Advertisement

Transgenic Research

, Volume 28, Issue 5–6, pp 465–478 | Cite as

Development of dairy herd of transgenic goats as biofactory for large-scale production of biologically active recombinant human lactoferrin

  • I. SemakEmail author
  • A. Budzevich
  • E. Maliushkova
  • V. Kuzniatsova
  • N. Popkov
  • I. Zalutsky
  • O. Ivashkevich
Original Paper

Abstract

The primary male-goats Lac-1 (human lactoferrin gene construct hLF5) and Lac-2 (human lactoferrin gene construct hLF3) with genome containing human lactoferrin gene were bred and the sperm bank of primary male-goats and their male descendents (F1–F7) was created. The herd of goats (200 transgenic females) that produced recombinant human lactoferrin (rhLF) in their milk at levels up to 16 g/L was obtained. The rhLF from milk of transgenic goats, natural human lactoferrin (hLF) from woman milk and natural goat lactoferrin (gLF) from milk of non-transgenic goats were purified using cation-exchange chromatography. It has been shown that rhLF is a glycoprotein and its physicochemical characteristics of rhLF are similar to hLf as revealed by different analytical methods including electron paramagnetic resonance, spectrophotometry, differential scanning calorimetry, mass spectrometry and peptide mapping. The high expression level of rhLF achieved in milk of transgenic goats provides a solid basis for developing an efficient and cost-effective downstream processing. The rhLF exhibited a prominent biological activity suggesting it as a promising biopharmaceutical and food supplements.

Keywords

Recombinant human lactoferrin Herd of transgenic goats 

Abbreviations

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

EPR

Electron paramagnetic resonance

DSC

Differential scanning calorimetry

FeNTA

Ferric nitrilotriacetate

HDL cholesterol

Cholesterol of high-density lipoproteins

LDL cholesterol

Low density lipoproteins

LF

Lactoferrin

hLF

Human lactoferrin

rhLF

Recombinant human lactoferrin

gLF

Natural goat lactoferrin

bLF

Bovine lactoferrin

PNGase

Peptide N-glycosidase

MWCO

Molecular weight cutoff

Notes

Acknowledgements

The authors acknowledge funding from the following: Union State Belarus-Russia, Ministry of Education and National Academy of Sciences of Belarus. The authors thank M.V.Serebryakova (Institute of Biomedical Chemistry, Moscow, Russia) for performing MALDI-TOF–MS analysis; I.Azarko (Belarusian State University, Minsk, Belarus) for performing EPR spectroscopy; D.Fima (Research Centre for Hematology and Transfusiology, Minsk, Belarus) for performing differential scanning calorimetry.

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest to declare.

Supplementary material

11248_2019_165_MOESM1_ESM.docx (436 kb)
Supplementary material 1 (DOCX 436 kb)

References

  1. Albar AH, Almehdar HA, Uversky VN, Redwan EM (2014) Structural heterogeneity and multifunctionality of lactoferrin. Curr Protein Pept Sci 15(8):778–797.  https://doi.org/10.2174/1389203715666140919124530 CrossRefPubMedGoogle Scholar
  2. Amini AA, Nair LS (2011) Lactoferrin: a biologically active molecule for bone regeneration. Curr Med Chem 18(8):1220–1229.  https://doi.org/10.2174/092986711795029744 CrossRefPubMedGoogle Scholar
  3. Baker HM, Baker EN (2004) Lactoferrin and iron: structural and dynamic aspects of binding and release. Biometals 17(3):209–216.  https://doi.org/10.1023/B:BIOM.0000027694.40260.70 CrossRefPubMedGoogle Scholar
  4. Baker EN, Baker HM (2005) Molecular structure, binding properties and dynamics of lactoferrin. Cell Mol Life Sci: CMLS 62(22):2531–2539.  https://doi.org/10.1007/s00018-005-5368-9 CrossRefPubMedGoogle Scholar
  5. Ballester M, Castelló A, Ibáñez E, Sánchez A, Folch JM (2004) Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals. Biotechniques 37(4):610–613.  https://doi.org/10.2144/04374ST06 CrossRefPubMedGoogle Scholar
  6. Berlutti F, Pantanella F, Natalizi T, Frioni A, Paesano R, Polimeni A, Valenti P (2011) Antiviral properties of lactoferrin—a natural immunity molecule. Molecules 16(8):6992–7018.  https://doi.org/10.3390/molecules16086992 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bertolini LR, Meade H, Lazzarotto CR, Martins LT, Tavares KC, Bertolini M, Murray JD (2016) The transgenic animal platform for biopharmaceutical production. Transgenic Res 25(3):329–343.  https://doi.org/10.1007/s11248-016-9933-9 CrossRefPubMedGoogle Scholar
  8. Chekhun VF, Zalutskii IV, Naleskina LA, Lukianova NY, Yalovenko TM, Borikun TV, Sobchenko SO, Semak IV, Lukashevich VS (2015) Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells. Exp Oncol 37(3):181–186CrossRefGoogle Scholar
  9. Chissov VI, Iakubovskaia RI, Nemtsova ER, Osipova NA, Edeleva NV, Utkin MM, Zviagin AA (2008) Antioxidants treatment of severe post-operative pyoinflammatory and septic complications. Khirurgiia 11:14–19Google Scholar
  10. Cooper CA, Maga EA, Murray JD (2015) Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future. Transgenic Res 24(4):605–614.  https://doi.org/10.1007/s11248-015-9885-5 CrossRefPubMedGoogle Scholar
  11. Drago-Serrano ME, de la Garza-Amaya M, Luna JS, Campos-Rodríguez R (2012) Lactoferrin-lipopolysaccharide (LPS) binding as key to antibacterial and antiendotoxic effects. Int Immunopharmacol 12(1):1–9.  https://doi.org/10.1016/j.intimp.2011.11.002 CrossRefPubMedGoogle Scholar
  12. García-Montoya IA, Cendón TS, Arévalo-Gallegos S, Rascón-Cruz Q (2012) Lactoferrin a multiple bioactive protein: an overview. Biochim Biophys Acta 1820(3):226–336.  https://doi.org/10.1016/j.bbagen.2011.06.018 CrossRefPubMedGoogle Scholar
  13. Goldman IL, Georgieva SG, YaG Gurskiy, Krasnov AN, Deykin AV, Popov AN, Ermolkevich TG, Budzevich AI, Chernousov AD, Sadchikova ER (2012) Production of human lactoferrin in animal milk. Biochem Cell Biol 90(3):513–519.  https://doi.org/10.1139/o11-088 CrossRefPubMedGoogle Scholar
  14. González-Chávez SA, Arévalo-Gallegos S, Rascón-Cruz Q (2009) Lactoferrin: structure, function and applications. Int J Antimicrob Agents 33(4):301.e1–301.e8.  https://doi.org/10.1016/j.ijantimicag.2008.07.020 CrossRefGoogle Scholar
  15. Hodgkinson AJ, Ross KM, Fahey SM, Prosser CG (2008) Quantification of lactoferrin in milk from New Zealand dairy goats. Proc NZ Soc Anim Prod 68:166–169Google Scholar
  16. Kanwar JR, Roy K, Patel Y, Zhou SF, Singh MR, Singh D, Nasir M, Sehgal R, Sehgal A, Singh RS, Garg S, Kanwar RK (2015) Multifunctional iron bound lactoferrin and nanomedicinal approaches to enhance its bioactive functions. Molecules 20(6):9703–9731.  https://doi.org/10.3390/molecules20069703 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kawakami H, Lönnerdal B (1991) Isolation and function of a receptor for human lactoferrin in human fetal intestinal brush-border membranes. Am J Physiol-Gastrointest Liver Physiol 261(5):841–846.  https://doi.org/10.1152/ajpgi.1991.261.5.G841 CrossRefGoogle Scholar
  18. Lai T, Yang Y, Ng SK (2013) Advances in mammalian cell line development technologies for recombinant protein production. Pharmaceuticals 6(5):579–603.  https://doi.org/10.3390/ph6050579 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Latorre D, Berlutti F, Valenti P, Gessani S, Puddu P (2012) LF immunomodulatory strategies: mastering bacterial endotoxin. Biochem Cell Biol 90(3):269–278.  https://doi.org/10.1139/o11-059 CrossRefPubMedGoogle Scholar
  20. Legrand D (2012) Lactoferrin, a key molecule in immune and inflammatory processes. Biochem Cell Biol 90(3):252–268.  https://doi.org/10.1139/o11-056 CrossRefPubMedGoogle Scholar
  21. Liao Y, Jiang R, Lönnerdal B (2012) Biochemical and molecular impacts of lactoferrin on small intestinal growth and development during early life. Biochem Cell Biol 90(3):476–484.  https://doi.org/10.1139/o11-075 CrossRefPubMedGoogle Scholar
  22. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25(4):402–408.  https://doi.org/10.1006/meth.2001.1262 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lukashevich VS, Budzevich AI, Semak IV, Kuznetsova VN, Malyushkova EV, Pyzh AE, Novakovskaya SA, Rudnichenko JA, Popkov NA, Ivashkevich OA, Zalutsky IV (2016) Production of recombinant human lactoferrin from the milk of goat-producers and its physiological effects. Doklady Natl Acad Sci Belarus 60(1):72–81Google Scholar
  24. Magnuson JS, Henry JF, Yip TT, Hutchens TW (1990) Structural homology of human, bovine, and porcine milk lactoferrins: evidence for shared antigenic determinants. Pediatr Res 28(2):176–181.  https://doi.org/10.1203/00006450-199008000-00019 CrossRefPubMedGoogle Scholar
  25. Mata L, Sánchez L, Headon D, Calvo M (1998) Thermal denaturation of human lactoferrin and its effect on the ability to bind iron. J Agric Food Chem 46(10):3964–3970.  https://doi.org/10.1021/jf980266d CrossRefGoogle Scholar
  26. Mayeur S, Spahis S, Pouliot Y, Levy E (2016) Lactoferrin, a pleiotropic protein in health and disease. Antioxid Redox Signal 24(14):813–836.  https://doi.org/10.1089/ars.2015.6458 CrossRefPubMedGoogle Scholar
  27. Mazurier J, Spik G (1980) Comparative study of the iron-binding properties of the human transferrins. I. Complete and sequential iron saturation and desaturation of the lactotransferrin. Biochim Biophys Acta 629:399–408.  https://doi.org/10.1016/0304-4165(80)90112-9 CrossRefPubMedGoogle Scholar
  28. Niemann H, Kues WA (2000) Transgenic livestock: premises and promises. Anim Reprod Sci 60:277–293.  https://doi.org/10.1016/S0378-4320(00)00091-9 CrossRefPubMedGoogle Scholar
  29. Ochoa TJ, Pezo A, Cruz K, Chea-Woo E, Cleary TG (2012) Clinical studies of lactoferrin in children. Biochem Cell Biol 90(3):457–467.  https://doi.org/10.1139/o11-087 CrossRefPubMedGoogle Scholar
  30. Patch MG, Carrano CJ (1981) The origin of the visible absorption in metal transferrins. Inorg Chim Acta 56:L71–L73CrossRefGoogle Scholar
  31. Peisach J, Blumberg WE, Lode ET, Coon MJ (1971) An analysis of the electron paramagnetic resonance spectrum of pseudomonas oleovorans rubredoxin: a method for determination of the liganids of ferric iron in completely rhombic sites. J Biol Chem 246(19):5877–5881PubMedGoogle Scholar
  32. Pham PV (2017) Medical biotechnology: techniques and applications. In: Barh D, Azevedo V (eds) Omics technologies and bio-engineering: towards improving quality of life, 1st edn. Elsevier, Amsterdam, pp 449–469Google Scholar
  33. Pierce A, Colavizza D, Benaissa M, Maes P, Tartar A, Montreuil J, Spik G (1991) Molecular cloning and sequence analysis of bovine lactotransferrin. FEBS J 196(1):177–184.  https://doi.org/10.1111/j.1432-1033.1991.tb15801.x CrossRefGoogle Scholar
  34. Rosa L, Cutone A, Lepanto MS, Paesano R, Valenti P (2017) Lactoferrin: a natural glycoprotein involved in iron and inflammatory homeostasis. Int J Mol Sci 18:1985.  https://doi.org/10.3390/ijms18091985 CrossRefPubMedCentralGoogle Scholar
  35. Rudnichenko YA, Lukashevich VS, Zalutsky IV (2016) Experimental study of the influence of recombinant human lactoferrin on the levels of androgens and basic parameters of lipid and protein metabolism. Biomed Khim. 62(5):566–571.  https://doi.org/10.18097/PBMC20166205566 CrossRefPubMedGoogle Scholar
  36. Suzuki YA, Shin K, Lönnerdal B (2001) Molecular cloning and functional expression of a human intestinal lactoferrin receptor. Biochemistry 40(51):15771–15779.  https://doi.org/10.1021/bi0155899 CrossRefPubMedGoogle Scholar
  37. Taguchi K, Yamasaki K, Seo H, Otagiri M (2015) Potential use of biological proteins for liver failure therapy. Pharmaceutics 7(3):255–274.  https://doi.org/10.3390/pharmaceutics7030255 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Tomita M, Wakabayashi H, Shin K, Yamauchi K, Yaeshima T, Iwatsuki K (2009) Twenty-five years of research on bovine lactoferrin applications. Biochimie 91(1):52–57.  https://doi.org/10.1016/j.biochi.2008.05.021 CrossRefPubMedGoogle Scholar
  39. Tsuda H, Kozu T, Iinuma G, Ohashi Y, Saito Y, Saito D, Akasu T, Alexander DB, Futakuchi M, Fukamachi K, Xu J, Kakizoe T, Iigo M (2010) Cancer prevention by bovine lactoferrin: from animal studies to human trial. Biometals 23(3):399–409.  https://doi.org/10.1007/s10534-010-9331-3 CrossRefPubMedGoogle Scholar
  40. Valenti P, Antonini G (2005) Lactoferrin: an important host defence against microbial and viral attack. Cell Mol Life Sci 62:2576–2587.  https://doi.org/10.1007/s00018-005-5372-0 CrossRefPubMedGoogle Scholar
  41. van Berkel PH, Geerts ME, van Veen HA, Kooiman PM, Pieper FR, de Boer HA, Nuijens JH (1995) Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis. Biochem J 312(1):107–114.  https://doi.org/10.1042/bj3120107 CrossRefPubMedPubMedCentralGoogle Scholar
  42. van Berkel PH, van Veen HA, Geerts ME, de Boer HA, Nuijens JH (1996) Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants. Biochem J 319(Pt1):117–122.  https://doi.org/10.1042/bj3190117 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Van Berkel PH, Welling MM, Geerts M, van Veen HA, Ravensbergen B, Salaheddine M, Pauwels EK, Pieper F, Nuijens JH, Nibbering PH (2002) Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat Biotechnol 20(5):484–487.  https://doi.org/10.1038/nbt0502-484 CrossRefPubMedGoogle Scholar
  44. Wakabayashi H, Yamauchi K, Takase M (2006) Lactoferrin research, technology and applications. Int Dairy J 16(11):1241–1251.  https://doi.org/10.1016/j.idairyj.2006.06.013 CrossRefGoogle Scholar
  45. Wakabayashi H, Oda H, Yamauchi K, Abe F (2014) Lactoferrin for prevention of common viral infections. J Infect Chemother 20(11):666–671.  https://doi.org/10.1016/j.jiac.2014.08.003 CrossRefPubMedGoogle Scholar
  46. Wang Y, Zhao S, Bai L, Fan J, Liu E (2013) Expression systems and species used for transgenic animal bioreactors. Biomed Res Int.  https://doi.org/10.1155/2013/580463 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Wang M, Sun Z, Yu T, Ding F, Li L, Wang X, Fu M, Wang H, Huang J, Li N, Dai Y (2017) Large-scale production of recombinant human lactoferrin from high-expression, marker-free transgenic cloned cows. Sci Rep. 7(1):10733.  https://doi.org/10.1038/s41598-017-11462-z CrossRefPubMedPubMedCentralGoogle Scholar
  48. Yemets AI, Tanasienko IV, Krasylenko YA, Blume YB (2014) Plant-based biopharming of recombinant human lactoferrin. Cell Biol Int 38(9):989–1002.  https://doi.org/10.1002/cbin.10304 CrossRefPubMedGoogle Scholar
  49. Zakharova ET, Sokolov AV, Pavlichenko NN, Kostevich VA, Abdurasulova IN, Chechushkov AV, Voynova IV, Elizarova AY, Kolmakov NN, Bass MG, Semak IV, Budevich AI, Kozhin PM, Zenkov NK, Klimenko VM, Kirik OV, Korzhevskii DE, Menshchikova EB, Vasilyev VB (2018) Erythropoietin and Nrf2: key factors in the neuroprotection provided by apo-lactoferrin. Biometals 31(3):425–443.  https://doi.org/10.1007/s10534-018-0111-9 CrossRefPubMedGoogle Scholar
  50. Zhang Y, Lima CF, Rodrigues LR (2014) Anticancer effects of lactoferrin: underlying mechanisms and future trends in cancer therapy. Nutr Rev 72(12):763–773.  https://doi.org/10.1111/nure.12155 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Belarusian State UniversityMinskBelarus
  2. 2.Scientific and Practical Centre on Animal Husbandry of the National Academy of Sciences of BelarusZhodinoBelarus
  3. 3.Institute of Physiology of the National Academy of Sciences of BelarusMinskBelarus

Personalised recommendations