Volume: 54 Issue: 1
Year: 2023, Page: 13-20, Doi: https://doi.org/10.51966/jvas.2023.54.1.13-20
Received: May 4, 2022 Accepted: Oct. 20, 2022 Published: March 31, 2023
Bovine trichomonosis is one of the most neglected venereal diseases of cattle. Trichomonas foetus, the causative organism was known over decades and is responsible for severe reproductive failure. Except for a few lab-based assays, to date, there are no point-of-care diagnostics developed to screen for the presence of infectious agents in cattle. In this study, we have identified cysteine protease 8 as a suitable antigenic protein for developing sero-diagnostics. A 960 bp Tf CP8 gene was cloned into methylotrophic Pichia pastoris X-33 by homologous recombination using a pPICZαA vector for recombinant protein expression. The traditional fed-batch method of induction with methanol resulted in inconsistent expression in 48h incubation, hence a novel single batch culture with 1% methanol induction for 24h was standardised and obtained optimal recovery of approximately 36 KDa recombinant protein secreted into media. To the best of our knowledge, this is the first report of cloning and expression of genes from Trichomonas foetus. This CP8 protein could be further optimised for developing lateral flow assays and ELISA as point-of-care tools.
Keywords: Fed-batch culture, His Tag, methylotrophic yeast
Ahmad, M., Hirz, M., Pichler, H., and Schwab, H. 2014. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl. Microbiol. Biotech. 98(12): 5301–5317.
Bryan, L. A., Campbell, J. R. and Gajadhar, A. A. 1999. Effects of temperature on the survival of Tritrichomonas foetus in transport, Diamond's and InPouch TF media. Vet. Rec. 144(9): 227–232. https://doi.org/10.1136/vr.144.9.227.
Casteriano, A., Molini, U., Kandjumbwa, K., Khaiseb, S., Frey, C. F. and Šlapeta, J. 2016. Novel genotype of Tritrichomonas foetus from cattle in Southern Africa. Parasitol. 143(14) : 1954–1959. https://doi.org/10.1017/S003118201600158X
Felleisen, R. S., Lambelet, N., Bachmann, P., Nicolet, J., Müller, N. and Gottstein, B. 1998 Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences. J. Clin. Microbiol. 36(2): 513–519.
Gleeson, M. A., White, C. E., Meininger, D. P. and Komives, E. A. 1998. Generation of protease-deficient strains and their use in heterologous protein expression. Methods Mol. Biol. (Clifton, N.J.), 103: 81–94.
Green, M. R. and Sambrook, J. 2016. Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Minipreps. Cold Spring Harb. Protoc (10): https://doi.org/10.1101/pdb.prot093344
Green, M. R. and Sambrook, J. 2018. The Basic Polymerase Chain Reaction (PCR). Cold Spring Harb. Protoc,2018 (5): 10.1101/pdb.prot095117. https://doi.org/10.1101/pdb.prot095117
Green, R. and Rogers, E. J. 2013. Transformation of chemically competent E. coli. Meth. Enzymol. 529: 329–336. https://doi.org/10.1016/B978-0-12-418687-3.00028-8
Huang, K. Y., Shin, J. W., Huang, P. J., Ku, F. M., Lin, W. C., Lin, R., Hsu, W. M. and Tang, P. 2013. Functional profiling of the Tritrichomonas foetus transcriptome and proteome. Mol. Biochem. Parasitol, 187(1): 60–71.
Karli G., Polava R. and Varada K. 2020. Comparative Omics Based Approach to Identify Putative Immunogenic Proteins of Trichomonas foetus. Learning and Analytics in Intelligent Systems, Springer, Cham.15: 583-592. https://doi.org/10.1007/978-3-030-46939-9_51.
Kawai, S., Hashimoto, W. and Murata, K. 2010. Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism. Bioeng. Bugs, 1(6): 395–403. https://doi.org/10.4161/bbug.1.6.13257
Koontz L. 2014. TCA precipitation. Meth. Enzymol. 541: 3–10. https://doi.org/10 .1016 / B978-0-12-420119-4.00001-X
Kõressaar, T., Lepamets, M., Kaplinski, L., Raime, K., Andreson, R. and Remm, M. 2018. Primer3_masker: integrating masking of template sequence with primer design software. Bioinformatics (Oxford, England), 34(11): 1937–1938.
Kurtzman C. P. 2009. Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. J. Ind. Microbiol. Biotech. 36(11): 1435–1438. https://doi.org/10.1007/s10295-009-0638-4.
Lee, P. Y., Costumbrado, J., Hsu, C. Y. and Kim, Y. H. 2012. Agarose gel electrophoresis for the separation of DNA fragments. JoVE, 62: 3923. https://doi.org/10.3791/3923
Lun, Z. R. and Gajadhar, A. A. 1999. A simple and rapid method for staining Tritrichomonas foetus and Trichomonas vaginalis. J. Vet. Diagn: official publication of the AAVLD, Inc, 11(5): 471–474. https://doi.org/10.1177/104063879901100516
Mallinson, D. J., Livingstone, J., Appleton, K. M., Lees, S. J., Coombs, G. H. and North, M. J. 1995. Multiple cysteine proteinases of the pathogenic protozoon Tritrichomonas foetus: identification of seven diverse and differentially expressed genes. Microbiol. (Reading, England), 141: 3077-3085.
Salamin, K., Sriranganadane, D., Léchenne, B., Jousson, O. and Monod, M. 2010. Secretion of an endogenous subtilisin by Pichia pastoris strains GS115 and KM71. Appl. Environ. Microbiol., 76(13): 4269–4276. https://doi.org/10.1128/AEM.00412-10
Shu, M., Shen, W., Yang, S., Wang, X., Wang, F., Wang, Y. and Ma, L. 2016. High-level expression and characterization of a novel serine protease in Pichia pastoris by multi-copy integration. Enzyme Microb. Technol., 92:56–66.
Singh, B. N., Hayes, G. R., Lucas, J. J., Beach, D. H. and Gilbert, R. O. 2005. In vitro cytopathic effects of a cysteine protease of Tritrichomonas foetus on cultured bovine uterine epithelial cells. Am. J. Vet. Res, 66(7): 1181–1186.
Singh, B. N., Lucas, J. J., Hayes, G. R., Kumar, I., Beach, D. H., Frajblat, M., Gilbert, R. O., Sommer, U. and Costello, C. E. 2004. Tritrichomonas foetus induces apoptotic cell death in bovine vaginal epithelial cells. Infect. Immun., 72(7): 4151–4158.
Smith, B. J. 1994. SDS polyacrylamide gel electrophoresis of proteins. Methods Mol. Biol (Clifton, N.J.), 32: 23–34. https://doi.org/10.1385/0-89603-268-X:23
Sun, Z., Stack, C. and Šlapeta, J. 2012. Sequence differences in the diagnostic region of the cysteine protease 8 gene of Tritrichomonas foetus parasites of cats and cattle. Vet. Parasitol., 186(3): 445–449. https://doi.org/10.1016/j.vetpar.2011.12.001
Thomford, J. W., Talbot, J. A., Ikeda, J. S. and Corbeil, L. B. 1996. Characterization of extracellular proteinases of Tritrichomonas foetus. J. Parasitol, 82(1): 112–117.
Trichomonosis. OIE reference manual Chapter. 3.04.15 accessed on May 2019
Vincze, T., Posfai, J. and Roberts, R. J. 2003. NEBcutter: A program to cleave DNA with restriction enzymes. Nucleic Acids Res, 31(13): 3688–3691.
Weidner, M., Taupp, M. and Hallam, S. J 2010. Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris. J. Vis Exp. 36: e1862, doi:10.3791/1862.
Zhang, Y., Liu, R. and Wu, X. 2007. The proteolytic systems and heterologous proteins degradation in the methylotrophic yeast Pichia pastoris. Ann. Microbiol. 57: 553.
© 2023 Geethanjali et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Geethanjali,K., Rathnagiri,P. and Kalarani,V. 2023. Heterologous expression of Cysteine Protease 8 from Trichomonas foetus in Pichia pastoris. J. Vet. Anim. Sci. 54(1):13-20
DOI: https://doi.org/10.51966/jvas.2023.54.1.13-20