Production and purification of recombinant bovine osteopontin using Escherichia coli as a Cell Factory

Producción Y Purificación De Osteopontina Bovina Recombinante Mediante Escherichia coli Como Fábrica Celular

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Angela Patricia Brijaldo Villamizar
María Camila Londoño-Méndez
Luis Fernando Arbeláez Ramírez
Fabian Rueda

Abstract

Animal reproduction and improvement programs require the optimization of biotechnological tools capable of favoring reproductive rates in various species. The use of protein additives that improve sperm cryopreservation and in vitro embryo production seems to be an interesting alternative. Osteopontin has been related to the fertilizing potential of the sperm and early embryonic development. The objective of this work was to determine the optimal conditions to produce recombinant Osteopontin (rOPN) by using Escherichia coli as a cell factory. For this, the OPN gene was inserted into an expression vector pET28(a+) inducible by IPTG, with resistance to Kanamycin and a histidine tail (6xHis-tag). The resulting construct was used to transform competent E. coli BL21-Star ™ cells. The transformed colonies were used to produce rOPN-H6 at 20, 30, and 37 °C, testing two concentrations of the inducer IPTG (1.0 and 0.1mM). A purification of rOPN-H6 was performed using imidazole affinity columns (10, 50, 200, 350, 500mM). The results showed that the production of rOPN-H6 was only successful at 37°C regardless of the concentration of IPTG used. Purification of rOPN-H6 was successful using imidazole at 200mM, with an apparent tendency to dimerization after obtaining purified protein. In this way, the best conditions to obtain recombinant OPN is concluded, suggesting its potential use in sperm cryopreservation assays and culture media for in vitro embryo production.

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Alvarez-gallardo, H., Kjelland, M. E., Moreno, J. F., Jr, T. H. W., Lara-sagaho, A. V, Randel, R. D., Lammoglia, M. A., Pe, M., Romo, S., & Espero, E. (2013). Gamete Therapeutics : Recombinant Protein Adsorption by Sperm for Increasing Fertility via Artificial Insemination. 8(6). https://doi.org/10.1371/journal.pone.0065083. DOI: https://doi.org/10.1371/journal.pone.0065083

Barrios, B., Pe, R., Gallego, M., Muin, T., & Cebria, A. (2000). Seminal Plasma Proteins Revert the Cold-Shock Damage on Ram Sperm Membrane 1 and Jose. 1537, 1531–1537. https://doi.org/10.1095/biolreprod63.5.1531 DOI: https://doi.org/10.1095/biolreprod63.5.1531

Boccia, L., Di Francesco, S., Neglia, G., De Blasi, M., Longobardi, V., Campanile, G., & Gasparrini, B. (2013). Osteopontin improves sperm capacitation and in vitro fertilization efficiency in buffalo (Bubalus bubalis). Theriogenology, 80(3), 212–217. https://doi.org/10.1016/j.theriogenology.2013.04.017. DOI: https://doi.org/10.1016/j.theriogenology.2013.04.017

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254. https://doi.org/10.1016/0003-2697(76)90527-3. DOI: https://doi.org/10.1016/0003-2697(76)90527-3

Bustamanete Filho, I. C. (2010). Cloning, expression and purification of proteins from bovine seminal plasma related with semen freezability. In Science. e [Trabajo de grado, Repositorio institucional Universidade Federal do Rio Grande do sul. https://lume.ufrgs.br/handle/10183/23020

Bustamante-Filho, I. C., Renato Menegassi, S., Ribas Pereira, G., Dias Salton, G., Mosena Munari, F., Roberto Schneider, M., Costa Mattos, R., Otávio Jardim Barcellos, J., Pereira Laurino, J., Obino Cirne-Lima, E., & Inês Mascarenhas Jobim, M. (2021). Bovine seminal plasma osteopontin: Structural modelling, recombinant expression and its relationship with semen quality. Andrologia, 53(1), 1–15. https://doi.org/10.1111/and.13905 DOI: https://doi.org/10.1111/and.13905

Bustamante-Filho, I., Salton, G. D., Munari, F. M., Schneider, M. R., Mattos, R. C., Laurino, J. P., Cirne-Lima, E. O., & Jobim, M. I. M. (2014). Recombinant expression and purification of the bovine acidic Seminal Fluid Protein. Animal Reproduction, 11(2), 96–103.

Cajazeiras, J. B., Melo, L. M., Albuquerque, E. S., Rádis-Baptista, G., Cavada, B. S., & Freitas, V. J. F. (2009). Analysis of protein expression and a new prokaryotic expression system for goat (Capra hircus) spermadhesin Bdh-2 cDNA. Genetics and Molecular Research : GMR, 8(3), 1147–1157. https://doi.org/10.4238/vol8-3gmr639 DOI: https://doi.org/10.4238/vol8-3gmr639

Cancel, a M., Chapman, D. a, & Killian, G. J. (1997). Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma. Biology of Reproduction, 57(6), 1293–1301. https://doi.org/10.1095/biolreprod57.6.1293 DOI: https://doi.org/10.1095/biolreprod57.6.1293

Carbonell, X., & Villaverde, A. (2002). Protein aggregated into bacterial inclusion bodies does not result in protection from proteolytic digestion. 1939–1944. https://doi.org/10.1023/A:1021173114193 DOI: https://doi.org/10.1023/A:1021173114193

Eduardo, C., Souza, A., Moura, A. A., & Killian, G. J. (2008). Binding patterns of bovine seminal plasma proteins A1 / A2 , 30 kDa and osteopontin on ejaculated sperm before and after incubation with isthmic and ampullary oviductal fluid ଝ , ଝଝ. 105, 72–89. https://doi.org/10.1016/j.anireprosci.2007.11.027 DOI: https://doi.org/10.1016/j.anireprosci.2007.11.027

Gabler, C., Chapman, D. A., & Killian, G. J. (2003). Expression and presence of osteopontin and integrins in the bovine oviduct during the oestrous cycle. Reproduction, 126(6), 721–729. https://doi.org/10.1530/rep.0.1260721 DOI: https://doi.org/10.1530/rep.0.1260721

García-Fraga, B., da Silva, A. F., López-Seijas, J., & Sieiro, C. (2015). Optimized expression conditions for enhancing production of two recombinant chitinolytic enzymes from different prokaryote domains. Bioprocess and Biosystems Engineering, 38(12), 2477–2486. https://doi.org/10.1007/s00449-015-1485-5 DOI: https://doi.org/10.1007/s00449-015-1485-5

Haglin, E. R., Yang, W., Briegel, A., & Thompson, L. K. (2017). His-Tag-Mediated Dimerization of Chemoreceptors Leads to Assembly of Functional Nanoarrays. Biochemistry, 56(44), 5874–5885. https://doi.org/10.1021/acs.biochem.7b00698 DOI: https://doi.org/10.1021/acs.biochem.7b00698

Icer, M. A., & Gezmen-Karadag, M. (2018). The multiple functions and mechanisms of osteopontin. Clinical Biochemistry, 59(July), 17–24. https://doi.org/10.1016/j.clinbiochem.2018.07.003 DOI: https://doi.org/10.1016/j.clinbiochem.2018.07.003

Liu, Q., Xie, Q. Z., Zhou, Y., & Yang, J. (2014). Osteopontin is expressed in the oviduct and promotes fertilization and preimplantation embryo development of mouse. Zygote, 760. https://doi.org/10.1017/S0967199414000483 DOI: https://doi.org/10.1017/S0967199414000483

Monaco, E., Gasparrini, B., Boccia, L., De Rosa, A., Attanasio, L., Zicarelli, L., & Killian, G. (2009). Effect of osteopontin (OPN) on in vitro embryo development in cattle. Theriogenology, 71(3), 450–457. https://doi.org/10.1016/j.theriogenology.2008.08.012 DOI: https://doi.org/10.1016/j.theriogenology.2008.08.012

Moura, A. A. (2005). Seminal plasma proteins and fertility indexes in the bull : The case for osteopontin. Anim Reprod, 2, March, 3–10.

Nature. (1970). Laemmli buffer Background Purpose of the Laemmli buffer Laemmli Buffer Recipe Preparation Recommended Storage Temperature of Laemmli buffer Laemmli buffer References. Nature.

Ono, B., Kimiduka, H., Kubota, M., Okuno, K., & Yabuta, M. (2007). Role of the ompT mutation in stimulated decrease in colony-forming ability due to intracellular protein aggregate formation in Escherichia coli strain BL21. Bioscience, Biotechnology, and Biochemistry, 71(2), 504–512. https://doi.org/10.1271/bbb.60541 DOI: https://doi.org/10.1271/bbb.60541

Peris, S. I., Bilodeau, J. F., Dufour, M., & Bailey, J. L. (2007). Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Molecular Reproduction and Development, 74(7), 878–892. https://doi.org/10.1002/mrd.20686 DOI: https://doi.org/10.1002/mrd.20686

Prince, C. W. (1989). Secondary structure predictions for rat osteopontin. Connective Tissue Research, 21(1–4), 15–20. https://doi.org/10.3109/03008208909049991 DOI: https://doi.org/10.3109/03008208909049991

Retnoningrum, D. S., Pramesti, H. T., Santika, P. Y., Valerius, O., Asjarie, S., & Suciati, T. (2012). Codon optimization for high level expression of human bone morphogenetic protein-2 in Escherichia coli. Protein Expression and Purification, 84(2), 188–194. https://doi.org/10.1016/j.pep.2012.05.010 DOI: https://doi.org/10.1016/j.pep.2012.05.010

Rueda, F., Cano-Garrido, O., Mamat, U., Wilke, K., Seras-Franzoso, J., García-Fruitós, E., & Villaverde, A. (2014). Production of functional inclusion bodies in endotoxin-free Escherichia coli. Applied Microbiology and Biotechnology, 98(22), 9229–9238. https://doi.org/10.1007/s00253-014-6008-9 DOI: https://doi.org/10.1007/s00253-014-6008-9

Rueda, F., Céspedes, M. V., Conchillo-Solé, O., Sánchez-Chardi, A., Seras-Franzoso, J., Cubarsi, R., Gallardo, A., Pesarrodona, M., Ferrer-Miralles, N., Daura, X., Vázquez, E., García-Fruitós, E., Mangues, R., Unzueta, U., & Villaverde, A. (2015). Bottom-Up Instructive Quality Control in the Biofabrication of Smart Protein Materials. Advanced Materials, 27(47), 7816–7822. https://doi.org/10.1002/adma.201503676 DOI: https://doi.org/10.1002/adma.201503676

Rueda, F., Garcés P, T., Herrera L, R., Arbeláez R, L., Peña J, M., Velásquez P, H., Hernández V, A., & Cardozo C, J. (2013). Seminal plasma proteins increase the post-thaw sperm viability of Sanmartinero bull’s semen. Revista MVZ Cordoba, 18(1), 3327–3335. DOI: https://doi.org/10.21897/rmvz.195

Sicherle, C. C., Maia, M. S., Bicudo, S. D., Rodello, L., & Azevedo, H. C. (2011). Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen supplemented with catalase or Trolox. Small Ruminant Research, 95(2–3), 144–149. https://doi.org/10.1016/j.smallrumres.2010.10.011 DOI: https://doi.org/10.1016/j.smallrumres.2010.10.011

Singh, B. P., Asthana, A., Basu, A., & Tangirala, R. (2020). Conserved core tryptophans of FnII domains are crucial for the membranolytic and chaperone-like activities of bovine seminal plasma protein PDC-109. 594, 509–518. https://doi.org/10.1002/1873-3468.13617 DOI: https://doi.org/10.1002/1873-3468.13617

Sivashanmugam, A., Murray, V., Cui, C., Zhang, Y., Wang, J., & Li, Q. (2009). Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. 18(1), 936–948. https://doi.org/10.1002/pro.102 DOI: https://doi.org/10.1002/pro.102

Thérien, I., Moreau, R., & Manjunath, P. (1999). Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm. Biology of Reproduction, 61, 590–598. https://doi.org/10.1095/biolreprod61.3.590 DOI: https://doi.org/10.1095/biolreprod61.3.590

Watson, P. F. (1995). Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reproduction, Fertility and Development, 7(4), 871–891. https://doi.org/10.1071/RD9950871 DOI: https://doi.org/10.1071/RD9950871

Wei, R., Wong, J. P. C., & Kwok, H. F. (2017). Osteopontin - A promising biomarker for cancer therapy. Journal of Cancer, 8(12), 2173–2183. https://doi.org/10.7150/jca.20480 DOI: https://doi.org/10.7150/jca.20480

Willforss, J., Morrell, J. M., Resjö, S., Hallap, T., Padrik, P., Siino, V., de Koning, D. J., Andreasson, E., Levander, F., & Humblot, P. (2021). Stable bull fertility protein markers in seminal plasma. Journal of Proteomics, 236, 104135. https://doi.org/10.1016/j.jprot.2021.104135 DOI: https://doi.org/10.1016/j.jprot.2021.104135

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