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Biotechnology and Bioprocess Engineering

, Volume 24, Issue 2, pp 298–307 | Cite as

Functional Secretion of Granulocyte Colony Stimulating Factor in Bacillus subtilis and Its Thermogenic Activity in Brown Adipocytes

  • Seung Woo Lee
  • Nam Hyeon Kang
  • Jang Won ChoiEmail author
Research Paper
  • 6 Downloads

Abstract

Granulocyte colony-stimulating factor (G-CSF) is involved in generation of colonies hematopoietic cells by stimulation of bone marrow cells, resulting in the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. To express and secrete G-CSF in Bacillus system, the G-CSF gene was amplified using gene-specific primers and template DNA (pUNO1-hGCSFb). As a result, the PCR product of 530 bp was obtained, sequenced, and analyzed using the BLAST program. The open reading frame encoded a 204-amino acid polypeptide precursor consisting of the 30-residue signal sequence and a 174-residue mature polypeptide. Recombinant secretion vector (pRBAS-CSF3, 6.96 kb) was constructed using pRBAS vector which contains promoter (0.45 kb) and signal sequence (87 bp) of alkaline protease gene (aprE), ribosome binding site (GGAGAGGG) and transferred to Bacillus subtilis LKS. G-CSF protein was secreted and optimized to increase the productivity (19% of total secreted proteins, 0.12 mg/ml). The culture broth was harvested and followed by metal chelating, ethanol precipitation, and gel chromatography. The purified G-CSF was tested for thermogenic activity in cultured brown adipocytes. G-CSF activated HIB1B brown adipocytes by increasing expression levels of brown fat-specific marker proteins (UCP1 and PRDM16) and genes (Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. This result suggests that G-CSF per se or G-CSF-containing foodstuff can be used to treat obesity.

Keywords

anti-obesity aprE promoter Bacillus subtilis brown adipocyte granulocyte-colony stimulating factor thermogenic activity 

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Notes

Acknowledgement

This study was supported by Daegu University Research Grant 2014.

References

  1. 1.
    Anderlini, P., D. Ptzepiorka, R. Champlin, and M. Korbling (1996) Bio-logic and clinic effects of granulocyte colony-stimulating factor in normal individuals. Blood 88: 2819–2825.Google Scholar
  2. 2.
    Deotare, U., G Al-Dawsari, S. Couban, and J. H. Lipton (2015) G-CSF-primed bone marrow as a source of stem cells for allografting: revisiting the concept. Bone Marrow Transplant. 50: 1150–1156.CrossRefGoogle Scholar
  3. 3.
    Tay, J., J. P. Levesque, and I. G. Winkler (2016) Cellular players of hematopoietic stem cell mobilization in the bone marrow niche. Int. J. Hematol. 105: 129–140.CrossRefGoogle Scholar
  4. 4.
    Nagata, S., M. Tsuchiya, S. Asano, O. Yamamoto, Y. Hirata, N. Kubota, M. Oheda, H. Nomura, and T. Yamazai (1986) The chromosomal structure and two mRNAs for human granulocyte colony-stimulating factor. EMBO J. 5: 575581.CrossRefGoogle Scholar
  5. 5.
    Putland, R. A., T. A. Sassinis, J. S. Harvey, P. Diamond, L. S. Coles, C. Y. Brown, and G. J. Goodall (2002) RNA destabilization by the granulocyte colony-stimulating factor stem-loop destabilizing element involves a single stem-loop that promotes deadenylation. Mol. Cell Biol. 22: 1664–1673.CrossRefGoogle Scholar
  6. 6.
    Weisdorf, D., N. Chao, J. K. Waselenko, N. Dainiak, J. O. Armitage, I. McNiece I, and D. Confer (2006) Acute radiation injury: contingency planning for triage, supportive care, and transplantation. Biol. Blood Marrow Transplant. 12: 672–682.CrossRefGoogle Scholar
  7. 7.
    Weinstock, D. M., C. Case, J. L. Bader, N. J. Chao, C. N. Coleman, R. J. Hatchett, D. J. Weisdorf, and D. L. Confer (2008) Radiologic and nuclear events: contingency planning for hematologists/oncologists. Blood 111: 5440–5445.CrossRefGoogle Scholar
  8. 8.
    Finkel, E. (2005) Stem cells: controversy on the frontiers of science. Crows Nest: ABC Books. ISBN 978-0-7333-1248-9.Google Scholar
  9. 9.
    Sanchez-Ramos, J., S. Song, V. Sava, B. Catlow, X. Lin, T. Mori, C. Cao, and G. W. Arendash (2009) Granulocyte colony stimulating factor decreases brain amyloid burden and reverses cognitive impairment in Alzheimer’s mice. Neuroscience 163: 55–72.CrossRefGoogle Scholar
  10. 10.
    Zhang, Y., L. Wang, Y. Fu, H. Song, H. Zhao, M. Deng, J. Zhang, and D. Fan (2009) Preliminary investigation of effect of granulocyte colony stimulating factor on amyotrophic lateral sclerosis. ALS J. 10: 430–431.Google Scholar
  11. 11.
    Acosta, S. A., N. Tajiri, K. Shinozuka, H. Ishikawa, P. R. Sanberg, J. Sanchez-Ramos, S. Song, Y. Kaneko, and C. V. Borlongan (2014) Combination therapy of human umbilical cord blood cells and granulocyte colony stimulating factor reduces histopathological and motor impairments in an experimental model of chronic traumatic brain injury. PLoS One 9: e90953.CrossRefGoogle Scholar
  12. 12.
    Schneider, A., C. Krüger, T. Steigleder, D. Weber, C. Pitzer, R. Laage, J. Aronowski, M. H. Maurer, N. Gassler, W. Mier, M. Hasselblatt, R. Kollmar, S. Schwab, C. Sommer, A. Bach, H. G. Kuhn, and W. R. Schäbitz (2005). The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis. J. Clin. Invest. 115: 2083–2098.CrossRefGoogle Scholar
  13. 13.
    Pitzer, C., C. Krüger, C. Plaas, F. Kirsch, T. Dittgen, R. Müller, R. Laage, S. Kastner, S. Suess, R. Spoelgen, A. Henriques, H. Ehrenreich, W. R. Schäbitz, A. Bach, and A. Schneider (2008) Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis. Brain 131: 3335–3347.CrossRefGoogle Scholar
  14. 14.
    Thomas, J., F. Liu, and D. C. Link (2002) Mechanisms of mobilization of hematopoietic progenitors with granulocyte colony-stimulating factor. Curr. Opin. Hematol. 9: 183–189.CrossRefGoogle Scholar
  15. 15.
    Kim, C. K., J. H. Choi, S. B. Lee, S. M. Lee, and J. W. Oh (2014) Expression and purification of recombinant human granulocyte colony-stimulating factor in fed-batch culture of E. coli. Appl. Biochem. Biotechnol. 172: 2425–2435.CrossRefGoogle Scholar
  16. 16.
    Karimi, Z., N. Nezafat, M. Negahdaripour, A. Berenjian, S. Hemmati, and Y. Ghasemi (2015) The effect of rare codons following the ATG start codon on expression of human granulocyte-colony stimulating factor in E. coli. Protein Expr. Purif. 114: 108114.CrossRefGoogle Scholar
  17. 17.
    Vemula, S., R. Thunuguntla, A. Dedaniya, S. Kokkiligadda, C. Palle, and S. R. Ronda (2015) Improved production and characterization of recombinant human granulocyte colony stimulating factor from E. coli under optimized downstream processes. Protein Expr. Purif. 108: 6272.CrossRefGoogle Scholar
  18. 18.
    Maity, N., A. Thawani, A. Sharma, A. Gautam, S. Mishra, and V. Sahai (2015) Expression and control of codon-optimized granulocyte colony-stimulating factor in Pichia pastoris. Appl. Biochem. Biotechnol. 178: 159172.Google Scholar
  19. 19.
    Sigar, M., N. Maity, and S. Mishra (2016) Enhancing granulocyte colony-stimulating factor expression in Pichia pastoris through fusion with human serum albumin. Prep. Biochem. Biotechnol. 47: 364370.Google Scholar
  20. 20.
    Talebkhan Y., T. Samadi, A. Samie, F. Barkhordari, M. Azizi, V. Khalaj, and E. Mirabzadeh (2016) Expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha. Iran J. Microbiol. 8: 21–28.Google Scholar
  21. 21.
    Tseng, Y. H., A. M. Cypess, and C. R. Kahn (2010) Cellular bioenergetics as a target for obesity therapy. Nat. Rev. Drug Discov 9: 465–482.CrossRefGoogle Scholar
  22. 22.
    Vernochet, C., A. Mourier, O. Bezy, Y. Macotela, J. Boucher, M. J. Rardin, D. An, K. Y. Lee, O. R. Ilkayeva, C. M. Zingaretti, B. Emanuelli, G. Smyth, S. Cinti, C. B. Newgard, B. W. Gibson, N. G. Larsson, and C. R. Kahn (2012) Adipose-specific deletion of TFAM increases mitochondrial oxidation and protects mice against obesity and insulin resistance. Cell Metab. 16: 765–776.CrossRefGoogle Scholar
  23. 23.
    Virtanen, K. A., M. E. Lidell, J. Orava, M. Heglind, R. Westerngren, T. Niemi, M. Taittonen, J. Laine, N. J. Savisto, S. Enerbäck, and P. Nuutila (2009) Functional brown adipose tissue in healthy adults. N. Engl. J. Med. 360: 1518–1525.CrossRefGoogle Scholar
  24. 24.
    Samocha-Bonet, D., V. D. Dixit, C. R. Kahn, R. L. Leibel, X. Lin, M. Nieuwdorp, K. H. Pietiläinen, R. Rabasa-Lhoret, M. Roden, P. E. Scherer, S. Klein, and E. Ravussin (2014) Metabolically healthy and unhealthy obese—the 2013 Stock Conference report. Obese. Rev. 15: 697–708.CrossRefGoogle Scholar
  25. 25.
    Lee, Y. H., E. P. Mottilo, and J. G. Granneman (2014) Adipose tissue plasticity from WAT to BAT and in between. Biochem. Biophys. Acta 1842: 358–369.Google Scholar
  26. 26.
    van Marken Lichtenbelt, W. D., J. W. Vanhommerig, N. M. Smulders, J. M. Drossaerts, G. J. Kemerink, N. D. Bouvy, P. Schrauwen, and G. J. Teule (2009) Cold-activated brown adipose tissue in healthy men. N. Engl. J. Med. 360: 1500–1508.CrossRefGoogle Scholar
  27. 27.
    Calderon-Dominguez, M., J. F. Mir, R. Fucho, M. Weber, D. Serra, and L. Herrero (2016) Fatty acid metabolism and the basis of brown adipose tissue function. Adipocyte 5: 98–118.CrossRefGoogle Scholar
  28. 28.
    Azhar, Y., A. Parmar, C. N. Miller, J. S. Samuels, and S. Rayalam (2016) Phytochemicals as novel agents for the induction of browning in white adipose tissue. Nutr. Metab. 13: 39.CrossRefGoogle Scholar
  29. 29.
    Vermaak, I., J. H. Hamman, and A. M. Vilijoen (2011) Hoodia gordonii: an up-to-date review of a commercially important anti-obesity plant. Planta Med. 77: 1149–1160.CrossRefGoogle Scholar
  30. 30.
    Jang, M. H., N. H. Kang, S. Mukherjee, and J. W. Yun (2018) Theobromine, a methylxanthine in cocoa bean, stimulates thermogenesis by inducing white fat browning and activating brown adipocytes. Biotechnol. Bioproc. Eng. 23: 617–626.CrossRefGoogle Scholar
  31. 31.
    Fruhbeck, G, S. Becerril, N. Sainz, P. Garrastachu, and M. J. Garcia-Velloso (2009) BAT: a new target for human obesity? Trends Pharmacol. Sci. 30: 387–396.CrossRefGoogle Scholar
  32. 32.
    Lim, Y. H., J. H. Joe, K. S. Jang, Y. S. Song, B. I. So, C. H. Fang, J. Shin, J. H. Kim, H. K. Lim, and K. S. Kim (2014) Anti-obesity effects of granulocyte-colony stimulating factor in Otsuka-Long-Evans-Tokushima fatty rats. PLoS One 9: e105603.CrossRefGoogle Scholar
  33. 33.
    Schumann, W. (2007) Production of recombinant proteins in Bacillus subtilis. Adv. Appl. Microbiol. 62: 137–189.CrossRefGoogle Scholar
  34. 34.
    Yao, Z., J. A. Kim, and J. H. Kim (2018) Gene cloning, expression, and properties of a fibrinolytic enzyme secreted by Bacillus pumilus BS15 isolated from Gul (Oyster) Jeotgal. Biotechnol. Bioproc. Eng. 23: 293–301.CrossRefGoogle Scholar
  35. 35.
    Li, S., N. Wang, Z. J. Du, and G. J. Chen (2018) Intergeneric hybridization between Streptomyces albulus and Bacillus subtilis facilitates production of ε-poly-L-lysine from corn starch residues. Biotechnol. Bioproc. Eng. 23: 580–587.CrossRefGoogle Scholar
  36. 36.
    Jeong, S. H. and J. W. Choi (2017) The secretion optimization of oligopeptide with his-pro repeats in Bacillus subtilis and its anti-diabetic effects. KSBB J. 32: 71–82.CrossRefGoogle Scholar
  37. 37.
    Sambrook, J., E.F. Fritsch, and T. Maniatis (1989). In Molecular cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press.Google Scholar
  38. 38.
    Kim, S. I., J. W. Choi, and S. Y. Lee (1997). Effects of pleiotrophic mutations, degU h and spoOA, on the production of foreign proteins using the heterologous secretion system of Bacillus subtilis. Mol. Cells 7: 158–164.Google Scholar
  39. 39.
    Lee, M. H., J. J. Song, Y. H. Choi, S. P. Hong, E. H. Rha, K. Kim, S. G. Lee, S. C. Lee, H. Poo, Y. B. Seu, and M. H. Sung (2003). High-Level Expression and Secretion of Bacillus pumilus lipase B26 in Bacillus subtilis chungkookjang. J. Microbiol. Biotechnol. 13: 892–896.Google Scholar
  40. 40.
    Tallent, S. M., K. M. Kristin, S. A. Errol, and B. W. Reginald (2012) Efficient isolation and identification of Bacillus cereus group. J. AOAC Int. 95: 446–451.CrossRefGoogle Scholar
  41. 41.
    Sadaie, Y. and T. Kada (1983) Formation of competent Bacillus cells. J. Bacteriol. 153: 813–821.Google Scholar
  42. 42.
    Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.CrossRefGoogle Scholar
  43. 43.
    Hollingshead, L. M. and K. L. Goa (1991) Recombinant granulocyte colony-stimulating factor (rG-CSF): a review of its pharmacological properties and prospective role in neutropenic conditions. Drugs 42: 300–330.CrossRefGoogle Scholar
  44. 44.
    Westers, L., D. S. Dijkstra, H. Wester, J. M. van Dijl, and W. J. Quax (2006) Secretion of functional human interleukin-3 from Bacillus subtilis. J. Biotechnol. 123: 211–224.CrossRefGoogle Scholar
  45. 45.
    Lam, K. H. E., K. C. Chow, and W. K. R. Wong (1998) Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins. J. Biotechnol. 63: 167–177.CrossRefGoogle Scholar
  46. 46.
    Brown, T. A (1991) Genomes and genes. In: Brown, T. A (Ed.), Molecular Biology Labfax. BIOS Scientific Publisher, Oxford. 235–254.Google Scholar
  47. 47.
    Simonen, M. and I. Palva (1993) Protein secretion in Bacillus species. Microbiol. Rev. 57: 109–137.Google Scholar
  48. 48.
    Daltro, P. S., P. S. Alves, M. F. Castro, C. M. Azevedo, J. F. Vasconcelos, K. J. Allahdadi, L. A. de Freitas, B. S. de Freitas Souza, R. R. Dos Santos, M. B. Soares, and S. G. Macambira (2015) Administration of granulocyte-colony stimulating factor accompanied with a balanced diet improves cardiac function alterations induced by high fat diet in mice. BMC Cardiovasc. Disord. 15: 162.CrossRefGoogle Scholar
  49. 49.
    Levine, J. A., M. D. Jensen, N. L. Eberhardt, and T. O’Brien (1998) Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth. J. Clin. Invest. 101: 1557–1564.CrossRefGoogle Scholar
  50. 50.
    Sugita, S., Y. Kamei, J. Oka, T. Suganami, and Y. Ogawa (2007) Macrophage-colony stimulating factor in obese adipose tissue: studies with heterozygous op/+ mice. Obesity 15: 1988–1995.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer 2019

Authors and Affiliations

  • Seung Woo Lee
    • 1
  • Nam Hyeon Kang
    • 2
  • Jang Won Choi
    • 1
    Email author
  1. 1.Department of BioindustryDaegu UniversityGyeongsanKorea
  2. 2.Department of BiotechnologyDaegu UniversityGyeongsanKorea

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