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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Shapiro TA, Englund PT (1995) The structure and replication of kinetoplast DNA. Annu Rev Microbiol 49:117–143
Clayton CE (1999) Genetic manipulation of kinetoplastida. Parasitol Today 15(9):372–378
Beverley SM (2003) Protozomics: trypanosomatid parasite genetics comes of age. Nat Rev Genet 4(1):11–19
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
Lipoldova M, Demant P (2006) Genetic susceptibility to infectious disease: lessons from mouse models of leishmaniasis. Nat Rev Genet 7(4):294–305
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
Kaye P, Scott P (2011) Leishmaniasis: complexity at the host-pathogen interface. Nat Rev Microbiol 9(8):604–615
WHO (2013) Sustaining the drive to overcome the global impact of neglected tropical diseases. Second WHO report on neglected tropical diseases
Elwasila M (1988) Leishmania tarentolae Wenyon, 1921 from the gecko Tarentola annularis in the Sudan. Parasitol Res 74(6):591–592
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
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
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
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
Teixeira SM (1998) Control of gene expression in Trypanosomatidae. Braz J Med Biol Res 31(12):1503–1516
Clayton CE (2002) Life without transcriptional control? From fly to man and back again. EMBO J 21(8):1881–1888
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
Lee MG, Van der Ploeg LH (1997) Transcription of protein-coding genes in trypanosomes by RNA polymerase I. Annu Rev Microbiol 51:463–489
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
Clayton C, Shapira M (2007) Post-transcriptional regulation of gene expression in trypanosomes and leishmanias. Mol Biochem Parasitol 156(2):93–101
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
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
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
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
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
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
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
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
Nazari R, Davoudi N (2011) Cloning and expression of truncated form of tissue plasminogen activator in Leishmania tarentolae. Biotechnol Lett 33(3):503–508
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
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
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
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
Kornberg RD (2007) The molecular basis of eukaryotic transcription. Proc Natl Acad Sci U S A 104(32):12955–12961
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
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
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
Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3(2):97–130
Varki A (2007) Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 446(7139):1023–1029
Elbein AD (1991) The role of N-linked oligosaccharides in glycoprotein function. Trends Biotechnol 9(10):346–352
Helenius A, Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73:1019–1049
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
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
Timpl R (1996) Macromolecular organization of basement membranes. Curr Opin Cell Biol 8(5):618–624
Miner JH, Yurchenco PD (2004) Laminin functions in tissue morphogenesis. Annu Rev Cell Dev Biol 20:255–284
Domogatskaya A, Rodin S, Tryggvason K (2012) Functional diversity of laminins. Annu Rev Cell Dev Biol 28:523–553
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
Aumailley M (2013) The laminin family. Cell Adh Migr 7(1):48–55
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
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
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
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
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
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
Marinkovich MP (2007) Tumour microenvironment: laminin 332 in squamous-cell carcinoma. Nat Rev Cancer 7(5):370–380
Beck K, Hunter I, Engel J (1990) Structure and function of laminin: anatomy of a multidomain glycoprotein. FASEB J 4(2):148–160
Bernhard F, Tozawa Y (2013) Cell-free expression-making a mark. Curr Opin Struct Biol 23(3):374–380
Rosenblum G, Cooperman BS (2014) Engine out of the chassis: cell-free protein synthesis and its uses. FEBS Lett 588(2):261–268
Harbers M (2014) Wheat germ systems for cell-free protein expression. FEBS Lett 588:2762–2773
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
Mureev S, Kovtun O, Nguyen UT, Alexandrov K (2009) Species-independent translational leaders facilitate cell-free expression. Nat Biotechnol 27(8):747–752
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
Kovtun O, Mureev S, Jung W, Kubala MH, Johnston W, Alexandrov K (2011) Leishmania cell-free protein expression system. Methods 55(1):58–64
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
Fernandez-Robledo JA, Vasta GR (2010) Production of recombinant proteins from protozoan parasites. Trends Parasitol 26(5):244–254
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
Acknowledgments
The preparation of this chapter was supported by JSPS KAKENHI Grant Numbers 18108003 and 26350959.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights 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
DOI: https://doi.org/10.1007/978-3-319-27216-0_10
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-27214-6
Online ISBN: 978-3-319-27216-0
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)