Lactoferrin isolation

Iron supplementation to meet nutritional requirements can seriously limit the shelf life of milk products especially infant formulas. lipid oxidation can be controlled in iron-supplemented milk by using lactoferrin, a non-heme ironbinding glycoprotein found in human (1.4 mg/ml) and bovine milk (0.1 mg/ ml). Lactoferrin in bovine milk is 22% saturated with iron compared to 4% in mature human milk. Compared to human milk, infant formulas are more susceptible to lipid oxidation because they are supplemented with greater amounts of iron and do not contain lactoferrin. This antioxidant protein in milk has an important activity by binding prooxidant iron ions. Commercially available bovine lactoferrin, isolated from cheese whey, inhibited lipid... 

The Gram-positive bacterium Streptococcus pneumoniae is an important cause of respiratory tract infections, bacteremia, and meningitis. In this strain, the cell wall anchored pneumococcal surface protein A (PspA) has been demonstrated to bind lactoferrin [181]. PspA and closely related proteins in a variety of pneumococcal isolates are most likely involved in the sequestration of iron from lactoferrins, and finally contribute to the virulence of these bacteria. However, the means by which the pneumococcus acquires iron at the mucosal surface during invasive infection is not well understood at the molecular level [182],... 

Tani, F., Iio, K., Chiba, H., and Yoshikawa, M. 1990. Isolation and characterization of opioid antagonist peptides derived from human lactoferrin. Agric. Biol. Chem. 54, 1803-1810. 

Qian, Z.Y., Jolles, P., Migliore-Samour, D., and Fiat, A.M. 1995. Isolation and characterization of sheep lactoferrin, an inhibitor of platelet aggregation and comparison with human lactoferrin. Biochim. Biophys. Acta 1243, 25-32. 

Recio, I. and Visser, S. 1999. Two ion-exchange methods for the isolation of antibacterial peptides from lactoferrin—in situ enzymatic hydrolysis on an ion-exchange membrane. J. Chromatogr. 831, 191-201. 

The successful isolation of primary cells is dependent on several factors, some of which are not subject to optimization, such as species, type of tissue, age and sex of donor, and presence of genetic modifications (e.g., knockout animals). Other factors such as the dissociation medium, enzymes and concentrations, temperature, and incubation times can be optimized to ensure the quality and consistency of a primary or cell line preparation. The identification and availability of key growth factors is an important determinant of which primary cells can be maintained in culture. For example lactoferrin is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. Recently it has been shown that lactoferrin at physiological concentrations can also promote bone growth, potently stimulating the proliferation and differentiation of primary osteoblasts (Naot, 2005). 

The recombinant whole molecules are both expressed in glycosylated form, although the glycosylation patterns differ from the proteins isolated from natural sources. The recombinant human transferrin binds to receptors both in its glycosylated form and as a nonglycosylated mutant, showing that the carbohydrate is not required for receptor binding (230). Recombinant human lactoferrin shows identical spectroscopic properties and shows an identical profile of pH-dependent iron release when compared with human milk lactoferrin (231). 

There have been special problems in the isolation of some lysozymes, particularly those from cow milk and the milk of monotremes. Isolation from colostrum has proved difficult because of the previously noted tendency of lysozyme to complex with other proteins (e.g., immunoglobulins). Some proteins, particularly lactoferrin and transferrin, may elute from ion exchangers similarly to lysozyme, and it is frequently necessary to take the precaution of using multiple passages of the lysozyme-containing product thereafter, through an appropriate gel filtration column. 

Lactoferrin has been isolated and identified from a wide variety of animal species. However, most of the studies on structure and iron-binding properties have involved either human or bovine proteins (2). Lactoferrin closely resembles transferrin in molecular weight of 75,000 to 90,000 and consists of a single polypeptide chain that binds two ferric ions. The pi of transferrin is 5.9 while that of lactoferrin is approximately 9.0 (8) and has an even higher association constant for iron-binding. Lactoferrin has the property of retaining its iron even in the presence of a relatively low-affir-nity iron chelator such as citrate below pH 4.0. Transferrin, on the other hand, looses its iron when the pH is lowered from 6 to 5 (7). There is extensive information in the literature concerning the physical properties of lactoferrin which will not be covered in this paper. 

In the meantime, in 1947, Laurell and Ingelman[17] had independently purified the red protein from pig plasma and in the same year proposed the name transferrin which has since been adopted as the generic name of the proteins of this family serotransferrin (instead of siderophilin) present in blood and some external secretions, ovotransferrin (instead of conalbumin) in avian egg-white, lactotransferrin (also called lactoferrin) from milk, external secretions and leukocytes and melanotransferrin (instead of p97) in melanocyte and normal cell plasma membrane. A dozen mammalian and some frog, fish and insect serotransferrins were later isolated and characterized. 

The transferrins are a class of iron-binding glycoproteins which have been found in the blood serum of a variety of vertebrates and are presumably also present in moth hemolymph [see (74) and references therein], These proteins would appear to mediate the absorption and distribution of iron (75), and could play some role in the movement of carbon dioxide in the body (76). Proteins of similar characteristics, lactoferrin and conalbumin, have been isolated from milk and avian egg white respectively. It has been proposed (77) that conalbumin could prevent bacterial contamination of the egg yolk by removing free iron. A similar bacteriostatic action could be performed by lactoferrin in milk, and it is possible that this protein is involved in controlling the intestinal flora in infants (78). 

Lactoferrin is a 703-amino-acid glycoprotein originally isolated from milk. Plasma lactoferrin is predominantly neutrophil-derived, but indications are that it may also be produced by other cells. Lactoferrin in body fluids is found in the iron-free form, the monoferric form, and in the difer-ric form. Three isoforms of lactoferrin have been isolated, i.e., two with RNAse activity (lactoferrin-beta and lactoferrin-gamma) and one without RNAse activity (lactoferrin-alpha). Receptors for lactoferrin can be found on intestinal tissue, monocytes/macrophages, neutrophils, lymphocytes, platelets, and on certain bacteria. A wide spectrum of functions is ascribed to lactoferrin. These range from a role in the control of iron availability to immune modulation. 

Ammons, M. C., Ward, L. S., Fisher, S. T., Wolcott, R. D., and James, G. A. (2009). In vitro susceptibility of established biofilms composed of a clinical wound isolate of Pseudomonas aeruginosa treated with lactoferrin and xyUtol. Int. J. Antimicrob. Agents 33,230-236. 

Perhaps the most exciting aspect of this proposed mechanism is the manner in which it correlates with recently-isolated lon-blndlng molecules called slderophores (129). The primary function of lactoferrin is to diminish the amount of extracellular free iron and thereby inhibit bacterial growth (130). Lactoferrin deposited by polymorphonuclear leukocytes is attached to the surface of monocytes and macrophages in inflammatory responses (130). Slderophores are synthesized by bacterial cells to sequester iron needed for growth, and... 

Law, B. A, and Reiter, B, (1977) The isolation and bacteriostatic properties of lactoferrin from bovine milk whey. 

Selective elution, also known as ion exchange chromatography, is the process most often used to make whey protein isolates. Using selective elution, whey protein solution is applied to an ion exchange column so that all of the proteins bind to the column. The column is then rinsed and the individual proteins are eluded one by one to produce highly purified whey proteins. This process can be used to both concentrate and fractionate the proteins. If done carefully, the native protein structures can be retained. However, some of the smaller peptides such as lactoferrin may have a decreased concentration, while the P-lactoglobulin protein fraction tends to increase in whey protein isolates. 

---