Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1996-10-16
1999-01-19
Degen, Nancy
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530395, 530400, 530413, 530832, C07K 120, C07K 14435, C07K 1447, C07K 1479
Patent
active
058614915
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention relates to the purification of lactoferrin from milk, particularly the purification of human lactoferrin from the milk of transgenic non-human animals expressing a human lactoferrin polypeptide encoded by a transgene.
BACKGROUND
Recent advances in the field of molecular biology allow the production of transgenic animals (i.e., non-human animals containing an exogenous DNA sequence in the genome of germline and somatic cells introduced by way of human intervention). Differences in the regulation of these foreign genes in different cell types make it possible to promote the differential expression of the foreign gene in a preselected tissue, such as the mammary gland, for ease of isolation of the protein encoded by the foreign gene, for a desired activity of the foreign gene product in the selected tissues, or for other reasons.
An advantage of transgenic animals and differential gene expression is the isolation of important proteins in large amounts, especially by economical purification methods. Such proteins are typically exogenous to the transgenic animal and may comprise pharmaceuticals, food additives, nutritional supplements, and the like. However, exogenous proteins are preferably expressed in tissues analogous to those in which they are naturally expressed. For example, exogenous milk proteins (e.g., lactoferrin) are preferably expressed in milk-forming tissues in the transgenic animal. As a result, difficult isolation problems are presented because the exogenous protein is often expressed in the tissue or bodily fluid containing an endogenous counterpart protein (if it exists), and possibly other undesired contaminant species which may have very similar physicochemical properties. Moreover, many exogenous proteins must be substantially purified from other species, frequently purified to homogeneity, prior to their use as pharmaceuticals or food additives.
For example, the production of transgenic bovine species containing a transgene encoding a human lactoferrin polypeptide targeted for expression in mammary secreting cells is described in WO91/08216, published Jun. 13, 1991. The purification of human lactoferrin (hLF) from a transgenic animal containing a functional endogenous bovine lactoferrin (bLF) gene and a transgene encoding the expression of hLF is complicated by the presence of endogenous bLF which has physicochemical properties similar to human lactoferrin. Even in a transgenic bovine lacking a functional endogenous bLF gene (e.g., as a result of homologous gene targeting to functionally disrupt the endogenous bLF alleles), it is frequently desirable and/or necessary to purify transgene-encoded hLF from other biological macromolecules and contaminant species. Since hLF has potential pharmaceutical uses and may be incorporated in human food products as a nutritive supplement, uses which typically require highly purified hLF, it is imperative that methods be developed to purify hLF from milk, especially from milk or milk fractions of transgenic nonhuman animals such as bovine species.
Human lactoferrin is a single-chain glycoprotein which binds ferric ions. Secreted by exocrine glands (Mason et al. (1978) J. Clin. Path. 31: 316; Tennovuo et al. (1986) Infect. Immunol. 51: 49) and contained in granules of neutrophilic leukocytes (Mason et al. (1969) J. EXP. Med. 130: 643), this protein functions as part of a host nonspecific defense system by inhibiting the growth of a diverse spectrum of bacteria. hLF exhibits a bacteriostatic effect by chelation of the available iron in the medium, making this essential metal inaccessible to the microorganisms (Bullen et al. (1972) Brit. Med. J. 1: 69; Griffiths et al. (1977) Infect. Immunol. 15: 396; Spik et al. (1978) Immunology 8: 663; Stewart et al. (1984) Int. J. Biochem. 16: 1043). The bacteriostatic effect may be blocked if ferric ions are present in excess of those needed to saturate the hLF binding sites.
Lactoferrin is a major protein in human milk (present at a concentration of about 1.5 mg/ml) and may pl
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Nuijens Jan H
Van Veen Harry H
Degen Nancy
Pharming B.V.
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