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BMC Proceedings

Open Access

Expression and purification of LipL32 recombinant protein of Leptospira interrogans in different E.coli strains

  • Victor Bastos1,
  • Jonny Yokosawa2 and
  • Paulo Ho3
BMC Proceedings20148(Suppl 4):P165

https://doi.org/10.1186/1753-6561-8-S4-P165

Published: 1 October 2014

Background

Leptospirosis is a zoonosis caused by the infection of pathogenic bacteria of the genera Leptospira, often transmitted by direct or indirect contact with urine of infected animal hosts [1]. In national territory, leptospirosis is considered an endemic disease, with high incidence in raining seasons, but occurring during the whole year [2]. From all the cases, 15% evolve to the icteric form, with serious clinic manifestations, as severe icteric, hemorrhages and kidney disease, including lethal onset in 50% of the cases [3].

From all membrane proteins LipL32 is the most abundant and found in all pathogenic species of Leptospira. Saprophytic strains do not express LipL32, making the detection of the protein or the detection of specific antibody a potential toll for the development of a laboratorial diagnosis [4].

Methods

DNA of pAE-lipL32 was utilized in the transformation of E. coli strains BL21-S1, with expression induced by NaCl, and BL21 Star(DE3)pLysS, with expression induced by IPTG. Eight transformed colonies of each strain were inoculated in culture medium, and after the induced expression and analysis, by SDS-PAGE, the results showed a high level of expression in BL21-SI strains. One of the BL21-SI clone was chosen to express the LipL32 protein in a higher volume, for purification in a anionic chromatographic column, using a crescent concentration of ambic elution buffer (0,05 to 1,0 M). The purification resulted in 295 fractions combined in 15 proteic pools according with the protein spikes observed by spectrophotometry. The 15 proteic pool were analyzed in SDS-PAGE.

Results and conclusions

The SDS-PAGE showed only one band corresponding to the LipL32 protein, from pools six to 15, although, pool six has a visible higher expression, and pools 14 and 15 showed the presence of contaminant proteins, probably due to the high concentration of the eluent. More purifications steps are needed, but the expression of LipL32 by E. coli strain BL21-SI, induced by NaCl, was successfully achieved.

Authors’ Affiliations

(1)
Universidade Federal de Uberlândia, UFU, Santa Monica
(2)
Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, UFU, Santa Monica
(3)
Centro de Biotecnologia, Instituto Butantan

References

  1. Guia de Leptospirose: Diagnóstico e Manejo Clínico. Secretaria De Vigilância Em Saúde/Ministério Da Saúde (SVS/MS). 2009Google Scholar
  2. 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.View ArticlePubMedGoogle Scholar
  3. Levett PN: Leptospirosis. Clin Microbiol Rev. 2001, 14 (2): 296-326. 10.1128/CMR.14.2.296-326.2001.PubMed CentralView ArticlePubMedGoogle Scholar
  4. Hauk P, Carvalho E, Ho PL: Expression and purification of the non-tagged Lipl32 of pathogenic Leptospira. Braz J Med Biol Res. 2011, 44 (4): 297-302. 10.1590/S0100-879X2011007500025.View ArticlePubMedGoogle Scholar

Copyright

© Bastos et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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