Purification of a 76-kDa iron-binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin

A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 ml of seminal plasma. Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate. It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin. When tested for its iron binding capacity, with Fe-NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm.     

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.     

Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus

1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity.     

Lactoferrin Is the Major Deoxyribonuclease of Human Milk

Lactoferrin is the major iron-transferring protein of human barrier fluids such as blood and milk. It is a polyfunctional protein capable of binding DNA exposed on the surface of various cells. Electrophoretically homogenous lactoferrin was prepared by sequential chromatography of human milk proteins on DEAE-cellulose, heparin-Sepharose, and Sepharose containing immobilized anti-lactoferrin antibodies. By subsequent chromatography on Blue Sepharose the resulting lactoferrin was fractionated into several subfractions with different affinity for the sorbent, and this was associated with separation of additional lactoferrin peaks with DNase activity from the main peak. By various techniques, in particular, by in situ testing the DNase activity of lactoferrin in a DNA-containing gel after SDS-electrophoresis, hydrolysis of DNA was for the first time shown to be an intrinsic property of lactoferrin. The substrate specificity of lactoferrin in hydrolysis of DNA was different from specificities of known human DNases. Hydrolysis of DNA was activated by bivalent metal ions and also by ATP and NAD. Unlike the main fraction of lactoferrin with the highest affinity for Blue Sepharose, all protein subfractions with DNase activity were cytotoxic and suppressed growth of human and mouse tumor cell lines.     

Purification of human milk bile salt-activated lipase by cholic acid-coupled sepharose 4B affinity chromatography

Sequential chromatography of human milk whey on concanavalin A—Sepharose 4B followed by cholate—Sepharose 4B yielded a bile salt-activated lipase with 150-fold purification. The lipase was not retained by concanavalin A—Sepharose 4B but was retained by the cholate—Sepharose 4B, from which it was eluted with 2% sodium cholate. The affinity chromatography procedure on cholate—Sepharose 4B was based on the specific structural requirement of the enzyme for a 7-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7-hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on urea-sodium dodecyl sulfate—polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 0.5–1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity.     

Very stable high molecular mass multiprotein complex with DNase and amylase activities in human milk

For breastfed infants, human milk is more than a source of nutrients; it furnishes a wide array of proteins, peptides, antibodies, and other components promoting neonatal growth and protecting infants from viral and bacterial infection. It has been proposed that most biological processes are performed by protein complexes. Therefore, identification and characterization of human milk components including protein complexes is important for understanding the function of milk. Using gel filtration, we have purified a stable high molecular mass (~1000 kDa) multiprotein complex (SPC) from 15 preparations of human milk. Light scattering and gel filtration showed that the SPC was stable in the presence of high concentrations of NaCl and MgCl₂ but dissociated efficiently under the conditions that destroy immunocomplexes (2 M MgCl₂, 0.5 M NaCl, and 10 mM DTT). Such a stable complex is unlikely to be a casual associate of different proteins. The relative content of the individual SPCs varied from 6% to 25% of the total milk protein. According to electrophoretic and mass spectrometry analysis, all 15 SPCs contained lactoferrin (LF) and α‐lactalbumin as major proteins, whereas human milk albumin and β‐casein were present in moderate or minor amounts; a different content of IgGs and sIgAs was observed. All SPCs efficiently hydrolyzed Plasmid supercoiled DNA and maltoheptaose. Some freshly prepared SPC preparations contained not only intact LF but also small amounts of its fragments, which appeared in all SPCs during their prolonged storage; the fragments, similar to intact LF, possessed DNase and amylase activities. LF is found in human epithelial secretions, barrier body fluids, and in the secondary granules of leukocytes. LF is a protein of the acute phase response and nonspecific defense against different types of microbial and viral infections. Therefore, LF complexes with other proteins may be important for its functions not only in human milk. Copyright © 2014 John Wiley & Sons, Ltd.     

Interaction of lactoferrin with ceruloplasmin

When added to human blood serum, the iron-binding protein lactoferrin (LF) purified from breast milk interacts with ceruloplasmin (CP), a copper-containing oxidase. Selective binding of LF to CP is evidenced by the results of polyacrylamide gel electrophoresis, immunodiffusion, gel filtration, and affinity chromatography. The molar stoichiometry of CP:LF in the complex is 1:2. Near-uv circular dichroism spectra of the complex showed that neither of the two proteins undergoes major structural perturbations when interacting with its counterpart. K(d) for the CP/LF complex was estimated from Scatchard plot as 1.8 x 10(-6) M. The CP/LF complex is found in various fluids of the human body. Upon injection into rat of human LF, the latter is soon revealed within the CP/LF complex of the blood plasma, from where the human protein is substantially cleared within 5 h.     

The role of lactoferrin released by phagocytosing neutrophils in the regulation of colony-stimulating activity production by human mononuclear cells

There remains a controversy about the alleged inhibitory effect of lactoferrin on production of colony-stimulating activity (C.S.A.) by mononuclear cells. We confirm the inhibitory action of both lactoferrin purified from human breast milk and that released from phagocytosing neutrophils. To show the inhibitory effect, it is necessary to plot the dose-response curve of medium conditioned by mononuclear cells with and without lactoferrin. Crowding cells to promote contact is essential for the fraction of C.S.A. production inhibitable by lactoferrin. Saturation of purified lactoferrin by addition of iron salts in vitro may introduce an artifact, as this lactoferrin retained its inhibitory activity at much greater dilutions than lactoferrin released from phagocytosing neutrophils.     

Isolation and physico-chemical properties of lactoferrin from swine neutrophils

Lactoferrin, a non-heme iron-binding protein was isolated from pig neutrophils. The purification procedure included initial extraction of the protein in the presence of cetyltrimethylammonium bromide followed by chromatography on carboxymethyl-cellulose and Sephadex G-100. The thus obtained protein was found to be homogeneous on polyacrylamide gel (PAAG) electrophoresis at acidic values of pH. PAAG electrophoresis in the presence of sodium dodecyl sulfate revealed a single component with a molecular weight of 75 000-80 000. The resulting protein is capable of binding two atoms of iron molecule. The absorbance spectra for the pig neutrophil lactoferrin are identical to those for cow milk lactoferrin in the visible region and have a maximum at 465 nm. The amino acid composition of pig lactoferrin was determined. Isoelectric focusing of the protein obtained in a PAAG stabilized pH gradient revealed a component with pI of about 6.8. A single precipitin line was observed with rabbit antipig lactoferrin when examined by immunodiffusion. No immunological cross-reactions were observed between pig lactoferrin and bovine lactoferrin.     

Cryptic domains of a 60 kDa heat shock protein of Helicobacter pylori bound to bovine lactoferrin

Abstract Bovine lactoferrin binds to a 60 kDa heat shock protein of Helicobacter pylori . Binding ability was related to human immunoglobulin G because bovine lactoferrin binding proteins were isolated by extraction of cell surface associated proteins with distilled water, applied on IgG-Sepharose and nickel sulphate chelate affinity chromatography. Binding was demonstrated by Western blot after purified protein was digested with α-chymotrypsin and incubated with peroxidase-labeled bovine lactoferrin. Binding was inhibited by bovine lactoferrin, lactose, rhamnose, galactose, and two iron-containing proteins, ferritin and haptoglobin. Helicobacter pylori binds ferritin and haptoglobin via charge or hydrophobic interactions because this binding was not inhibited by specific and various glycoproteins or carbohydrates. Carbohydrate moieties of bovine lactoferrin molecules seem to be involved in binding because glycoproteins with similar carbohydrate structures strongly inhibited binding. Scatchard plot analysis of the binding of peroxidase-labeled bovine lactoferrin to H. pylori cells yielded a k _d 2.88 × 10⁻⁶ M. In addition, binding of H. pylori cells to bovine lactoferrin was enhanced when bacteria treated with pepsin or α-chymotrypsin after isolation from iron-restricted and iron-containing media.     

Zinc binding in cow''s milk and human milk

In both cow's milk and human milk, zinc was associated with proteins of high molecular weight (greater than 100 000), as judged by analysis with Sephadex G-75. Precipitation of the casein at pH 4.6 and filtration of the resultant acid whey on Sephadex G-25 led, however, to the recovery of about 90% of the zinc as a compound of low molecular weight, which was tentatively identified as zinc citrate. Over 95% of the zinc of cow's milk was sedimented with the casein micelles on ultracentrifugation. Filtration of these micellar caseins on Sephadex G-150 gave two peaks containing zinc, which corresponded to aggregates of alpha-casein-kappa-casein and of alpha-casein-beta-casein. Ultracentrifugation of human milk sedimented only approx. 40% of total zinc. Analysis of sediment and supernatant on Sephadex G-150, however, indicated that about 85% of the zinc was associated with a protein complex of molecular weight greater than 150 000. The major protein of this complex was identified as lactoferrin. A minor zinc-binding component of average molecular weight 30 000 was also observed in the supernatant. The results indicated that zinc is bound to different macromolecules in cow's and human milk. This may be a factor affecting the bioavailability to the human infant of zinc from the two milks, and it is suggested that in human milk lactoferrin may be involved in the uptake of zinc.     

Improved purification of beta-lactoglobulin from acid whey by means of ceramic hydroxyapatite chromatography with sodium fluoride as a displacer

The successful separation of beta-lactoglobulin from other bovine whey proteins was performed by ceramic hydroxyapatite chromatography with a fluoride ion gradient in phosphate buffer as displacement agent. The method was applied to acid whey originating from milk of healthy as well as of mastitic cows. beta-Lactoglobulin was completely eluted in one peak at a fluoride concentration of about 0.6 mol/l. The purity of beta-lactoglobulin in this fraction was at least 96% if whey from healthy milk was processed. Co-eluted contaminants are traces of immunoglobulin G, serum albumin and lactoferrin. In case of mastitic whey the proportion of beta-lactoglobulin is diminished as the amounts of immunglobulin G, serum albumin and lactoferrin are increased within this fraction. Size exclusion chromatography on Superdex 75 pg effectively removed contaminants resulting in a purity for beta-lactoglobulin from normal whey of approximately 99%. The yield of beta-lactoglobulin from physiological whey was 50-55% referring to the fraction highly enriched with beta-lactoglobulin by hydroxyapatite chromatography. In case of mastitic milk the higher amounts of contaminants were also removed successfully by size exclusion chromatography.     

Iron-binding Proteins in Milk and Resistance to Escherichia coli Infection in Infants

Human milk contains large quantities of iron-binding protein, of which the greater proportion is lactoferrin, though small amounts of transferrin are also present. Three samples of human milk with unsaturated iron-binding capacities of between 56 and 89% had a powerful bacteriostatic effect on Escherichia coli O111/B4. The bacteriostatic properties of milk were abolished if the iron-binding proteins were saturated with iron. Purified human lactoferrin, in combination with specific E. coli antibody, strongly inhibited the growth of E. coli, and this effect was also abolished by saturating the lactoferrin with iron.Guinea-pig milk also contains lactoferrin and transferrin. Newly born guinea-pigs fed on an artificial diet and dosed with E. coli O111 had higher counts of E. coli O111 in the intestine than suckled animals. The apparent suppressive effect of guinea-pig milk on E. coli in the intestine could be reversed by feeding the iron compound haematin. It seems that iron-binding proteins in milk may play an important part in resistance to infantile enteritis caused by E. coli.     

High-level expression of human lactoferrin in the milk of goats by using replication-defective adenoviral vectors

The expression of human lactoferrin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce lactorferrin in the milk of transgenic animals resulted in very high cost and uncertain results. In this paper, we have directly infused replication-defective adenovirus encoding human lactoferrin cDNA into the mammary gland of goats via the teat canal. In this way, we obtained a high level of expressed human lactoferrin up to 2g/L in the milk of goats. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. A approximately 80-kDa protein was visualized after viral vector infection. Our results demonstrate that intraductal injection of recombinant replication-defective adenovirus vectors may provide a very useful tool for large-scale production of recombinant proteins of biopharmaceutical interest.     

Expression,purification, and characterization of equine lactoferrin in Pichia pastoris

Lactoferrin is an 80kDa iron-binding glycoprotein. It is secreted by exocrine glands. Many functions such as iron sequestering, anti-bacterial activity, regulation of gene expression, and immunomodulation are attributed to it. In the present study, we report the production of recombinant equine lactoferrin (ELF) in the methylotropic yeast Pichia pastoris using pPIC9K vector. The recombinant protein was purified by one-step affinity chromatography using heparin-Sepharose column. The purified protein has a molecular weight of 80kDa and reacted with antibody raised against the native equine lactoferrin. Its N-terminal sequence was identical to that of the native ELF. The iron-binding behavior and circular dichroism studies of the purified protein indicate that it has folded properly. The recombinant protein appears to be hyperglycosylated by the host strain, GS115. This is the first heterologous expression of equine lactoferrin and also the first report of intact lactoferrin expression using P. pastoris system. An yield of 40mg/l obtained in shake-flask cultures with this system, which is higher than the reported values for other systems.     

The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct

Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3kb hLF minigene to the regulatory elements of the β-casein gene. The transgenic goat produced more than 30mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future.     

Bovine lactoferrin and lactoferricin derived from milk: production and applications.

Bovine lactoferrin is produced on an industrial scale from cheese whey or skim milk. The safety of purified lactoferrin has been confirmed from the results of a reverse mutation test using bacteria, a 13-week oral repeated-dose toxicity study in rats, and clinical studies. In order to apply active lactoferrin to various products, a process for its pasteurization was developed. Subsequently, lactoferrin has been used in a wide variety of products since it was first added to infant formula in 1986. A pepsin hydrolysate of lactoferrin is also used in infant formula. This hydrolysate contains a potent antimicrobial peptide named lactoferricin that is derived from the lactoferrin molecule by pepsin digestion. Semilarge-scale purification of lactoferricin can be performed by hydrophobic interaction chromatography. Lactoferricin also exhibits several biological actions and appears to be the functional domain of lactoferrin. Recent studies have demonstrated that oral administration of lactoferrin or lactoferricin exerts a host-protective effect in various animals and in humans. The results of these studies strongly suggest that the effects of oral lactoferrin are mediated by modulation of the immune system. Further elucidation of the clinical efficacy and mechanism of action of lactoferrin will increase the value of lactoferrin-containing products.     

Purification of novel peptide antibiotics from human milk

A strategy was established for the identification of novel antimicrobial peptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avoid the purification of the known abundant antimicrobial milk protein lysozyme, it was identified in HPLC fractions by its enzymatic activity and by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS). On condition that lysozyme was not detectable and antibacterial activity of HPLC fractions was caused by a peptide, which was confirmed by proteolytic cleavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. The new antimicrobial peptide resembles a proteolytic fragment of human casein-κ (residues 63–117) and inhibits the growth of Gram-positive, Gram-negative bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolysis and may play an important role in the host defense system of the newborn.     

Studies of the ceruloplasmin-lactoferrin complex.

We have previously shown that iron-containing human lactoferrin (LF) purified from breast milk is able to form both in vitro and in vivo a complex with ceruloplasmin (CP), the copper-containing protein of human plasma. Here we present evidence that the CP-LF complex is dissociated by high concentrations of NaCl, CaCl2, or EDTA, or by decreasing the pH to 4.7. In addition, DNA, bacterial lipopolysaccharide, and heparin can displace CP from its complex with LF. Antibodies to either of the two proteins also cause dissociation of the complex.