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Biochemistry (Moscow)

, Volume 84, Issue 1, pp 20–32 | Cite as

Recombinant Human Erythropoietin Proteins Synthesized in Escherichia coli Cells: Effects of Additional Domains on the in vitro and in vivo Activities

  • A. S. KaryaginaEmail author
  • T. M. Grunina
  • A. M. Lyaschuk
  • E. V. Voronina
  • R. A. Marigin
  • S. A. Cherepushkin
  • I. N. Trusova
  • A. V. Grishin
  • M. S. Poponova
  • P. A. Orlova
  • V. N. Manskikh
  • N. V. Strukova
  • M. S. Generalova
  • K. E. Nikitin
  • L. A. Soboleva
  • I. S. Boksha
  • A. V. Gromov
Article
  • 1 Downloads

Abstract

The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use.

Keywords

erythropoietin heparin-binding domain proliferation of human erythroleukemia TF-1 cells vascularization of surrounding tissues 

Abbreviation

a.a.

amino acid residue

BMP-2

bone morphogenetic protein-2

DBM

demineralized bone matrix

DTT

dithiothreitol

EPO(R)

erythropoietin (receptor)

HBD

heparin-binding domain

6His

six histidine amino acid residues

s-tag

15-a.a. oligopeptide from bovine pancreatic ribonuclease A

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References

  1. 1.
    Krantz, S. B. (1991) Erythropoietin, Blood, 77, 419–434.Google Scholar
  2. 2.
    Shiozawa, Y., Jung, Y., Ziegler, A. M., Pedersen, E. A., Wang, J., Wang, Z., Song, J., Wang, J., Lee, C. H., Sud, S., Pienta, K. J., Krebsbach, P. H., and Taichman, R. S. (2010) Erythropoietin couples hematopoiesis with bone forma–tion, PLoS One, 5, e10853.Google Scholar
  3. 3.
    Wu, C., Giaccia, A. J., and Rankin, E. B. (2014) Osteoblasts: a novel source of erythropoietin, Curr. Osteoporos. Rep., 4, 428–432.Google Scholar
  4. 4.
    Li, C., Shi, C., Kim, J., Chen, Y., Ni, S., Jiang, L., Zheng, C., Li, D., Hou, J., Taichman, R. S., and Sun, H. (2015) Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling, J. Dent. Res., 94, 455–463.Google Scholar
  5. 5.
    Holstein, J. H., Menger, M. D., Scheuer, C., Meier, C., Culemann, U., Wirbel, R. J., Garcia, P., and Pohlemann, T. (2007) Erythropoietin (EPO): EPO–receptor signaling improves early endochondral ossification and mechanical strength in fracture healing, Life Sci., 80, 893–900.Google Scholar
  6. 6.
    Holstein, J. H., Orth, M., Scheuer, C., Tami, A., Becker, S. C., Garcia, P., Histing, T., Morsdorf, P., Klein, M., Pohlemann, T., and Menger, M. D. (2011) Erythropoietin stimulates bone formation, cell proliferation, and angio–genesis in a femoral segmental defect model in mice, Bone, 49, 1037–1045.Google Scholar
  7. 7.
    Garcia, P., Speidel, V., Scheuer, C., Laschke, M. W., Holstein, J. H., Histing, T., Pohlemann, T., and Menger, M. D. (2011) Low dose erythropoietin stimulates bone healing in mice, J. Orthop. Res., 29, 165–172.Google Scholar
  8. 8.
    Rolfing, J. H. D., Bendtsen, M., Jensen, J., Stiehler, M., Foldager, C. B., Hellfritzsch, M. B., and Bunger, C. (2012) Erythropoietin augments bone formation in a rabbit pos–terolateral spinal fusion model, J. Orthop. Res., 30, 1083–1088.Google Scholar
  9. 9.
    Sun, H., Jung, Y., Shiozawa, Y., Taichman, R. S., and Krebsbach, P. H. (2012) Erythropoietin modulates the structure of bone morphogenetic protein 2–engineered cra–nial bone, Tissue Eng. Part A, 18, 2095–20105.Google Scholar
  10. 10.
    Rolfing, J. H., Jensen, J., Jensen, J. N., Greve, A. S., Lysdahl, H., Chen, M., Rejnmark, L., and Bunger, C. (2014) A single topical dose of erythropoietin applied on a collagen carrier enhances calvarial bone healing in pigs, Acta Orthop., 85, 201–209.Google Scholar
  11. 11.
    Patel, J. J., Modes, J. E., Flanagan, C. L., and Krebsbach, P. H. (2015) Dual delivery of EPO and BMP2 from a novel modular poly–ε–caprolactone construct to increase the bone formation in prefabricated bone flaps, Tissue Eng. Part C Methods, 21, 889–897.Google Scholar
  12. 12.
    Omlor, G. W., Kleinschmidt, K., Gantz, S., Speicher, A., Guehring, T., and Richter, W. (2016) Increased bone for–mation in a rabbit long–bone defect model after single local and single systemic application of erythropoietin, Acta Orthop., 87, 425–431.Google Scholar
  13. 13.
    Wang, Y. J., Liu, Y. D., Chen, J., Hao, S. J., Hu, T., Ma, G. H., and Su, Z. G. (2010) Efficient preparation and PEGylation of recombinant human non–glycosylated ery–thropoietin expressed as inclusion body in E. coli, Int. J. Pharm., 386, 156–164.Google Scholar
  14. 14.
    Jeong, T. H., Son, Y. J., Ryu, H. B., Koo, B. K., Jeong, S. M., Hoang, P., Do, B. H., Song, J. A., Chong, S. H., Robinson, R. C., and Choe, H. (2014) Soluble expression and partial purification of recombinant human erythropoi–etin from E. coli, Protein Expr. Purif., 95, 211–218.Google Scholar
  15. 15.
    Boissel, J. P., Lee, W. R., Presnell, S. R., Cohen, F. E., and Bunn, H. F. (1993) Erythropoietin structure–function rela–tionships. Mutant proteins that test a model of tertiary structure, J. Biol. Chem., 268, 5983–5993.Google Scholar
  16. 16.
    Narhi, L. O., Arakawa, T., Aoki, K., Wen, J., Elliott, S., Boone, T., and Cheetham, J. (2001) Asn to Lys mutations at three sites which are N–glycosylated in the mammalian protein decrease the aggregation of Escherichia coli–derived erythropoietin, Protein Eng., 14, 135–140.Google Scholar
  17. 17.
    Grunina, T. M., Demidenko, A. V., Lyaschuk, A. M., Poponova, M. S., Galushkina, Z. M., Soboleva, L. A., Cherepushkin, S. A., Polyakov, N. B., Grumov, D. A., Solovyev, A. I., Zhukhovitsky, V. G., Boksha, I. S., Subbotina, M. E., Gromov, A. V., Lunin, V. G., and Karyagina, A. S. (2017) Recombinant human erythropoi–etin with additional processable protein domains: purifica–tion of protein synthesized in Escherichia coli heterolo–gous expression system, Biochemistry (Moscow), 82, 1285–1294.Google Scholar
  18. 18.
    Karyagina, A. S., Grunina, T. M., Poponova, M. S., Orlova, P. A., Manskikh, V. N., Demidenko, A. V., Strukova, N. V., Manukhina, M. S., Lyaschuk, A. M., Galushkina, Z. M., Cherepushkin, S. A., Polyakov, N. B., Solovyev, A. I., Zhukhovitsky, V. G., Tretyak, D. A., Boksha, I. S., Gromov, A. V., and Lunin, V. G. (2017) Synthesis in Escherichia coli and characterization of human recombinant erythropoietin with additional heparin–bind–ing domain, Biochemistry (Moscow), 83, 1504–1522.Google Scholar
  19. 19.
    McCullough, P. A., Barnhart, H. X., Inrig, J. K., Reddan, D., Sapp, S., Patel, U. D., Singh, A. K., Szczech, L. A., and Califf, R. M. (2013) Cardiovascular toxicity of epoet–in–alfa in patients with chronic kidney disease, Am. J. Nephrol., 37, 549–558.Google Scholar
  20. 20.
    Douros, A., Jobski, K., Kollhorst, B., Schink, T., and Garbe, E. (2016) Risk of venous thromboembolism in can–cer patients treated with epoetins or blood transfusions, Br. J. Clin. Pharmacol., 82, 839–848.Google Scholar
  21. 21.
    Ruppert, R., Hoffmann, E., and Sebald, W. (1996) Human bone morphogenetic protein 2 contains a heparin–binding site, which modifies its biological activity, Eur. J. Biochem., 237, 295–302.Google Scholar
  22. 22.
    Karyagina, A. S., Boksha, I. S., Grunina, T. M., Demidenko, A. V., Poponova, M. S., Sergienko, O. V., Lyaschuk, A. M., Galushkina, Z. M., Soboleva, L. A., Osidak, E. O., Semikhin, A. S., Gromov, A. V., and Lunin, V. G. (2016) Optimization of rhBMP–2 active–form pro–duction in a heterologous expression system using microbi–ological and molecular genetic approaches, Mol. Genet. Microbiol. Virol., 31, 208–213.Google Scholar
  23. 23.
    Bartov, M. S., Gromov, A. V., Poponova, M. S., Savina, D. M., Nikitin, K. E., Grunina, T. M., Manskikh, V. N., Gra, O. A., Lunin, V. G., Karyagina, A. S., and Gintsburg, A. L. (2016) Modern approaches to research of new osteogenic biomaterials on the model of regeneration of cranial criti–cal–sized defects in rats, Bull. Exp. Biol. Med., 162, 273–276.Google Scholar
  24. 24.
    Chen, X., Zaro, J. L., and Shen, W. C. (2013) Fusion pro–tein linkers: property, design and functionality, Adv. Drug. Deliv. Rev., 65, 1357–1369.Google Scholar
  25. 25.
    Brines, M., Grasso, G., Fiordaliso, F., Sfacteria, A., Ghezzi, P., Fratelli, M., Latini, R., Xie, Q. W., Smart, J., Su–Rick, C. J., Pobre, E., Diaz, D., Gomez, D., Hand, C., Coleman, T., and Cerami, A. (2004) Erythropoietin medi–ates tissue protection through an erythropoietin and com–mon beta–subunit heteroreceptor, Proc. Natl. Acad. Sci. USA, 101, 14907–14912.Google Scholar
  26. 26.
    Rolfing, J., Baatrup, A., Stiehler, M., Jensen, J., Lysdahl, H., and Bunger, C. (2014) The osteogenic effect of erythro–poietin on human mesenchymal stromal cells is dose–dependent and involves non–hematopoietic receptors and multiple intracellular signaling pathways, Stem Cell Rev., 10, 69–78.Google Scholar
  27. 27.
    Kim, J., Jung, Y., Sun, H., Joseph, J., Mishra, A., Shiozawa, Y., Wang, J., Krebsbach, P. H., and Taichman, R. S. (2012) Erythropoietin mediated bone formation is regulated by mTOR signaling, J. Cell Biochem., 113, 220–228.Google Scholar
  28. 28.
    Garcia, P., Speidel, V., Scheuer, C., Laschke, M. W., Holstein, J. H., Histing, T., Pohlemann, T., and Menger, M. D. (2011) Low dose erythropoietin stimulates bone healing in mice, J. Orthop. Res., 29, 165–172.Google Scholar
  29. 29.
    Holstein, J. H., Orth, M., Scheuer, C., Tami, A., Becker, S. C., Garcia, P., Histing, T., Morsdorf, P., Klein, M., Pohlemann, T., and Menger, M. D. (2011) Erythropoietin stimulates bone formation, cell proliferation, and angio–genesis in a femoral segmental defect model in mice, Bone, 49, 1037–1045.Google Scholar
  30. 30.
    Li, C., Shi, C., Kim, J., Chen, Y., Ni, S., Jiang, L., Zheng, C., Li, D., Hou, J., Taichman, R. S., and Sun, H. (2015) Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling, J. Dent. Res., 94, 455–463.Google Scholar
  31. 31.
    Karyagina, A. S., Boksha, I. S., Grunina, T. M., Demidenko, A. V., Poponova, M. S., Sergienko, O. V., Lyashchuk, A. M., Galushkina, Z. M., Soboleva, L. A., Osidak, E. O., Bartov, M. S., Gromov, A. V., and Lunin, V. G. (2017) Two variants of recombinant human bone morphogenetic protein 2 (rhBMP–2) with additional pro–tein domains: synthesis in an Escherichia coli heterolo–gous expression system, Biochemistry (Moscow), 82, 613–624.Google Scholar
  32. 32.
    Concepcion, J., Witte, K., Wartchow, C., Choo, S., Yao, D., Persson, H., Wei, J., Li, P., Heidecker, B., Ma, W., Varma, R., Zhao, L.–S., Perillat, D., Carricato, G., Recknor, M., Du, K., Ho, H., Ellis, T., Gamez, J., Howes, M., Phi–Wilson, J., Lockard, S., Zuk, R., and Tan, H. (2009) Label–free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization, Comb. Chem. High Throughput Screen., 12, 791–800.Google Scholar
  33. 33.
    Gromov, A. V., Nikitin, K. E., Karpova, T. A., Zaitsev, V. V., Sidirova, E. I., Andreeva, E. V., Bartov, M. S., Mishina, D. M., Subbotina, M. E., Shevlyagina, N. V., Sergienkov, M. A., Soboleva, L. A., Kotnova, A. P., Sharapova, N. E., Semikhin, A. S., Didenko, L. V., Karyagina, A. S., and Lunin, V. G. (2012) Development of a method for prepara–tion of osteoplastic material based on demineralized bone matrix with maximum content of native bone tissue growth factors, Biotekhnologiya, 5, 66–75.Google Scholar
  34. 34.
    Heidenhain, M. (1905) Zeitschrift fur wissenschaftliche Mikroskopie und fur mikroskopische Technik, S. Hirzel, Leipzig, 22, 339.Google Scholar
  35. 35.
    Zwingenberger, S., Langanke, R., Vater, C., Lee, G., Niederlohmann, E., Sensenschmidt, M., Jacobi, A., Bernhardt, R., Muders, M., Rammelt, S., Knaack, S., Gelinsky, M., Gunther, K. P., Goodman, S. B., and Stiehler, M. (2016) The effect of SDF–1α on low dose BMP–2 mediated bone regeneration by release from heparinized mineralized collagen type I matrix scaffolds in a murine critical size bone defect model, J. Biomed. Mater. Res. A, 104, 2126–2134.Google Scholar
  36. 36.
    Hwang, C. J., Vaccaro, A. R., Lawrence, J. P., Hong, J., Schellekens, H., Alaoui–Ismaili, M. H., and Falb, D. (2009) Immunogenicity of bone morphogenetic proteins, J. Neurosurg. Spine, 10, 443–451.Google Scholar

Copyright information

© Pleiades Publishing, Inc. 2019

Authors and Affiliations

  • A. S. Karyagina
    • 1
    • 2
    • 3
    Email author
  • T. M. Grunina
    • 1
  • A. M. Lyaschuk
    • 1
  • E. V. Voronina
    • 4
  • R. A. Marigin
    • 4
  • S. A. Cherepushkin
    • 4
  • I. N. Trusova
    • 4
  • A. V. Grishin
    • 1
    • 2
  • M. S. Poponova
    • 1
  • P. A. Orlova
    • 1
  • V. N. Manskikh
    • 1
    • 3
  • N. V. Strukova
    • 1
  • M. S. Generalova
    • 1
  • K. E. Nikitin
    • 1
  • L. A. Soboleva
    • 1
  • I. S. Boksha
    • 1
    • 5
  • A. V. Gromov
    • 1
  1. 1.Gamaleya National Research Center of Epidemiology and MicrobiologyMinistry of Healthcare of the Russian FederationMoscowRussia
  2. 2.All-Russia Research Institute of Agricultural BiotechnologyMoscowRussia
  3. 3.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  4. 4.State Research Institute of Genetics and Selection of Industrial MicroorganismsKurchatov Institute National Research CenterMoscowRussia
  5. 5.Research Center of Mental HealthMoscowRussia

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