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.
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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
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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.
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Semak, I., Budzevich, A., Maliushkova, E. et al. Development of dairy herd of transgenic goats as biofactory for large-scale production of biologically active recombinant human lactoferrin. Transgenic Res 28, 465–478 (2019). https://doi.org/10.1007/s11248-019-00165-y
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DOI: https://doi.org/10.1007/s11248-019-00165-y