Human proinsulin production in the milk of transgenic cattle.

Autor: Monzani PS; Center for Biological and Health Sciences, University of Northern Paraná, Londrina, Paraná, Brazil.; Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA., Sangalli JR; Department of Veterinary Medicine, University of São Paulo, Pirassununga, São Paulo, Brazil., Sampaio RV; Department of Veterinary Medicine, University of São Paulo, Pirassununga, São Paulo, Brazil., Guemra S; Center for Biological and Health Sciences, University of Northern Paraná, Londrina, Paraná, Brazil., Zanin R; Laffranchi Agriculture, Tamarana, Paraná, Brazil., Adona PR; Center for Biological and Health Sciences, University of Northern Paraná, Londrina, Paraná, Brazil., Berlingieri MA; Center for Biological and Health Sciences, University of Northern Paraná, Londrina, Paraná, Brazil., Cunha-Filho LFC; Center for Biological and Health Sciences, University of Northern Paraná, Londrina, Paraná, Brazil., Mora-Ocampo IY; Department of Biological Sciences, State University of Santa Cruz (UESC), Ilhéus, Bahia, Brazil., Pirovani CP; Department of Biological Sciences, State University of Santa Cruz (UESC), Ilhéus, Bahia, Brazil., Meirelles FV; Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA., Wheeler MB; Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA., Ohashi OM; Institute of Biological Sciences, Federal University of Pará, Belém, Pará, Brazil.
Jazyk: angličtina
Zdroj: Biotechnology journal [Biotechnol J] 2024 Mar; Vol. 19 (3), pp. e2300307.
DOI: 10.1002/biot.202300307
Abstrakt: Background: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk.
Methods and Results: Pseudo-lentivirus containing the bovine β-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein.
Conclusion: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.
(© 2024 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)
Databáze: MEDLINE
Externí odkaz: