Leishmania tarentolae Cell-Free System for Filamentous Protein Expression
Recombinant Protein Expression Using L. tarentolae
DOI:
https://doi.org/10.69750/dmls.01.010.079Keywords:
Expression system, Leishmania tarentolae, proteins of Toxoplasma gondii, SAG1, Cyclophilin 18Abstract
Background: In earlier studies, Leishmania tarentolae is known as an expression system due to its post-translational modification. SAG1 and Cyc18 proteins of T. gondii was evaluated for protection against toxoplasmosis. A surface glycoprotein of T. gondii, is a promoter of the immune response and previously studied for vaccine development during the infection. Cyc18 induces IL-12, which can drive Th1 and CD8+ T cell development when bound to CCR5, it rolled to adjuvant the immune response of T. gondii surface protein.
Objectives: This study was performed to evaluate the efficiency of SAG1 (Surface Antigen 1) and TgCyc18 (Cyclophilin 18) considered a a CCR5 chemochine for Toxoplasma gondii vaccine when expressed in L. tarentolae.
Methods: SAG1 and TgCyc18 genes were cloned and transfected into L. tarentolae. Protein expression and secretion were effectively validated by PCR, immunoblotting, enzyme activity, ELISA, and immunofluor. And features of SAG1 were evaluated mass spectrometry.
Results: Successful transfection of SAG1 and TgCyc18 plasmids and transfected into Leishmania tarentolae, and was confirmed by two steps of PCR. Current study verified that the culture supernatant included positive expression and secretion of the T.gondii protein (SAG1) into the media, indicated by acid phosphatase assay. The characteristics of the SAG1 protein were established using immunofluorescence, immunoblot, ELISA, and mass spectrometry.
Conclusion: This study confirms that L. tarentolae effectively can produces and secretes recombinant T. gondii proteins (SAG1 and TgCyc18), offering a potential platform for vaccine development against toxoplasmosis.
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References
Klatt S, Simpson L, Maslov DA, et al. Leishmania tarentolae: Taxonomic classification and its application as a promising biotechnological expression host. PLoS Negl Trop Dis 2019; 13: e0007424.
Taylor VM, Muñoz DL, Cedeño DL, et al. Leishmania tarentolae: Utility as an in vitro model for screening of antileishmanial agents. Exp Parasitol 2010; 126: 471–475.
Li J, Zheng Z-W, Natarajan G, et al. The first successful report of the in vitro life cycle of Chinese Leishmania: the in vitro conversion of Leishmania amastigotes has been raised to 94% by testing 216 culture medium compounds. Acta Parasitol 2017; 62: 154–163.
Grebowski J, Studzian M, Bartosz G, et al. Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter. Biochim Biophys Acta - Biomembr 2016; 1858: 2617–2624.
Akram J, Mojgan B, Bahram K, et al. Compound serum and hemin free medium for cultivation of Leishmania tarentolae: A recombinant protein expression system. African J Biotechnol 2015; 14: 2130–2134.
Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet 2018; 392: 951–970.
Bolhassani A, Shirbaghaee Z, Agi E, et al. VLP production in Leishmania tarentolae: a novel expression system for purification and assembly of HPV16 L1. Protein Expr Purif 2015; 116: 7–11.
Kalef DA. Leishmania mexicana recombinant filamentous acid phosphatase as a carrier for Toxoplasma gondii surface antigen 1 expression in Leishmania tarentolae. J Parasit Dis 2021; 45: 1135–1144.
Hunter DJB, Bhumkar A, Giles N, et al. Unexpected instabilities explain batch‐to‐batch variability in cell‐free protein expression systems. Biotechnol Bioeng 2018; 115: 1904–1914.
Wiese M, Görcke I, Overath P. Expression and species-specific glycosylation of Leishmania mexicana secreted acid phosphatase in Leishmania major. Mol Biochem Parasitol 1999; 102: 325–329.
Ilg T, Stierhof YD, Craik D, et al. Purification and structural characterization of a filamentous, mucin-like proteophosphoglycan secreted by Leishmania parasites. J Biol Chem 1996; 271: 21583–21596.
Stierhof Y-D, Wiese M, Ilg T, et al. Structure of a filamentous phosphoglycoprotein polymer: the secreted acid phosphatase of Leishmania mexicana11Edited by W. Baumeister. J Mol Biol 1998; 282: 137–148.
Lekutis C, Ferguson DJP, Grigg ME, et al. Surface antigens of Toxoplasma gondii: variations on a theme. Int J Parasitol 2001; 31: 1285–1292.
He X, Grigg ME, Boothroyd JC, et al. Structure of the immunodominant surface antigen from the Toxoplasma gondii SRS superfamily. Nat Struct Biol 2002; 9: 606–11.
Wang Y, Yin H. Research progress on surface antigen 1 (SAG1) of Toxoplasma gondii. Parasit Vectors 2014; 7: 180.
Abdizadeh R, Maraghi S, Ghadiri AA, et al. Cloning and expression of major surface antigen 1 gene of Toxoplasma gondii RH strain using the expression vector pVAX1 in Chinese Hamster Ovary cells. Jundishapur J Microbiol 2015; 8: 1–10.
Ibrahim HM, Nishimura M, Tanaka S, et al. Overproduction of Toxoplasma gondii cyclophilin-18 regulates host cell migration and enhances parasite dissemination in a CCR5-independent manner. BMC Microbiol 2014; 14: 76.
Fachado A, Rodriguez A, Molina J, et al. Long-term protective immune response elicited by vaccination with an expression genomic library of Toxoplasma gondii. Infect Immun 2003; 71: 5407–5411.
Badirzadeh A, Montakhab-Yeganeh H, Miandoabi T. Arginase/nitric oxide modifications using live non-pathogenic Leishmania tarentolae as an effective delivery system inside the mammalian macrophages. J Parasit Dis 2021; 45: 65–71.
Fritsche C, Sitz M, Weiland N, et al. Characterization of the growth behavior of Leishmania tarentolae - A new expression system for recombinant proteins. J Basic Microbiol 2007; 47: 384–393.
Wingfield PT. Protein Precipitation Using Ammonium Sulfate. Curr Protoc Protein Sci 2016; 1–10.
Wingfield P. Protein precipitation using ammonium sulfate. Curr Protoc Protein Sci 2001; Appendix 3: Appendix 3F.
Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides 2015; 72: 4–15.
Efstathiou A, Smirlis D. Leishmania Protein Kinases: Important Regulators of the Parasite Life Cycle and Molecular Targets for Treating Leishmaniasis. Microorganisms; 9. Epub ahead of print 2021. DOI: 10.3390/microorganisms9040691.
Shakarian AM, Joshi MB, Ghedin E, et al. Molecular dissection of the functional domains of a unique, tartrate-resistant, surface membrane acid phosphatase in the primitive human pathogen Leishmania donovani. J Biol Chem 2002; 277: 17994–18001.
Lai JY, Klatt S, Lim TS. Potential application of Leishmania tarentolae as an alternative platform for antibody expression. Crit Rev Biotechnol 2019; 39: 380–394.
Raymond F de´ ric, Boisvert S bastien, Roy G tan, et al. Genome sequencing of the lizard parasite Leishmania tarentolae reveals the loss of genes associated with the intracellular stage of human pathogenic species. 2012; 40: 1131–1147.
Cantacessi C, Dantas-Torres F, Nolan MJ, et al. The past, present, and future of Leishmania genomics and transcriptomics. Trends Parasitol 2015; 31: 100–108.
Gehl J. Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand 2003; 177: 437–447.
de Oliveira TA, da Silva W, da Rocha Torres N, et al. Application of the LEXSY Leishmania tarentolae system as a recombinant protein expression platform: A review. Process Biochem 2019; 87: 164–173.
Sanchez-Garcia L, Martín L, Mangues R, et al. Recombinant pharmaceuticals from microbial cells: a 2015 update. Microb Cell Fact 2016; 15: 33.
Hartmann MWW, Breitling R. Suspension Culture of Protozoan Organisms. In: Industrial Scale Suspension Culture of Living Cells. John Wiley & Sons, Ltd, pp. 294–347.
Kianmehr A, Mahrooz A, Oladnabi M, et al. Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae. Mol Biotechnol 2016; 58: 566–572.
Basile G, Peticca M. Recombinant Protein Expression in Leishmania tarentolae. Mol Biotechnol 2009; 43: 273–278.
Clayton CE. Genetic Manipulation of Kinetoplastida. Parasitol Today 1999; 15: 372–378.
Teixeira SM. Control of gene expression in Trypanosomatidae. Braz J Med Biol Res 1998; 31: 1503–16.
Garg G, Singh K, Ali V. Proteomic approaches to unravel the intricacy of secreted proteins of Leishmania: an updated review. Biochim Biophys Acta (BBA)-Proteins Proteomics 2018; 1866: 913–923.
Dorsey BM, Cass CL, Cedeño DL, et al. Effects of Specific Electric Field Stimulation on the Release and Activity of Secreted Acid Phosphatases from Leishmania tarentolae and Implications for Therapy. Pathogens; 7. Epub ahead of print 2018. DOI: 10.3390/pathogens7040077.
Koteshwara A, Philip N V, Aranjani JM, et al. A set of simple methods for detection and extraction of laminarinase. Sci Rep 2021; 11: 2489.
Halliday C. The role of the flagellum attachment zone in Leishmania mexicana flagellar pocket architecture. Oxford Brookes University, 2021.
Nakayasu ES, Gritsenko M, Piehowski PD, et al. Tutorial: Best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation. Nat Protoc 2021; 16: 3737–3760.
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