Skip to main content

Advertisement

Log in

Systematic Comparison of Strategies to Achieve Soluble Expression of Plasmodium falciparum Recombinant Proteins in E. coli

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Constructs containing partial coding sequences of myosin A, myosin B, and glideosome-associated protein (50 kDa) of Plasmodium falciparum were used to challenge several strategies designed in order to improve the production and solubility of recombinant proteins in Escherichia coli. Assays were carried out inducing expression in a late log phase culture, optimizing the inductor concentration, reducing the growth temperature for induced cultures, and supplementing additives in the lysis buffer. In addition, recombinant proteins were expressed as fusion proteins with three different tags (6His, GST, and MBP) in four different E. coli strains. We found that the only condition that consistently produced soluble proteins was the use of MBP as a fusion tag, which became a valuable tool for detecting the proteins used in this study and did not caused any interference in protein–protein interaction assays (Far Western Blot). Besides, we found that BL21-pG-KJE8 strain did not improve the solubility of any of the recombinant protein produced, while the BL21-CodonPlus(DE3)-RIL strain improved the expression of some of them independent of the rare codon content. Proteins with rare codons occurring at high frequencies (» 10%) were expressed efficiently in strains that do not supplement tRNAs for these triplets.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. World Health Organization. (2016) World malaria report 2015. Geneva: World Health Organization.

    Google Scholar 

  2. Meissner, M., Breinich, M. S., Gilson, P. R., & Crabb, B. S. (2007). Molecular genetic tools in Toxoplasma and Plasmodium: Achievements and future needs. Current Opinion in Microbiology, 10, 349–356.

    Article  CAS  Google Scholar 

  3. Baum, J., Papenfuss, A. T., Mair, G. R., Janse, C. J., Vlachou, D., Waters, A. P., Cowman, A. F., Crabb, B. S., & De Koning-Ward, T. F. (2009). Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites. Nucleic Acids Research, 37, 3788–3798.

    Article  CAS  Google Scholar 

  4. Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology, 5, 172.

    PubMed  PubMed Central  Google Scholar 

  5. Mehlin, C., Boni, E. F., Buckner, S., Engel, L., Feist, T., Gelb, M. H., Haji, L., Kim, D., Liu, C., Mueller, N., Myler, P. J., Reddy, J. T., Sampson, J. N., Subramanian, E., Van Voorhis, W. C., Worthey, E., Zucker, F., & Hol, W. G. (2006). Heterologous expression of proteins from Plasmodium falciparum: Results from 1000 genes. Molecular and Biochemical Parasitology, 148, 144–160.

    Article  CAS  Google Scholar 

  6. Myler, P. J., Stacy, R., Stewart, L., Staker, B. L., Van Voorhis, W. C., Varani, G., & Buchko, G. W. (2009). The seattle structural genomics center for infectious disease (SSGCID). Infectious Disorders Drug Targets, 9, 493–506.

    Article  CAS  Google Scholar 

  7. Seattle Structural Genomics Center for Infectious Disease (SSGCID) (2017). https://www.ssgcid.org/target-status/. Accessed 17 Nov 2017.

  8. Hernández, P. C., Morales, L., Castellanos, I. C., Wasserman, M., & Chaparro-Olaya, J. (2017). Myosin B of Plasmodium falciparum (PfMyoB): In silico prediction of its three-dimensional structure and its possible interaction with MTIP. Parasitology Research, 116, 1373–1382.

    Article  Google Scholar 

  9. Guerra, ÁP., Calvo, E. P., Wasserman, M., & Chaparro-Olaya, J. (2016). Production of recombinant proteins from Plasmodium falciparum in Escherichia coli. Biomédica, 36, 97–108.

    Article  Google Scholar 

  10. Nakamura, Y., Gojobori, T., & Ikemura, T. (2000). Codon usage tabulated from international DNA sequence databases: Status for the year 2000. Nucleic Acids Research, 28, 292–292.

    Article  CAS  Google Scholar 

  11. Wasserman, M., Contreras, J., Pinilla, G., Rojas, M. O., Páez, A., & Caminos, E. (1995). Plasmodium falciparum: Characterization of a 0.7-kbp, moderately repetitive sequence. Experimental Parasitology, 81, 165–171.

    Article  CAS  Google Scholar 

  12. Pinder, J. C., Fowler, R. E., Dluzewski, A. R., Bannister, L. H., Lavin, F. M., Mitchell, G. H., Wilson, R. J., & Gratzer, W. B. (1998). Actomyosin motor in the merozoite of the malaria parasite, Plasmodium falciparum: Implications for red cell invasion. Journal of Cell Science, 111, 1831–1839.

    PubMed  CAS  Google Scholar 

  13. Chaparro-Olaya, J., Dluzewski, A. R., Margos, G., Wasserman, M. M., Mitchell, G. H., Bannister, L. H., & Pinder, J. C. (2003). The multiple myosins of malaria: The smallest malaria myosin, Plasmodium falciparum myosin-B (Pfmyo-B) is expressed in mature schizonts and merozoites. European Journal of Protistology, 39, 423–427.

    Article  Google Scholar 

  14. Redinbaugh, M. G., & Campbell, W. H. (1985). Adaptation of the dye-binding protein assay to microtiter plates. Analytical Biochemistry, 147, 144–147.

    Article  CAS  Google Scholar 

  15. Wu, Y., Li, Q., & Chen, X. Z. (2007). Detecting protein-protein interactions by Far western blotting. Nature Protocols, 2, 3278–3284.

    Article  CAS  Google Scholar 

  16. Bergman, L. W., Kaiser, K., Fujioka, H., Coppens, I., Daly, T. M., Fox, S., Matuschewski, K., Nussenzweig, V., & Kappe, S. H. (2003). Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites. Journal of Cell Science, 116, 39–49.

    Article  CAS  Google Scholar 

  17. Bosch, J., Turley, S., Daly, T. M., Bogh, S. M., Villasmil, M. L., Roach, C., Zhou, N., Morrisey, J. M., Vaidya, A. B., Bergman, L. W., & Hol, W. G. (2006). Structure of the MTIP-MyoA complex, a key component of the malaria parasite invasion motor. Proceedings of the National Academy of Sciences of the United States of America, 103, 4852–4857.

    Article  CAS  Google Scholar 

  18. Green, J. L., Martin, S. R., Fielden, J., Ksagoni, A., Grainger, M., Yim, B., Lim, Y., Molloy, J. E., & Holder, A. A. (2006). The MTIP–myosin A complex in blood stage malaria parasites. Journal of Molecular Biology, 355, 933–941.

    Article  CAS  Google Scholar 

  19. Thomas, J. C., Green, J. L., Howson, R. I., Simpson, P., Moss, D. K., Martin, S. R., Holder, A. A., Cota, E., & Tate, E. W. (2010). Interaction and dynamics of the Plasmodium falciparum MTIP–MyoA complex, a key component of the invasion motor in the malaria parasite. Molecular BioSystems, 6(3), 494–498.

    Article  CAS  Google Scholar 

  20. Bosch, J., Turley, S., Roach, C. M., Daly, T. M., Bergman, L. W., & Hol, W. G. (2007). The closed MTIP-myosin A-tail complex from the malaria parasite invasion machinery. Journal of Molecular Biology, 372, 77–88.

    Article  CAS  Google Scholar 

  21. Magnan, C. N., Randall, A., & Baldi, P. (2009). SOLpro: Accurate sequence-based prediction of protein solubility. Bioinformatics, 25, 2200–2207.

    Article  CAS  Google Scholar 

  22. Smialowski, P., Doose, G., Torkler, P., Kaufmann, S., & Frishman, D. (2012). PROSO II—A new method for protein solubility prediction. The FEBS Journal, 279, 2192–2200.

    Article  CAS  Google Scholar 

  23. Chaparro-Olaya, J., Margos, G., Coles, D. J., Dluzewski, A. R., Mitchell, G. H., Wasserman, M. M., & Pinder, J. C. (2005). Plasmodium falciparum myosins: Transcription and translation during asexual parasite development. Cell Motility and the Cytoskeleton, 60, 200–213.

    Article  CAS  Google Scholar 

  24. Flick, K., Ahuja, S., Chene, A., Bejarano, M. T., & Chen, Q. (2004) Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli. Malaria Journal, 3(1), 3–50.

    Article  CAS  Google Scholar 

  25. Leibly, D. J., Nguyen, T. N., Kao, L. T., Hewitt, S. N., Barrett, L. K., & Van Voorhis, W. C. (2012). Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins. PLoS ONE, 7, e52482.

    Article  CAS  Google Scholar 

  26. Donovan, R. S., Robinson, C. W., & Glick, B. R. (1996). Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. Journal of Industrial Microbiology, 16, 145–154.

    Article  CAS  Google Scholar 

  27. Vera, A., González-Montalbán, N., Arís, A., & Villaverde, A. (2007). The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnology and Bioengineering, 96, 1101–1106.

    Article  CAS  Google Scholar 

  28. De Marco, A., Deuerling, E., Mogk, A., Tomoyasu, T., & Bukau, B. (2007) Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnology, 7(1), 7–32.

    Article  CAS  Google Scholar 

  29. Baca, A. M., & Hol, W. G. (2000). Overcoming codon bias: A method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli. International Journal for Parasitology, 30, 113–118.

    Article  CAS  Google Scholar 

  30. Zhou, Z. P., Schnake, L., & Xiao, A. A. (2004). Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization. Protein Expression and Purification, 34, 87–94.

    Article  CAS  Google Scholar 

  31. Jeong, H., Barbe, V., Lee, C. H., Vallenet, D., Yu, D. S., Choi, S. H., Couloux, A., Lee, S. W., Yoon, S. H., Cattolico, L., Hur, C. G., Park, H. S., Ségurens, B., Kim, S. C., Oh, T. K., Lenski, R. E., Studier, F. W., Daegelen, P., & Kim, J. F. (2009). Genome sequences of Escherichia coli B strains REL606 and BL21 (DE3). Journal of Molecular Biology, 394, 644–652.

    Article  CAS  Google Scholar 

  32. Brinkmann, U., Mattes, R. E., & Buckel, P. (1989). High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene, 85, 109–114.

    Article  CAS  Google Scholar 

  33. Del Tito, B. J. Jr., Ward, J. M., Hodgson, J., Gershater, C. J., Edwards, H., Wysocki, L. A., Watson, F. A., Sathe, G., & Kane, J. F. (1995). Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli. Journal of Bacteriology, 177, 7086–7091.

    Article  Google Scholar 

  34. Kane, J. F. (1995). Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Current Opinion in Biotechnology, 6, 494–500.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by COLCIENCIAS (projects 110152128729 and 130834319109) and Universidad El Bosque (projects UB-271-2010 and PCI-2011-264). Funding organizations had no role in study design, data analysis, decision to publish, or preparation of the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jacqueline Chaparro-Olaya.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Morales, L., Hernández, P. & Chaparro-Olaya, J. Systematic Comparison of Strategies to Achieve Soluble Expression of Plasmodium falciparum Recombinant Proteins in E. coli. Mol Biotechnol 60, 887–900 (2018). https://doi.org/10.1007/s12033-018-0125-0

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-018-0125-0

Keywords

Navigation