italic>Escherichia coli<\/jats:italic> have demonstrated promising results, albeit with variable efficacy. <jats:italic>Pichia pastoris<\/jats:italic> is an alternative host with several advantages for the production of recombinant proteins.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>The vaccine candidates LigANI and LipL32 were cloned and expressed in <jats:italic>P. pastoris<\/jats:italic> as secreted proteins. Large-scale expression resulted in a yield of 276 mg\/L for LigANI and 285 mg\/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>The expression of LigANI and LipL32 in <jats:italic>P. pastoris<\/jats:italic> resulted in a significant increase in yield compared to expression in <jats:italic>E. coli<\/jats:italic>. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1475-2859-9-98","type":"journal-article","created":{"date-parts":[[2010,12,7]],"date-time":"2010-12-07T07:14:28Z","timestamp":1291706068000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast Pichia pastoris"],"prefix":"10.1186","volume":"9","author":[{"given":"Daiane D","family":"Hartwig","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tha\u00eds L","family":"Oliveira","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fabiana K","family":"Seixas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karine M","family":"Forster","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Caroline","family":"Rizzi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cl\u00e1udia P","family":"Hartleben","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alan JA","family":"McBride","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Odir A","family":"Dellagostin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2010,12,6]]},"reference":[{"issue":"12","key":"488_CR1","doi-asserted-by":"publisher","first-page":"757","DOI":"10.1016\/S1473-3099(03)00830-2","volume":"3","author":"AR Bharti","year":"2003","unstructured":"Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, et al.: Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis. 2003, 3 (12): 757-771. 10.1016\/S1473-3099(03)00830-2.","journal-title":"Lancet Infect Dis"},{"issue":"3-4","key":"488_CR2","doi-asserted-by":"publisher","first-page":"287","DOI":"10.1016\/j.vetmic.2009.03.012","volume":"140","author":"B Adler","year":"2010","unstructured":"Adler B, de la Pena Moctezuma A: Leptospira and leptospirosis. Vet Microbiol. 2010, 140 (3-4): 287-296. 10.1016\/j.vetmic.2009.03.012.","journal-title":"Vet Microbiol"},{"key":"488_CR3","volume-title":"Leptospira and leptospirosis","author":"SB Faine","year":"1999","unstructured":"Faine SB, Adler B, Bolin C, Perolat P: Leptospira and leptospirosis. 1999, Melbourne: MediSci, 2","edition":"2"},{"issue":"4","key":"488_CR4","doi-asserted-by":"publisher","first-page":"e228","DOI":"10.1371\/journal.pntd.0000228","volume":"2","author":"RB Reis","year":"2008","unstructured":"Reis RB, Ribeiro GS, Felzemburgh RD, Santana FS, Mohr S, Melendez AX, Queiroz A, Santos AC, Ravines RR, Tassinari WS, et al.: Impact of environment and social gradient on leptospira infection in urban slums. PLoS Negl Trop Dis. 2008, 2 (4): e228- 10.1371\/journal.pntd.0000228.","journal-title":"PLoS Negl Trop Dis"},{"issue":"5","key":"488_CR5","doi-asserted-by":"publisher","first-page":"376","DOI":"10.1097\/01.qco.0000178824.05715.2c","volume":"18","author":"AJ McBride","year":"2005","unstructured":"McBride AJ, Athanazio DA, Reis MG, Ko AI: Leptospirosis. Curr Opin Infect Dis. 2005, 18 (5): 376-386. 10.1097\/01.qco.0000178824.05715.2c.","journal-title":"Curr Opin Infect Dis"},{"issue":"3","key":"488_CR6","doi-asserted-by":"publisher","first-page":"505","DOI":"10.3201\/eid1403.071064","volume":"14","author":"EL Gouveia","year":"2008","unstructured":"Gouveia EL, Metcalfe J, de Carvalho AL, Aires TS, Villasboas-Bisneto JC, Queirroz A, Santos AC, Salgado K, Reis MG, Ko AI: Leptospirosis-associated Severe Pulmonary Hemorrhagic Syndrome, Salvador, Brazil. Emerg Infect Dis. 2008, 14 (3): 505-508. 10.3201\/eid1403.071064.","journal-title":"Emerg Infect Dis"},{"issue":"3","key":"488_CR7","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1086\/427110","volume":"40","author":"ER Segura","year":"2005","unstructured":"Segura ER, Ganoza CA, Campos K, Ricaldi JN, Torres S, Silva H, Cespedes MJ, Matthias MA, Swancutt MA, Lopez Linan R, et al.: Clinical spectrum of pulmonary involvement in leptospirosis in a region of endemicity, with quantification of leptospiral burden. Clin Infect Dis. 2005, 40 (3): 343-351. 10.1086\/427110.","journal-title":"Clin Infect Dis"},{"issue":"12","key":"488_CR8","doi-asserted-by":"crossref","first-page":"6572","DOI":"10.1128\/IAI.67.12.6572-6582.1999","volume":"67","author":"DA Haake","year":"1999","unstructured":"Haake DA, Mazel MK, McCoy AM, Milward F, Chao G, Matsunaga J, Wagar EA: Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection. Infect Immun. 1999, 67 (12): 6572-6582.","journal-title":"Infect Immun"},{"issue":"11","key":"488_CR9","doi-asserted-by":"publisher","first-page":"2277","DOI":"10.1016\/j.vaccine.2009.12.071","volume":"28","author":"W Yan","year":"2010","unstructured":"Yan W, Faisal SM, McDonough SP, Chang CF, Pan MJ, Akey B, Chang YF: Identification and characterization of OmpA-like proteins as novel vaccine candidates for Leptospirosis. Vaccine. 2010, 28 (11): 2277-2283. 10.1016\/j.vaccine.2009.12.071.","journal-title":"Vaccine"},{"key":"488_CR10","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1186\/1475-2859-6-39","volume":"6","author":"Z Wang","year":"2007","unstructured":"Wang Z, Jin L, Wegrzyn A: Leptospirosis vaccines. Microb Cell Fact. 2007, 6: 39- 10.1186\/1475-2859-6-39.","journal-title":"Microb Cell Fact"},{"issue":"11-12","key":"488_CR11","doi-asserted-by":"publisher","first-page":"1545","DOI":"10.1016\/j.vaccine.2003.10.007","volume":"22","author":"N Koizumi","year":"2004","unstructured":"Koizumi N, Watanabe H: Leptospiral immunoglobulin-like proteins elicit protective immunity. Vaccine. 2004, 22 (11-12): 1545-1552. 10.1016\/j.vaccine.2003.10.007.","journal-title":"Vaccine"},{"issue":"2","key":"488_CR12","doi-asserted-by":"publisher","first-page":"230","DOI":"10.1016\/j.micinf.2008.11.008","volume":"11","author":"W Yan","year":"2009","unstructured":"Yan W, Faisal SM, McDonough SP, Divers TJ, Barr SC, Chang CF, Pan MJ, Chang YF: Immunogenicity and protective efficacy of recombinant Leptospira immunoglobulin-like protein B (rLigB) in a hamster challenge model. Microbes Infect. 2009, 11 (2): 230-237. 10.1016\/j.micinf.2008.11.008.","journal-title":"Microbes Infect"},{"issue":"33","key":"488_CR13","doi-asserted-by":"publisher","first-page":"6277","DOI":"10.1016\/j.vaccine.2007.05.053","volume":"25","author":"EF Silva","year":"2007","unstructured":"Silva EF, Medeiros MA, McBride AJ, Matsunaga J, Esteves GS, Ramos JG, Santos CS, Croda J, Homma A, Dellagostin OA, et al.: The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis. Vaccine. 2007, 25 (33): 6277-6286. 10.1016\/j.vaccine.2007.05.053.","journal-title":"Vaccine"},{"issue":"3","key":"488_CR14","doi-asserted-by":"publisher","first-page":"1745","DOI":"10.1128\/IAI.74.3.1745-1750.2006","volume":"74","author":"RU Palaniappan","year":"2006","unstructured":"Palaniappan RU, McDonough SP, Divers TJ, Chen CS, Pan MJ, Matsumoto M, Chang YF: Immunoprotection of recombinant leptospiral immunoglobulin-like protein A against Leptospira interrogans serovar Pomona infection. Infect Immun. 2006, 74 (3): 1745-1750. 10.1128\/IAI.74.3.1745-1750.2006.","journal-title":"Infect Immun"},{"issue":"9","key":"488_CR15","doi-asserted-by":"publisher","first-page":"796","DOI":"10.1590\/S0100-879X2009005000013","volume":"42","author":"CY Feng","year":"2009","unstructured":"Feng CY, Li QT, Zhang XY, Dong K, Hu BY, Guo XK: Immune strategies using single-component LipL32 and multi-component recombinant LipL32-41-OmpL1 vaccines against leptospira. Braz J Med Biol Res. 2009, 42 (9): 796-803. 10.1590\/S0100-879X2009005000013.","journal-title":"Braz J Med Biol Res"},{"issue":"1","key":"488_CR16","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1016\/j.vaccine.2007.10.052","volume":"26","author":"FK Seixas","year":"2007","unstructured":"Seixas FK, da Silva EF, Hartwig DD, Cerqueira GM, Amaral M, Fagundes MQ, Dossa RG, Dellagostin OA: Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge. Vaccine. 2007, 26 (1): 88-95. 10.1016\/j.vaccine.2007.10.052.","journal-title":"Vaccine"},{"issue":"7","key":"488_CR17","doi-asserted-by":"publisher","first-page":"4062","DOI":"10.1128\/IAI.73.7.4062-4069.2005","volume":"73","author":"C Branger","year":"2005","unstructured":"Branger C, Chatrenet B, Gauvrit A, Aviat F, Aubert A, Bach JM, Andre-Fontaine G: Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1. Infect Immun. 2005, 73 (7): 4062-4069. 10.1128\/IAI.73.7.4062-4069.2005.","journal-title":"Infect Immun"},{"issue":"1","key":"488_CR18","doi-asserted-by":"publisher","first-page":"86","DOI":"10.1016\/S0264-410X(00)00129-8","volume":"19","author":"C Sonrier","year":"2000","unstructured":"Sonrier C, Branger C, Michel V, Ruvoen-Clouet N, Ganiere JP, Andre-Fontaine G: Evidence of cross-protection within Leptospira interrogans in an experimental model. Vaccine. 2000, 19 (1): 86-94. 10.1016\/S0264-410X(00)00129-8.","journal-title":"Vaccine"},{"key":"488_CR19","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1186\/1475-2859-9-27","volume":"9","author":"HP Sorensen","year":"2010","unstructured":"Sorensen HP: Towards universal systems for recombinant gene expression. Microb Cell Fact. 2010, 9: 27- 10.1186\/1475-2859-9-27.","journal-title":"Microb Cell Fact"},{"key":"488_CR20","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1186\/1475-2859-5-17","volume":"5","author":"O Cos","year":"2006","unstructured":"Cos O, Ramon R, Montesinos JL, Valero F: Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: a review. Microb Cell Fact. 2006, 5: 17- 10.1186\/1475-2859-5-17.","journal-title":"Microb Cell Fact"},{"issue":"1","key":"488_CR21","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1385\/MB:16:1:23","volume":"16","author":"JM Cregg","year":"2000","unstructured":"Cregg JM, Cereghino JL, Shi J, Higgins DR: Recombinant protein expression in Pichia pastoris. Mol Biotechnol. 2000, 16 (1): 23-52. 10.1385\/MB:16:1:23.","journal-title":"Mol Biotechnol"},{"issue":"2","key":"488_CR22","doi-asserted-by":"publisher","first-page":"277","DOI":"10.1016\/j.vaccine.2007.10.029","volume":"26","author":"SM Faisal","year":"2008","unstructured":"Faisal SM, Yan W, Chen CS, Palaniappan RU, McDonough SP, Chang YF: Evaluation of protective immunity of Leptospira immunoglobulin like protein A (LigA) DNA vaccine against challenge in hamsters. Vaccine. 2008, 26 (2): 277-287. 10.1016\/j.vaccine.2007.10.029.","journal-title":"Vaccine"},{"issue":"3","key":"488_CR23","doi-asserted-by":"publisher","first-page":"378","DOI":"10.1016\/j.vaccine.2008.10.089","volume":"27","author":"SM Faisal","year":"2009","unstructured":"Faisal SM, Yan W, McDonough SP, Chang YF: Leptospira immunoglobulin-like protein A variable region (LigAvar) incorporated in liposomes and PLGA microspheres produces a robust immune response correlating to protective immunity. Vaccine. 2009, 27 (3): 378-387. 10.1016\/j.vaccine.2008.10.089.","journal-title":"Vaccine"},{"issue":"47","key":"488_CR24","doi-asserted-by":"publisher","first-page":"6537","DOI":"10.1016\/j.vaccine.2009.08.051","volume":"27","author":"SM Faisal","year":"2009","unstructured":"Faisal SM, Yan W, McDonough SP, Chang CF, Pan MJ, Chang YF: Leptosome-entrapped leptospiral antigens conferred significant higher levels of protection than those entrapped with PC-liposomes in a hamster model. Vaccine. 2009, 27 (47): 6537-6545. 10.1016\/j.vaccine.2009.08.051.","journal-title":"Vaccine"},{"key":"488_CR25","volume-title":"Curr Microbiol","author":"A Habarta","year":"2010","unstructured":"Habarta A, Abreu PA, Olivera N, Hauk P, Cedola MT, Ferrer MF, Ho PL, Gomez RM: Increased Immunogenicity to LipL32 of Leptospira interrogans when Expressed as a Fusion Protein with the Cholera Toxin B Subunit. Curr Microbiol. 2010"},{"issue":"10","key":"488_CR26","doi-asserted-by":"publisher","first-page":"736","DOI":"10.1038\/nrmicro2208","volume":"7","author":"AI Ko","year":"2009","unstructured":"Ko AI, Goarant C, Picardeau M: Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol. 2009, 7 (10): 736-747. 10.1038\/nrmicro2208.","journal-title":"Nat Rev Microbiol"},{"issue":"4","key":"488_CR27","doi-asserted-by":"publisher","first-page":"929","DOI":"10.1046\/j.1365-2958.2003.03619.x","volume":"49","author":"J Matsunaga","year":"2003","unstructured":"Matsunaga J, Barocchi MA, Croda J, Young TA, Sanchez Y, Siqueira I, Bolin CA, Reis MG, Riley LW, Haake DA, et al.: Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol Microbiol. 2003, 49 (4): 929-945. 10.1046\/j.1365-2958.2003.03619.x.","journal-title":"Mol Microbiol"},{"issue":"11","key":"488_CR28","doi-asserted-by":"publisher","first-page":"5924","DOI":"10.1128\/IAI.70.11.5924-5930.2002","volume":"70","author":"RU Palaniappan","year":"2002","unstructured":"Palaniappan RU, Chang YF, Jusuf SS, Artiushin S, Timoney JF, McDonough SP, Barr SC, Divers TJ, Simpson KW, McDonough PL, et al.: Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans. Infect Immun. 2002, 70 (11): 5924-5930. 10.1128\/IAI.70.11.5924-5930.2002.","journal-title":"Infect Immun"},{"issue":"4","key":"488_CR29","doi-asserted-by":"publisher","first-page":"472","DOI":"10.1139\/w06-138","volume":"53","author":"FK Seixas","year":"2007","unstructured":"Seixas FK, Fernandes CH, Hartwig DD, Conceicao FR, Aleixo JA, Dellagostin OA: Evaluation of different ways of presenting LipL32 to the immune system with the aim of developing a recombinant vaccine against leptospirosis. Can J Microbiol. 2007, 53 (4): 472-479. 10.1139\/W06-138.","journal-title":"Can J Microbiol"},{"issue":"2","key":"488_CR30","doi-asserted-by":"publisher","first-page":"119","DOI":"10.1002\/jmr.687","volume":"18","author":"R Daly","year":"2005","unstructured":"Daly R, Hearn MT: Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit. 2005, 18 (2): 119-138. 10.1002\/jmr.687.","journal-title":"J Mol Recognit"},{"issue":"2","key":"488_CR31","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1006\/prep.1998.0933","volume":"14","author":"R Montesino","year":"1998","unstructured":"Montesino R, Garcia R, Quintero O, Cremata JA: Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris. Protein Expr Purif. 1998, 14 (2): 197-207. 10.1006\/prep.1998.0933.","journal-title":"Protein Expr Purif"},{"issue":"5","key":"488_CR32","doi-asserted-by":"publisher","first-page":"421","DOI":"10.1038\/nbt0590-421","volume":"8","author":"CF Goochee","year":"1990","unstructured":"Goochee CF, Monica T: Environmental effects on protein glycosylation. Biotechnology (NY). 1990, 8 (5): 421-427. 10.1038\/nbt0590-421.","journal-title":"Biotechnology (NY)"},{"issue":"8","key":"488_CR33","doi-asserted-by":"publisher","first-page":"975","DOI":"10.1038\/nbt0896-975","volume":"14","author":"N Jenkins","year":"1996","unstructured":"Jenkins N, Parekh RB, James DC: Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol. 1996, 14 (8): 975-981. 10.1038\/nbt0896-975.","journal-title":"Nat Biotechnol"},{"issue":"9181","key":"488_CR34","doi-asserted-by":"publisher","first-page":"820","DOI":"10.1016\/S0140-6736(99)80012-9","volume":"354","author":"AI Ko","year":"1999","unstructured":"Ko AI, Galvao Reis M, Ribeiro Dourado CM, Johnson WD, Riley LW: Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet. 1999, 354 (9181): 820-825. 10.1016\/S0140-6736(99)80012-9.","journal-title":"Lancet"},{"key":"488_CR35","volume-title":"Molecular Cloning: A Laboratory Manual","author":"J Sambrook","year":"2000","unstructured":"Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual. 2000, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 3","edition":"3"}],"container-title":["Microbial Cell Factories"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/1475-2859-9-98.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,9,1]],"date-time":"2021-09-01T04:40:36Z","timestamp":1630471236000},"score":1,"resource":{"primary":{"URL":"https:\/\/microbialcellfactories.biomedcentral.com\/articles\/10.1186\/1475-2859-9-98"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2010,12]]},"references-count":35,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2010,12]]}},"alternative-id":["488"],"URL":"https:\/\/doi.org\/10.1186\/1475-2859-9-98","relation":{},"ISSN":["1475-2859"],"issn-type":[{"value":"1475-2859","type":"electronic"}],"subject":[],"published":{"date-parts":[[2010,12]]},"assertion":[{"value":"25 October 2010","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"6 December 2010","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"6 December 2010","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}}],"article-number":"98"}}