Skip to main content

Leishmania tarentolae for the Production of Multi-subunit Complexes

  • Chapter
  • First Online:
Advanced Technologies for Protein Complex Production and Characterization

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 896))

Abstract

Multi-subunit protein complexes are involved in a wide variety of cellular processes including DNA replication, transcriptional regulation, signal transduction, protein folding and degradation. A better understanding of the function of these protein complexes requires structural insights into the molecular arrangement and interactions of their constituent subunits. However, biochemical and structural analysis of multi-subunit protein complexes is still limited because of technical difficulties with their recombinant expression and reconstitution. This chapter presents an overview of a novel protein expression system based on Leishmania tarentolae, a unicellular protozoan parasite of lizards, and practical considerations for the production of multi-subunit protein complexes. The Leishmania tarentolae expression system offers fully eukaryotic protein expression with post-translational modifications but with ease of handling similar to bacteria. This chapter also summarizes studies on the production of laminins, large heterotrimeric glycoproteins of the extracellular matrix, using this expression system. In addition, a recently developed Leishmania tarentolae-based cell-free translation system is briefly described.

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

Access this chapter

Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Shapiro TA, Englund PT (1995) The structure and replication of kinetoplast DNA. Annu Rev Microbiol 49:117–143

    Article  CAS  PubMed  Google Scholar 

  2. Clayton CE (1999) Genetic manipulation of kinetoplastida. Parasitol Today 15(9):372–378

    Article  CAS  PubMed  Google Scholar 

  3. Beverley SM (2003) Protozomics: trypanosomatid parasite genetics comes of age. Nat Rev Genet 4(1):11–19

    Article  CAS  PubMed  Google Scholar 

  4. Liu B, Liu Y, Motyka SA, Agbo EE, Englund PT (2005) Fellowship of the rings: the replication of kinetoplast DNA. Trends Parasitol 21(8):363–369

    Article  CAS  PubMed  Google Scholar 

  5. Lipoldova M, Demant P (2006) Genetic susceptibility to infectious disease: lessons from mouse models of leishmaniasis. Nat Rev Genet 7(4):294–305

    Article  CAS  PubMed  Google Scholar 

  6. Banuls AL, Hide M, Prugnolle F (2007) Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans. Adv Parasitol 64:1–109

    Article  PubMed  Google Scholar 

  7. Kaye P, Scott P (2011) Leishmaniasis: complexity at the host-pathogen interface. Nat Rev Microbiol 9(8):604–615

    Article  CAS  PubMed  Google Scholar 

  8. WHO (2013) Sustaining the drive to overcome the global impact of neglected tropical diseases. Second WHO report on neglected tropical diseases

    Google Scholar 

  9. Elwasila M (1988) Leishmania tarentolae Wenyon, 1921 from the gecko Tarentola annularis in the Sudan. Parasitol Res 74(6):591–592

    Article  CAS  PubMed  Google Scholar 

  10. Breton M, Tremblay MJ, Ouellette M, Papadopoulou B (2005) Live nonpathogenic parasitic vector as a candidate vaccine against visceral leishmaniasis. Infect Immun 73(10):6372–6382

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tamar S, Dumas C, Papadopoulou B (2000) Chromosome structure and sequence organization between pathogenic and non-pathogenic Leishmania spp. Mol Biochem Parasitol 111(2):401–414

    Article  CAS  PubMed  Google Scholar 

  12. Mizbani A, Taslimi Y, Zahedifard F, Taheri T, Rafati S (2011) Effect of A2 gene on infectivity of the nonpathogenic parasite Leishmania tarentolae. Parasitol Res 109(3):793–799

    Article  PubMed  Google Scholar 

  13. Raymond F, Boisvert S, Roy G, Ritt JF, Legare D, Isnard A, Stanke M, Olivier M, Tremblay MJ, Papadopoulou B, Ouellette M, Corbeil J (2012) Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species. Nucleic Acids Res 40(3):1131–1147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Teixeira SM (1998) Control of gene expression in Trypanosomatidae. Braz J Med Biol Res 31(12):1503–1516

    Article  CAS  PubMed  Google Scholar 

  15. Clayton CE (2002) Life without transcriptional control? From fly to man and back again. EMBO J 21(8):1881–1888

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Martinez-Calvillo S, Vizuet-de-Rueda JC, Florencio-Martinez LE, Manning-Cela RG, Figueroa-Angulo EE (2010) Gene expression in trypanosomatid parasites. J Biomed Biotechnol 2010:525241

    Article  PubMed  PubMed Central  Google Scholar 

  17. Lee MG, Van der Ploeg LH (1997) Transcription of protein-coding genes in trypanosomes by RNA polymerase I. Annu Rev Microbiol 51:463–489

    Article  CAS  PubMed  Google Scholar 

  18. Teixeira SM, de Paiva RM, Kangussu-Marcolino MM, Darocha WD (2012) Trypanosomatid comparative genomics: contributions to the study of parasite biology and different parasitic diseases. Genet Mol Biol 35(1):1–17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Clayton C, Shapira M (2007) Post-transcriptional regulation of gene expression in trypanosomes and leishmanias. Mol Biochem Parasitol 156(2):93–101

    Article  CAS  PubMed  Google Scholar 

  20. Breitling R, Klingner S, Callewaert N, Pietrucha R, Geyer A, Ehrlich G, Hartung R, Muller A, Contreras R, Beverley SM, Alexandrov K (2002) Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expr Purif 25(2):209–218

    Article  CAS  PubMed  Google Scholar 

  21. Kushnir S, Gase K, Breitling R, Alexandrov K (2005) Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae. Protein Expr Purif 42(1):37–46

    Article  CAS  PubMed  Google Scholar 

  22. Kushnir S, Cirstea IC, Basiliya L, Lupilova N, Breitling R, Alexandrov K (2011) Artificial linear episome-based protein expression system for protozoon Leishmania tarentolae. Mol Biochem Parasitol 176(2):69–79

    Article  CAS  PubMed  Google Scholar 

  23. Soleimani M, Mahboudi F, Davoudi N, Amanzadeh A, Azizi M, Adeli A, Rastegar H, Barkhordari F, Mohajer-Maghari B (2007) Expression of human tissue plasminogen activator in the trypanosomatid protozoan Leishmania tarentolae. Biotechnol Appl Biochem 48(Pt 1):55–61

    CAS  PubMed  Google Scholar 

  24. Ben-Abdallah M, Bondet V, Fauchereau F, Beguin P, Goubran-Botros H, Pagan C, Bourgeron T, Bellalou J (2011) Production of soluble, active acetyl serotonin methyl transferase in Leishmania tarentolae. Protein Expr Purif 75:114–118

    Article  CAS  PubMed  Google Scholar 

  25. Gazdag EM, Cirstea IC, Breitling R, Lukes J, Blankenfeldt W, Alexandrov K (2010) Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae. Acta Crystallogr Sect F: Struct Biol Cryst Commun 66(Pt 8):871–877

    Article  CAS  Google Scholar 

  26. Dadashipour M, Fukuta Y, Asano Y (2011) Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems. Protein Expr Purif 77(1):92–97

    Article  CAS  PubMed  Google Scholar 

  27. Dortay H, Schmockel SM, Fettke J, Mueller-Roeber B (2011) Expression of human c-reactive protein in different systems and its purification from Leishmania tarentolae. Protein Expr Purif 78(1):55–60

    Article  CAS  PubMed  Google Scholar 

  28. Nazari R, Davoudi N (2011) Cloning and expression of truncated form of tissue plasminogen activator in Leishmania tarentolae. Biotechnol Lett 33(3):503–508

    Article  CAS  PubMed  Google Scholar 

  29. Baechlein C, Meemken D, Pezzoni G, Engemann C, Grummer B (2013) Expression of a truncated hepatitis E virus capsid protein in the protozoan organism Leishmania tarentolae and its application in a serological assay. J Virol Methods 193(1):238–243

    Article  CAS  PubMed  Google Scholar 

  30. Jorgensen ML, Friis NA, Just J, Madsen P, Petersen SV, Kristensen P (2014) Expression of single-chain variable fragments fused with the Fc-region of rabbit IgG in Leishmania tarentolae. Microb Cell Fact 13:9

    Article  PubMed  PubMed Central  Google Scholar 

  31. Chang CS, Chang KP (1985) Heme requirement and acquisition by extracellular and intracellular stages of Leishmania mexicana amazonensis. Mol Biochem Parasitol 16(3):267–276

    Article  CAS  PubMed  Google Scholar 

  32. Fritsche C, Sitz M, Weiland N, Breitling R, Pohl HD (2007) Characterization of the growth behavior of Leishmania tarentolae: a new expression system for recombinant proteins. J Basic Microbiol 47(5):384–393

    Article  CAS  PubMed  Google Scholar 

  33. Kornberg RD (2007) The molecular basis of eukaryotic transcription. Proc Natl Acad Sci U S A 104(32):12955–12961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wiese M, Ilg T, Lottspeich F, Overath P (1995) Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase. EMBO J 14(6):1067–1074

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Basak A, Shervani NJ, Mbikay M, Kolajova M (2008) Recombinant proprotein convertase 4 (PC4) from Leishmania tarentolae expression system: purification, biochemical study and inhibitor design. Protein Expr Purif 60(2):117–126

    Article  CAS  PubMed  Google Scholar 

  36. Mureev S, Kushnir S, Kolesnikov AA, Breitling R, Alexandrov K (2007) Construction and analysis of Leishmania tarentolae transgenic strains free of selection markers. Mol Biochem Parasitol 155(2):71–83

    Article  CAS  PubMed  Google Scholar 

  37. Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3(2):97–130

    Article  CAS  PubMed  Google Scholar 

  38. Varki A (2007) Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 446(7139):1023–1029

    Article  CAS  PubMed  Google Scholar 

  39. Elbein AD (1991) The role of N-linked oligosaccharides in glycoprotein function. Trends Biotechnol 9(10):346–352

    Article  PubMed  Google Scholar 

  40. Helenius A, Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73:1019–1049

    Article  CAS  PubMed  Google Scholar 

  41. Klatt S, Rohe M, Alagesan K, Kolarich D, Konthur Z, Hartl D (2013) Production of glycosylated soluble amyloid precursor protein alpha (sAPPalpha) in Leishmania tarentolae. J Proteome Res 12(1):396–403

    Article  CAS  PubMed  Google Scholar 

  42. Phan HP, Sugino M, Niimi T (2009) The production of recombinant human laminin-332 in a Leishmania tarentolae expression system. Protein Expr Purif 68(1):79–84

    Article  CAS  PubMed  Google Scholar 

  43. Timpl R (1996) Macromolecular organization of basement membranes. Curr Opin Cell Biol 8(5):618–624

    Article  CAS  PubMed  Google Scholar 

  44. Miner JH, Yurchenco PD (2004) Laminin functions in tissue morphogenesis. Annu Rev Cell Dev Biol 20:255–284

    Article  CAS  PubMed  Google Scholar 

  45. Domogatskaya A, Rodin S, Tryggvason K (2012) Functional diversity of laminins. Annu Rev Cell Dev Biol 28:523–553

    Article  CAS  PubMed  Google Scholar 

  46. Aumailley M, Bruckner-Tuderman L, Carter WG, Deutzmann R, Edgar D, Ekblom P, Engel J, Engvall E, Hohenester E, Jones JC, Kleinman HK, Marinkovich MP, Martin GR, Mayer U, Meneguzzi G, Miner JH, Miyazaki K, Patarroyo M, Paulsson M, Quaranta V, Sanes JR, Sasaki T, Sekiguchi K, Sorokin LM, Talts JF, Tryggvason K, Uitto J, Virtanen I, von der Mark K, Wewer UM, Yamada Y, Yurchenco PD (2005) A simplified laminin nomenclature. Matrix Biol 24(5):326–332

    Article  CAS  PubMed  Google Scholar 

  47. Aumailley M (2013) The laminin family. Cell Adh Migr 7(1):48–55

    Article  PubMed  PubMed Central  Google Scholar 

  48. Miyazaki T, Futaki S, Hasegawa K, Kawasaki M, Sanzen N, Hayashi M, Kawase E, Sekiguchi K, Nakatsuji N, Suemori H (2008) Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem Biophys Res Commun 375(1):27–32

    Article  CAS  PubMed  Google Scholar 

  49. Rodin S, Domogatskaya A, Strom S, Hansson EM, Chien KR, Inzunza J, Hovatta O, Tryggvason K (2010) Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat Biotechnol 28(6):611–615

    Article  CAS  PubMed  Google Scholar 

  50. Miyazaki T, Futaki S, Suemori H, Taniguchi Y, Yamada M, Kawasaki M, Hayashi M, Kumagai H, Nakatsuji N, Sekiguchi K, Kawase E (2012) Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat Commun 3:1236

    Article  PubMed  PubMed Central  Google Scholar 

  51. Nakagawa M, Taniguchi Y, Senda S, Takizawa N, Ichisaka T, Asano K, Morizane A, Doi D, Takahashi J, Nishizawa M, Yoshida Y, Toyoda T, Osafune K, Sekiguchi K, Yamanaka S (2014) A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep 4:3594

    PubMed  PubMed Central  Google Scholar 

  52. Tsubota Y, Mizushima H, Hirosaki T, Higashi S, Yasumitsu H, Miyazaki K (2000) Isolation and activity of proteolytic fragment of laminin-5 alpha3 chain. Biochem Biophys Res Commun 278(3):614–620

    Article  CAS  PubMed  Google Scholar 

  53. Kariya Y, Yasuda C, Nakashima Y, Ishida K, Tsubota Y, Miyazaki K (2004) Characterization of laminin 5B and NH2-terminal proteolytic fragment of its alpha3B chain: promotion of cellular adhesion, migration, and proliferation. J Biol Chem 279(23):24774–24784

    Article  CAS  PubMed  Google Scholar 

  54. Marinkovich MP (2007) Tumour microenvironment: laminin 332 in squamous-cell carcinoma. Nat Rev Cancer 7(5):370–380

    Article  CAS  PubMed  Google Scholar 

  55. Beck K, Hunter I, Engel J (1990) Structure and function of laminin: anatomy of a multidomain glycoprotein. FASEB J 4(2):148–160

    CAS  PubMed  Google Scholar 

  56. Bernhard F, Tozawa Y (2013) Cell-free expression-making a mark. Curr Opin Struct Biol 23(3):374–380

    Article  CAS  PubMed  Google Scholar 

  57. Rosenblum G, Cooperman BS (2014) Engine out of the chassis: cell-free protein synthesis and its uses. FEBS Lett 588(2):261–268

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Harbers M (2014) Wheat germ systems for cell-free protein expression. FEBS Lett 588:2762–2773

    Article  CAS  PubMed  Google Scholar 

  59. Phan HP, Ezure T, Ito M, Kadowaki T, Kitagawa Y, Niimi T (2008) Expression and chain assembly of human laminin-332 in an insect cell-free translation system. Biosci Biotechnol Biochem 72(7):1847–1852

    Article  CAS  PubMed  Google Scholar 

  60. Mureev S, Kovtun O, Nguyen UT, Alexandrov K (2009) Species-independent translational leaders facilitate cell-free expression. Nat Biotechnol 27(8):747–752

    Article  CAS  PubMed  Google Scholar 

  61. Kovtun O, Mureev S, Johnston W, Alexandrov K (2010) Towards the construction of expressed proteomes using a Leishmania tarentolae based cell-free expression system. PLoS One 5(12), e14388

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Kovtun O, Mureev S, Jung W, Kubala MH, Johnston W, Alexandrov K (2011) Leishmania cell-free protein expression system. Methods 55(1):58–64

    Article  CAS  PubMed  Google Scholar 

  63. Guo Z, Johnston W, Kovtun O, Mureev S, Brocker C, Ungermann C, Alexandrov K (2013) Subunit organisation of in vitro reconstituted HOPS and CORVET multisubunit membrane tethering complexes. PLoS One 8(12), e81534

    Article  PubMed  PubMed Central  Google Scholar 

  64. Fernandez-Robledo JA, Vasta GR (2010) Production of recombinant proteins from protozoan parasites. Trends Parasitol 26(5):244–254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Fernandez FJ, Vega MC (2013) Technologies to keep an eye on: alternative hosts for protein production in structural biology. Curr Opin Struct Biol 23(3):365–373

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The preparation of this chapter was supported by JSPS KAKENHI Grant Numbers 18108003 and 26350959.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tomoaki Niimi .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Niimi, T. (2016). Leishmania tarentolae for the Production of Multi-subunit Complexes. In: Vega, M. (eds) Advanced Technologies for Protein Complex Production and Characterization. Advances in Experimental Medicine and Biology, vol 896. Springer, Cham. https://doi.org/10.1007/978-3-319-27216-0_10

Download citation

Publish with us

Policies and ethics