Production of human lactoferrin in animal milk.

Institute of Gene Biology, Russian Academy of Sciences, 119334, Vavilova str, 34/5, Moscow, Russia.
Biochemistry and Cell Biology (Impact Factor: 2.35). 02/2012; 90(3):513-9. DOI: 10.1139/o11-088
Source: PubMed

ABSTRACT Genetic constructs containing the human lactoferrin (hLf) gene were created within a joint program of Russian and Belorussian scientists. Using these constructs, transgenic mice were bred (the maximum hLf concentration in their milk was 160 g/L), and transgenic goats were also generated (up to 10 g/L hLf in their milk). Experimental goatherds that produced hLf in their milk were also bred, and the recombinant hLf was found to be identical to the natural protein in its physical and chemical properties. These properties included electrophoretic mobility, isoelectric point, recognition by polyclonal and monoclonal antibodies, circular dichroic spectra, interaction with natural ligands (DNA, lipopolysaccharides, and heparin), the binding of iron ions, the sequence of the 7 terminal amino acids, and its biological activity. The latter was assessed by the agglutination of Micrococcus luteus protoplasts, bactericidal activity against Escherichia coli and Listeria monocytogenes , and fungicidal activity against Candida albicans . We also demonstrated a significant increase in the activity of antibiotics when used in combination with Lf.

1 Follower
  • [Show abstract] [Hide abstract]
    ABSTRACT: Single-domain antibody generation technology was applied to make new Sepharose-bound ligands for affinity separation of closely related proteins, such as human and goat lactoferrin. We generated recombinant antibodies that can selectively bind/recognize only lactoferrins having amino acid sequences identical to that of human natural lactoferrin (anti-hLF Ab). Selected and purified histidine-tagged single-domain antibodies were used as ligands, and different lactoferrins were used as analytes in the kinetics analysis of lactoferrin binding to captured anti-hLF Abs using the Bio-Rad ProteOn XPR36 protein interaction array system. The data obtained were consistent with a 1:1 binding model with very high affinity, practically equal in the case of hLF and rec-hLF (calculated KD varied from 0.43nM to 3.7nM). Interaction of captured fsdAbs with goat LF was significantly weaker and not detectable under the same analysis conditions. We demonstrated the high efficiency of the recombinant human lactoferrin purification from goat lactoferrin and other proteins using the obtained single domain antibody-based affinity ligands. We believe this approach can be used for the generation of single-domain antibody-based affinity media for the efficient separation/purification of a wide spectrum of other highly homologous proteins.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 01/2014; 949-950C:48-57. DOI:10.1016/j.jchromb.2013.12.034 · 2.69 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: During the past two decades, there have been numerous attempts at using animals in order to produce recombinant human proteins and monoclonal antibodies. However, it is only recently that the first two therapeutic agents isolated from the milk of transgenic animals, C1 inhibitor (Ruconest) and antithrombin (ATryn), appeared on the market. This inspires hope that a considerable number of new recombinant proteins created using such technology could become available for practical use in the near future. In this review, the methods applied to produce transgenic animals are described and the advantages and drawbacks related to their use for producing recombinant human proteins and monoclonal antibodies are discussed.
    01/2013; 5(1):33-46.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Expression in milk of transgenic farm animals is one of the most effective ways to produce eukaryotic recombinant proteins. In this review, we discuss the problems of the appropriateness of selecting this expression system for the production of proteins and the development characteristics of appropriate production processes.
    Applied Biochemistry and Microbiology 12/2013; 49(9). DOI:10.1134/S0003683813090020 · 0.66 Impact Factor