Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars
,
Abstract
Background and Objectives: One of the highly conserved outer membrane proteins expressed only by pathogenic Leptospires is Loa22. The study aims is to achieve the optimum conditions for high expression of recombinant Loa22 (rLoa22) protein.
Materials and Methods: Complete coding sequence of loa22 gene sub-cloned into a pET32a (+) expression vector. BL21 competent E. coli (pLysS) used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers. Then expression conditions optimized by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration.
Results: SDS-PAGE analysis showed that high production of rLoa22 protein obtained when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1 M and 5 h in 2×TY medium respectively. The purification of rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration.
Conclusion: The findings of the study led to high production of pure Loa22 protein, which can form the basis for future investigation on the design of rapid diagnostic tests and more effective vaccine candidates for leptospirosis.
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10. Raja V, Natarajaseenivasan K. Pathogenic, diagnostic and vaccine potential of leptospiral outer membrane proteins (OMPs). Crit Rev Microbiol 2015; 41: 1-17.
11. Lin Xa, Chen Y, Yan J. Recombinant multiepitope protein for diagnosis of leptospirosis. Clin Vaccine Immunol 2008; 15: 1711-1714.
12. Dellagostin OA, Grassmann AA, Hartwig DD, Felix SR, da Silva EF, McBride AJ. Recombinant vaccines against leptospirosis. Hum Vaccin 2011; 7: 1215-1224.
13. Cilia G, Bertelloni F, Albini S, Fratini F. Insight into the epidemiology of leptospirosis: a review of Leptospira isolations from “Unconventional” Hosts. Animals (Basel) 2021; 11: 191.
14. Domingos RF, Vieira ML, Romero EC, Gonçales AP, de Morais ZM, Vasconcellos SA, et al. Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions. BMC Microbiol 2012; 12: 50.
15. Oliveira R, de Morais ZM, Gonçales AP, Romero EC, Vasconcellos SA, Nascimento AL. Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen. PLoS One 2011; 6(7): e21962.
16. Zhang Y, Bao L, Zhu H, Huang B, Zhang H. OmpA-like protein Loa22 from Leptospira interrogans serovar Lai is cytotoxic to cultured rat renal cells and promotes inflammatory responses. Acta Biochim Biophys Sin (Shanghai) 2010; 42: 70-79.
17. Choy HA, Kelley MM, Chen TL, Møller AK, Matsunaga J, Haake DA. Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen. Infect Immun 2007; 75: 2441-2450.
18. Ristow P, Bourhy P, da Cruz McBride FW, Figueira CP, Huerre M, Ave P, et al. The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathog 2007; 3(7): e97.
19. Hsu S-H, Chang M-Y, Lin S-M, Ko Y-C, Chou L-F, Tian Y-C, et al. Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition. Sci Rep 2021; 11: 1064.
20. Balamurugan V, Thirumalesh SRA, Alamuri A, SowjanyaKumari S, Vinod Kumar K, Linshamol L, et al. Evaluation of the diagnostic potential of recombinant leptospiral OMP A‐like protein (Loa22) and transmembrane (OmpL37) protein in latex agglutination test for serodiagnosis of leptospirosis in animals. Lett Appl Microbiol 2021; 72: 730-740.
21. Ye C, Yan W, Xiang H, He H, Yang M, Ijaz M, et al. Recombinant antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 for serological diagnosis of leptospirosis by enzyme-linked immunosorbent assays in dogs. PLoS One 2014; 9(12): e111367.
22. Adler B. Vaccines against leptospirosis. Curr Top Microbiol Immunol 2015; 387: 251-272.
23. Grassmann AA, Souza JD, McBride AJA. A universal vaccine against leptospirosis: are we going in the right direction? Front Immunol 2017; 8: 256.
24. Bashiru G, Bahaman AR. Advances & challenges in leptospiral vaccine development. Indian J Med Res 2018; 147: 15-22.
25. Mohammadi YM, Khaki P, Bidhendi SM, Nofeli M. Evaluation of the presence of gene encoding Loa22 in pathogenic Leptospira serovars. Iran J Med Microbiol 2022; 16: 392-398.
26. QiaExpressionist T. A handbook for high-level expression and purification of 6xhis-tagged proteins. Qiagen, Chatsworth, Calif, USA. 2003.
27. Kielkopf CL, Bauer W, Urbatsch IL. Bradford assay for determining protein concentration. Cold Spring Harb Protoc 2020; 2020: 102269.
28. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, et al. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003; 3: 757-771.
29. Haake DA, Zückert WR. The leptospiral outer membrane. Curr Top Microbiol Immunol 2015; 387: 187-221.
30. Kaur D, Verma R, Kumar BVS, Deka D, Agrawal RK. Cloning, Phylogenetic analysis and expression of recombinant LipL41, Loa22 and LipL21 proteins from Leptospira interrogans. Int J Agric Environ Biotechnol 2014; 7: 409-420.
31. Silveira MM, Oliveira TL, Schuch RA, McBride AJA, Dellagostin OA, Hartwig DD. DNA vaccines against leptospirosis: A literature review. Vaccine 2017; 35: 5559-5567.
32. Golab N, Khaki P, Harzandi N, Esmaelizad M, Tebianian M. Expression and purification of the Lipl41, a surface-exposed lipoprotein antigen of pathogenic Leptospira spp. Vet Arh 2020; 90: 297-305.
33. Zhao X, Wang J, Ge Y-M, Lin X-A, Yan J, Sun A-H. Characterization and immunogenicity of rLipL32/1-LipL21-OmpL1/2 fusion protein as a novel immunogen for a vaccine against Leptospirosis. Arch Biol Sci 2015; 67: 41-55.
34. Fraser T, Brown PD. Temperature and oxidative stress as triggers for virulence gene expression in pathogenic Leptospira spp. Front Microbiol 2017; 8: 783.
2. Karpagam KB, Ganesh B. Leptospirosis: a neglected tropical zoonotic infection of public health importance—an updated review. Eur J of Clin Microbiol Infect Dis 2020;39:835-846.
3. Widiasih DA, Isozumi R, Yoshimatsu K, Arikawa J, editors. Serological and Molecular Biological Diagnosis for Leptospirosis. Proceeding of the 1st International Conference on Tropical Agriculture; 2017, pp. 533-547.
4. Nascimento AL, Ko AI, Martins EA, Monteiro-Vitorello CB, Ho PL, Haake DA, et al. Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. J Bacteriol 2004; 186: 2164-2172.
5. Chalayon P, Chanket P, Boonchawalit T, Chattanadee S, Srimanote P, Kalambaheti T. Leptospirosis serodiagnosis by ELISA based on recombinant outer membrane protein. Trans R Soc Trop Med Hyg 2011; 105: 289-297.
6. Esfandiari B, Pourshafie MR, Gouya MM, Khaki P, Mostafavi E, Darvish J, et al. An epidemiological comparative study on diagnosis of rodent leptospirosis in Mazandaran Province, northern Iran. Epidemiol Health 2015; 37: e2015012.
7. Soltani Majd N, Khodaverdi Darian E, Khaki P, Moradi Bidhendi S, Yahaghi E, Mirnejad R. Epidemiological patterns of Leptospira spp. among slaughterhouse workers in Zanjan–Iran. Asian Pac J Trop Dis 2012; 2: S550-S552.
8. Nogueira SV, Backstedt BT, Smith AA, Qin J-H, Wunder EA Jr, Ko A, et al. Leptospira interrogans enolase is secreted extracellularly and interacts with plasminogen. PLoS One 2013; 8(10): e78150.
9. Adler B, de la Peña Moctezuma A. Leptospira and leptospirosis. Vet Microbiol 2010; 140: 287-296.
10. Raja V, Natarajaseenivasan K. Pathogenic, diagnostic and vaccine potential of leptospiral outer membrane proteins (OMPs). Crit Rev Microbiol 2015; 41: 1-17.
11. Lin Xa, Chen Y, Yan J. Recombinant multiepitope protein for diagnosis of leptospirosis. Clin Vaccine Immunol 2008; 15: 1711-1714.
12. Dellagostin OA, Grassmann AA, Hartwig DD, Felix SR, da Silva EF, McBride AJ. Recombinant vaccines against leptospirosis. Hum Vaccin 2011; 7: 1215-1224.
13. Cilia G, Bertelloni F, Albini S, Fratini F. Insight into the epidemiology of leptospirosis: a review of Leptospira isolations from “Unconventional” Hosts. Animals (Basel) 2021; 11: 191.
14. Domingos RF, Vieira ML, Romero EC, Gonçales AP, de Morais ZM, Vasconcellos SA, et al. Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions. BMC Microbiol 2012; 12: 50.
15. Oliveira R, de Morais ZM, Gonçales AP, Romero EC, Vasconcellos SA, Nascimento AL. Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen. PLoS One 2011; 6(7): e21962.
16. Zhang Y, Bao L, Zhu H, Huang B, Zhang H. OmpA-like protein Loa22 from Leptospira interrogans serovar Lai is cytotoxic to cultured rat renal cells and promotes inflammatory responses. Acta Biochim Biophys Sin (Shanghai) 2010; 42: 70-79.
17. Choy HA, Kelley MM, Chen TL, Møller AK, Matsunaga J, Haake DA. Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen. Infect Immun 2007; 75: 2441-2450.
18. Ristow P, Bourhy P, da Cruz McBride FW, Figueira CP, Huerre M, Ave P, et al. The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathog 2007; 3(7): e97.
19. Hsu S-H, Chang M-Y, Lin S-M, Ko Y-C, Chou L-F, Tian Y-C, et al. Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition. Sci Rep 2021; 11: 1064.
20. Balamurugan V, Thirumalesh SRA, Alamuri A, SowjanyaKumari S, Vinod Kumar K, Linshamol L, et al. Evaluation of the diagnostic potential of recombinant leptospiral OMP A‐like protein (Loa22) and transmembrane (OmpL37) protein in latex agglutination test for serodiagnosis of leptospirosis in animals. Lett Appl Microbiol 2021; 72: 730-740.
21. Ye C, Yan W, Xiang H, He H, Yang M, Ijaz M, et al. Recombinant antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 for serological diagnosis of leptospirosis by enzyme-linked immunosorbent assays in dogs. PLoS One 2014; 9(12): e111367.
22. Adler B. Vaccines against leptospirosis. Curr Top Microbiol Immunol 2015; 387: 251-272.
23. Grassmann AA, Souza JD, McBride AJA. A universal vaccine against leptospirosis: are we going in the right direction? Front Immunol 2017; 8: 256.
24. Bashiru G, Bahaman AR. Advances & challenges in leptospiral vaccine development. Indian J Med Res 2018; 147: 15-22.
25. Mohammadi YM, Khaki P, Bidhendi SM, Nofeli M. Evaluation of the presence of gene encoding Loa22 in pathogenic Leptospira serovars. Iran J Med Microbiol 2022; 16: 392-398.
26. QiaExpressionist T. A handbook for high-level expression and purification of 6xhis-tagged proteins. Qiagen, Chatsworth, Calif, USA. 2003.
27. Kielkopf CL, Bauer W, Urbatsch IL. Bradford assay for determining protein concentration. Cold Spring Harb Protoc 2020; 2020: 102269.
28. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, et al. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003; 3: 757-771.
29. Haake DA, Zückert WR. The leptospiral outer membrane. Curr Top Microbiol Immunol 2015; 387: 187-221.
30. Kaur D, Verma R, Kumar BVS, Deka D, Agrawal RK. Cloning, Phylogenetic analysis and expression of recombinant LipL41, Loa22 and LipL21 proteins from Leptospira interrogans. Int J Agric Environ Biotechnol 2014; 7: 409-420.
31. Silveira MM, Oliveira TL, Schuch RA, McBride AJA, Dellagostin OA, Hartwig DD. DNA vaccines against leptospirosis: A literature review. Vaccine 2017; 35: 5559-5567.
32. Golab N, Khaki P, Harzandi N, Esmaelizad M, Tebianian M. Expression and purification of the Lipl41, a surface-exposed lipoprotein antigen of pathogenic Leptospira spp. Vet Arh 2020; 90: 297-305.
33. Zhao X, Wang J, Ge Y-M, Lin X-A, Yan J, Sun A-H. Characterization and immunogenicity of rLipL32/1-LipL21-OmpL1/2 fusion protein as a novel immunogen for a vaccine against Leptospirosis. Arch Biol Sci 2015; 67: 41-55.
34. Fraser T, Brown PD. Temperature and oxidative stress as triggers for virulence gene expression in pathogenic Leptospira spp. Front Microbiol 2017; 8: 783.
| Files | ||
| Issue | Vol 15 No 5 (2023) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v15i5.13873 | |
| Keywords | ||
| Leptospirosis; Outer membrane proteins; Gene expression; Recombinant protein Loa22 | ||
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How to Cite
1.
Gharakhani M, Faezi Ghasemi M, Khaki P, Esmaelizad M, Tebianian M. Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars. Iran J Microbiol. 2023;15(5):674-684.
