Optimizations of expression and purification of recombinant HIV-1 CRF01_AE p24 protein in Escherichia coli for development of immunodiagnostic assay

  • Ade P.R. Simaremare Biomedical Programme, Faculty of Medicine, Universitas Indonesia, Jakarta
  • Budiman Bela Institute of Human Virology and Cancer Biology, Faculty of Medicine, Universitas Indonesia, Jakarta
  • Andi Yasmon Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta
  • Fera Ibrahim Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta
DOI: https://doi.org/10.13181/mji.v24i1.1166
Keywords: expression, HIV-1 CRF01_AE, p24 protein, purification, recombinant protein
Abstract viewed: 1618 times
PDF downloaded: 1195 times

Abstract

Background: Conventional method for confirmation of HIV infection is Western blot. However, it has limitations because of contamination by human cellular antigen and genetic diversity among the HIV-1 subtypes that show indeterminate result and inaccuracy for the diagnosis of different strains. Most of Western blot developed are based on HIV-1 B subtype. In Indonesia HIV-1 CRF01_AE subtype is dominantly circulated. Therefore, we optimized the expression, purification of the recombinant HIV-1 CRF01_AE p24 protein for development of immunodiagnostic assay.

Methods: Optimization of protein expression in Escherichia coli strain BL21CP was performed including induction time, isopropyl-1-thio-d-galactopyranoside (IPTG) and immidazole consentrations, and induction temperature. Purification of the recombinant p24 protein was used by using Ni-NTA (Qiagen) purification system in native condition.

Results: Expression and purification of HIV-1 CRF01_AE p24 protein have been performed. Confirmation of the recombinant protein by Western blot showed the expression and purification of recombinant p24 protein has been optimized well and reactive with sera of patients with HIV-1 CRF01_AE subtype positive.

Conclusion: The recombinant HIV-1 CRF01_AE p24 protein has been expressed and purified successfully, and it is potential to be used as antigen for immunodiagnostic assay.

Author Biography

Ade P.R. Simaremare, Biomedical Programme, Faculty of Medicine, Universitas Indonesia, Jakarta
Microbiology department

References

  1. Abbas AK, Lichtman AH, Pillai S. Cellular and molecular immunology. 6th ed. Philadelphia: Saunders Elsevier; 2010.

  2. Osmanov S, Pattou C, Walker N, Schwardländer B, Esparza J, WHO-UNAIDS Network for HIV Isolation and Characterization. Estimated global distribution and regional spread of HIV-1 genetic subtypes in the year 2000. J Acquir Immune Defic Syndr. 2002;29(2):184-90. http://dx.doi.org/10.1097/00042560-200202010-00013

  3. Knipe DM, Howley PM. Fields Virology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2007.

  4. Lau KA, Wang B, Saksena NK. Emerging trends of HIV epidemiology in Asia. AIDS Rev. 2007;9(4):218-29.

  5. Merati TP, Ryan CE, Spelmen T, Wirawan DN, Bakta IM, Otto B, et al. CRF01_AE dominates the HIV-1 epidemic in Indonesia. Sex Health. 2012;9(5):414-21. http://dx.doi.org/10.1071/SH11121

  6. Sahbandar IN, Takahashi K, Djoerban Z, Firmansyah I, Naganawa S, Motomura K, et al. Current HIV Type 1 Molecular epidemiology profile and identification of unique recombinant forms in Jakarta, Indonesia. AIDS Res Hum Retroviruses. 2009;25(7):637-46. http://dx.doi.org/10.1089/aid.2008.0266

  7. Roselinda R, Jekti RP. Genotype and transmission human immunodeficiency virus-1 in seven provinces in Indonesia. Health Science Indones. 2012;3(2):27-31.

  8. World Health Organization. Global report: UNAIDS report on the global AIDS epidemic 2010. UNAIDS; 2011.

  9. Direktorat Jenderal Pengendalian Penyakit dan Penyehatan Lingkungan Kemenkes RI. Statistik Kasus HIV/AIDS di Indonesia. Jakarta; 2013. Indonesian.

  10. Luft S, Seme K, Poljak M. Laboratory diagnosis of human immunodeficiency virus infection. Acta Dermatoven APA. 2004;13(2):43-9.

  11. Buttò S, Suligoi B, Fanales-Belasio E, Raimondo M. Laboratory diagnostics for HIV infection. Ann Ist Super Sanita. 2010;46(1):24-33. http://dx.doi.org/10.1590/S0021-25712010000100004

  12. Lakshmi V, Ponamgi SP. Evaluation of an indigenous western blot kit for human immunodeficiency virus. Indian J Med Microbiol. 2002;20(4):200-5.

  13. Lal RB, Chakrabarti S, Yang C. Impact of genetic diversity of HIV-1 on diagnosis, antiretroviral therapy & vaccine development. Indian J Med Res. 2005;121(4):287-314.

  14. Icardi GC, Gabutti G. Multi-centre evaluation of RIBA⢠HIV-1/HIV-2 SIA compared to HIV-1 Western blot. Journal of Preventive Medicine and Hygiene. 1998;39:57-64.

  15. Phillips S, Granade TC, Pau CP, Candal D, Hu DJ, Parekh BS. Diagnosis of human immunodeficiency virus type 1 infection with different subtypes using rapid tests. Clin Diagn Lab Immunol. 2000;7(4):698-9. http://dx.doi.org/10.1128/CDLI.7.4.698-699.2000

  16. Stratagene. BL21-Codon Plus Competent Cell. 2012. Available at: www.stratagene.com.

  17. Zhang B, Liu D, Bao Z, Chen B, Li C, Jiang H, et al. High level soluble expression, one-step purification and characterization of HIV-1 p24 protein. Virol J. 2011;8:316. http://dx.doi.org/10.1186/1743-422X-8-316

  18. Singh SC, Vasudevan M, Richa, Sinha RP. Novel method for the detection of HIV using rp24 recombinant protein from E. coli BL-21. Pharmacognosy. 2010;1(4):287-94.

  19. The QIAexpressionist. A handbook for high-level expression and purification of 6xHis-tagged protein. 5th ed. Valencia: Qiagen; 2003. p.126.

  20. Bornhorst JA, Falke JJ. Purification of proteins using polyhistidine affinity tags. Methods Enzymol. 2000;326:245-54. http://dx.doi.org/10.1016/S0076-6879(00)26058-8

Published
2015-03-12
How to Cite
1.
Simaremare AP, Bela B, Yasmon A, Ibrahim F. Optimizations of expression and purification of recombinant HIV-1 CRF01_AE p24 protein in Escherichia coli for development of immunodiagnostic assay. Med J Indones [Internet]. 2015Mar.12 [cited 2024Apr.19];24(1):14-8. Available from: http://mji.ui.ac.id/journal/index.php/mji/article/view/1166
Issue
Vol. 24 No. 1 (2015): March
Section
Basic Medical Research

Copyright (c) 2015 Ade P.R. Simaremare, Budiman Bela, Andi Yasmon, Fera Ibrahim

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Authors who publish with Medical Journal of Indonesia agree to the following terms:

  1. Authors retain copyright and grant Medical Journal of Indonesia right of first publication with the work simultaneously licensed under a Creative Commons Attribution-NonCommercial License that allows others to remix, adapt, build upon the work non-commercially with an acknowledgment of the work’s authorship and initial publication in Medical Journal of Indonesia.
  2. Authors are permitted to copy and redistribute the journal's published version of the work non-commercially (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in Medical Journal of Indonesia.

Most read articles by the same author(s)