Advertisement

Biochemistry (Moscow)

, Volume 82, Issue 11, pp 1285–1294 | Cite as

Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system

  • T. M. Grunina
  • A. V. Demidenko
  • A. M. Lyaschuk
  • M. S. Poponova
  • Z. M. Galushkina
  • L. A. Soboleva
  • S. A. Cherepushkin
  • N. B. Polyakov
  • D. A. Grumov
  • A. I. Solovyev
  • V. G. Zhukhovitsky
  • I. S. Boksha
  • M. E. Subbotina
  • A. V. Gromov
  • V. G. Lunin
  • A. S. Karyagina
Article

Abstract

Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.

Keywords

erythropoietin heterologous expression Escherichia coli 

Abbreviations

a.a.

amino acid residue

BMP-2

bone morphogenetic protein-2

DTT

dithiothreitol

EPO

erythropoietin

6His

six histidine amino acid residues

MBP

maltose-binding protein

rhEPO

recombinant human erythropoietin

s-tag

15-a.a. oligonucleotide derived from bovine pancreatic ribonuclease A

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

10541_2017_511_MOESM1_ESM.pdf (84 kb)
Supplementary material, approximately 85 KB.

References

  1. 1.
    Cazzola, M., Mercuriali, F., and Brugnara, C. (1997) Use of recombinant human erythropoietin outside the setting of uremia, Blood, 89, 4248–4267.PubMedGoogle 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 formation, 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.CrossRefGoogle 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.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    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, 2095–2105.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lai, P. H., Everett, R., Wang, F. F., Arakawa, T., and Goldwasser, E. (1986) Structural characterization of human erythropoietin, J. Biol. Chem., 261, 3116–3121.PubMedGoogle Scholar
  8. 8.
    Recny, M. A., Scoble, H. A., and Kim, Y. (1987) Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of desarginine 166 erythropoietin, J. Biol. Chem., 262, 17156–17163.PubMedGoogle Scholar
  9. 9.
    Helenius, A., and Aebi, M. (2001) Intracellular functions of N-linked glycans, Science, 291, 2364–2369.CrossRefPubMedGoogle Scholar
  10. 10.
    Higuchi, M., Oh-eda, M., Kuboniwa, H., Tomonoh, K., Shimonaka, Y., and Ochi, N. (1992) Role of sugar chains in the expression of the biological activity of human erythropoietin, J. Biol. Chem., 267, 7703–7709.PubMedGoogle Scholar
  11. 11.
    Egrie, J. C., and Browne, J. K. (2001) Development and characterization of novel erythropoiesis stimulating protein (NESP), Br. J. Cancer, 84, 3–10.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    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 erythropoietin from E. coli, Protein Express. Purif., 95, 211–218.CrossRefGoogle Scholar
  13. 13.
    Boissel, J. P., Lee, W. R., Presnell, S. R., Cohen, F. E., and Bunn, H. F. (1993) Erythropoietin structurefunction relationships. Mutant proteins that test a model of tertiary structure, J. Biol. Chem., 268, 5983–5993.Google Scholar
  14. 14.
    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.CrossRefPubMedGoogle Scholar
  15. 15.
    Gasparian, M. E., Ostapchenko, V. G., Schulga, A. A., Dolgikh, D. A., and Kirpichnikov, M. P. (2003) Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli, Protein Express. Purif., 31, 133–139.CrossRefGoogle Scholar
  16. 16.
    Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., and Mann, M. (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes, Nat. Protoc., 1, 2856–2860.CrossRefPubMedGoogle Scholar
  17. 17.
    Sun, P., Tropea, J. E., and Waugh, D. S. (2011) Enhancing the solubility of recombinant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding protein as a fusion partner, Methods Mol. Biol., 705, 259–274.CrossRefPubMedGoogle Scholar
  18. 18.
    Raran-Kurussi, S., and Waugh, D. S. (2016) A dual protease approach for expression and affinity purification of recombinant proteins, Anal. Biochem., 504, 30–37.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    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 protein domains: synthesis in an Escherichia coli heterologous expression system, Biochemistry (Moscow), 82, 613–624.CrossRefGoogle Scholar
  20. 20.
    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 erythropoietin expressed as inclusion body in E. coli, Int. J. Pharm., 386, 156–164.CrossRefPubMedGoogle Scholar
  21. 21.
    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., Semikhin, A. S., Gromov, A. V., and Lunin, V. G. (2016) Optimization of rhBMP 2 active form production in a heterologous expression system using microbiological and molecular genetic approaches, Mol. Genet. Microbiol. Virol., 31, 208–213.CrossRefGoogle Scholar
  22. 22.
    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 studies of new osteogenic biomaterials on the model of regeneration of critical-size cranial defects in rats, Bull. Exp. Biol. Med., 162, 273–276.CrossRefPubMedGoogle Scholar
  23. 23.
    Gaifullin, N. M., Karyagina, A. S., Gromov, A. V., Terpilovsky, A. A., Malanin, D. A., Demeshchenko, M. V., and Novochadov, V. V. (2016) Morphological characteristics of osteointegration after application of titanium implants with bioactive coating and recombinant bone morphogenetic protein rhBMP-2, Morfologiya, 149, 77–84.Google Scholar
  24. 24.
    Zakharov, V. D., Zairat’yants, O. V., Andreev, A. Yu., Osidak, E. O., Borzenok, S. A., Krasheninnikov, S. V., Karyagina, A. S., and Domogatsky, S. P. (2016) Effect of rhBMP-2 growth factor in composition with collagen carrier on morphological and mechanical properties of cornea, Oftal’mokhirurgiya, 4, 20–28.Google Scholar
  25. 25.
    Zakharov, V. D., Andreev, A. Yu., Zairat’yants, O. V., Osidak, E. O., Borzenok, S. A., Krasheninnikov, S. V., Karyagina, A. S., and Domogatsky, S. P. (2016) Morphological changes in rabbit cornea caused by the bone and cartilage growth factor rhBMP-2 used as a component of intracorneal implant, Klin. Eksp. Morfol., 4, 36–42.Google Scholar
  26. 26.
    Bartov, M. S., Gromov, A. V., Manskikh, V. N., Makarova, E. B., Rubshteyn, A. P., Poponova, M. S., Savina, D. M., Savin, K. S., Nikitin, K. E., Grunina, T. M., Boksha, I. S., Orlova, P. A., Krivozubov, M. S., Subbotina, M. E., Lunin, V. G., Karyagina, A. S., and Gintsburg, A. L. (2018) Recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) with additional protein domain produced by synthesis in Escherichia coli: in vivo activity in models on small and large laboratory animals, Bull. Exp. Biol. Med., in press.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2017

Authors and Affiliations

  • T. M. Grunina
    • 1
  • A. V. Demidenko
    • 1
  • A. M. Lyaschuk
    • 1
  • M. S. Poponova
    • 1
  • Z. M. Galushkina
    • 1
  • L. A. Soboleva
    • 1
  • S. A. Cherepushkin
    • 2
  • N. B. Polyakov
    • 1
    • 3
  • D. A. Grumov
    • 1
  • A. I. Solovyev
    • 1
  • V. G. Zhukhovitsky
    • 1
    • 4
  • I. S. Boksha
    • 1
    • 5
  • M. E. Subbotina
    • 1
    • 6
  • A. V. Gromov
    • 1
  • V. G. Lunin
    • 1
    • 6
  • A. S. Karyagina
    • 1
    • 6
    • 7
  1. 1.Gamaleya National Research Center of Epidemiology and MicrobiologyMinistry of Health of the Russian FederationMoscowRussia
  2. 2.State Research Institute of Genetics and Selection of Industrial MicroorganismsMoscowRussia
  3. 3.Vernadsky Institute of Geochemistry and Analytical ChemistryRussian Academy of SciencesMoscowRussia
  4. 4.Sechenov Moscow State Medical UniversityMoscowRussia
  5. 5.Mental Health Research CenterMoscowRussia
  6. 6.All-Russia Research Institute of Agricultural BiotechnologyMoscowRussia
  7. 7.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia

Personalised recommendations