Purification of Bovine Lactoferricin from Cows Milk - Research Paper Example

Report Layout Summary Introduction Materials and Methods Results Discussion Future analysis Conclusion References Page No. 3 4 13 16 18 18 19 20 Purification of bovine lactoferricin from cow’s milk Summary Bovine lactoferrin from cow’s milk was hydrolysed using porcine pepsin. The peptic digest was subjected to chromatography on the weak cation-exchange resin, Macro-prep CM. Two protein fractions F1 and F2 corresponding to two eluate peaks were collected using 10% acetic acid as the eluent. The fractions, after neutralization, were subjected to freeze drying. The freeze dried samples F1 and F2, and lactoferrin samples before and after digestion were tested by the plate assay technique for antibacterial activity against Eschericia coli 0111 using Polymyxin B as the positive control and 0.1% acetic acid as the negative control. Results indicated antibacterial activity to be present in pepsin-digested lactoferrin and F2 sample. Thus, purification of lactoferricin obtained through peptic digestion of bovine lactoferrin was accomplished by fractionation of the hydrolysate using cation exchange chromatography. Introduction Lactoferrin (Lf) is an iron-binding glycoprotein and, as the name suggests, is a constituent of milk. It is also found, to a lesser extent, in various mucosal (exocrine) secretions of mammals that are commonly exposed to normal flora such as tears, nasal exudate, saliva, bronchial mucus, gastrointestinal fluids, cervicovaginal mucus and seminal fluid (Weinberg, 2003). Also, Lf is produced by secondary granules of polymorphonuclear neutrophils for deposition at septic sites. The granular contents of neutrophils can be released into inflammatory fluids after neutrophil death, by what is known as "holocrine secretion". The protein is closely related to transferrin, the iron-transport protein present in the plasma. Lactoferrin is a multifunctional innate-defense protein, known to exert a broad-spectrum primary defense activity against microbes including bacteria, fungi, protozoa and viruses (Orsi, 2004), and even some antibiotic-resistant pathogens (Wakabayashi et al., 2003). Lf is found in high concentrations in breast milk (~3–7 mg/ml) and tear fluid (1–4 mg/ml) (Rogan et al., 2006). Among the many constituents of milk which have revealed antimicrobial activity, lactoferrin exhibits both bacteriostatic and bactericidal activity against a wide array of microorganisms, including those causing gastroenteric infections, food poisoning, listeriosis and mastitis (Dionysius et al., 1993). Recently, clinical trials have demonstrated that bovine lactoferrin (bLF) administration can reduce the risk of colon carcinogenesis in humans (Tsuda et al., 2010). A key role of Lf is to scavenge non-protein-bound iron in body fluids and inflamed areas thereby suppressing free radical-mediated damage and, furthermore, decrease availability of the ferric ion to invading bacterial, fungal and neoplastic cells (Weinberg, 2003). On account of its iron-binding properties, lactoferrin plays a role in iron uptake by the intestinal mucosa and acts as a bacteriostatic agent by withholding iron from iron-requiring bacteria. Some of the possible functions of Lf are depicted in Figure 1. Lactoferrin comprises a single polypeptide chain of about 690 amino acid residues and has a molecular weight of 80-kDa. The molecule is folded into homologous N- and C-terminal lobes, each comprising two domains that include a conserved iron binding site. Cooperative interactions between the lobes influence iron binding and release by the lactoferrin molecule (Baker and Baker, 2005). It exists in both iron-replete and iron-depleted forms. The iron-depleted form of lactoferrin is its more biologically active form. The structural region responsible for the antimicrobial properties of lactoferrin is present near the N-terminus of lactoferrin in a region distinct from its iron-binding sites (Bellamy et al., 1992a). The authors isolated the active domain, lactoferricin (LFcin), after gastric pepsin cleavage of both human and bovine lactoferrins. Human lactoferricin (lactoferricin H) contains 47 amino acid residues while the bovine form (lactoferricin B) is made up of 25 residues ( Fig. 2). The lactoferricin moiety contains a high proportion of basic amino acid residues, in similarity with many other antimicrobial peptides having membrane-disruptive properties. Sequence determination by automated Edman degradation revealed the single active peptide, comprising the domain, to consist of a loop of 18 amino acid residues formed by a disulfide bridge between cysteine residues at positions 20 and 37 in the human lactoferrin, or 19 and 36 of bovine lactoferrin (Fig. 2). The molecule is folded into homologous N- and C-terminal lobes, each comprising two domains that enclose a conserved iron binding site. Lactoferrin has strong anti-inflammatory properties as it possesses two basic sequences at residues 1 to 5 and 28 to 34 of the N-terminal end that can bind anionic molecules such as lipopolysaccharide (LPS), heparin and heparin sulfates. Fig. 1. The proposed functions of lactoferrin. (Modified from Brock, 2002) Figure 2. Structure of human and bovine forms of lactoferricin with 47 and 25 amino acid residues, respectively (Source: Bellamy et al., 1992a). The active peptide of bovine Lf was more effective than the corresponding human Lf peptide against a physiologically diverse range Gram-negative and Gram-positive bacteria. The concentrations of LFcin required to cause complete inhibition of growth depend on the strain and the culture medium used. On the other hand, Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were found to be highly resistant to the bovine peptide, lactoferricin B (Bellamy et al., 1992b). The bacteriostatic effect of Lf is mainly due to its iron sequestering property. By binding the iron, it limits the availability of free iron which is an essential growth factor for microorganisms. Bacterial pathogens can counteract this by two mechanisms: either by producing siderophores with high affinity for binding ferric ion and transport it into cells, or by expressing specific receptors at the bacterial surface that bind the iron directly from lactoferrin (Orsi, 2004). In fact, many Neisseriaceae and Moraxellaceae families express surface receptors that specifically binding host lactoferrin and extract the iron from lactoferrin as a source of iron for growth (Beddek and Schryvers, 2010). Lactoferricin displays strong membrane-disruptive properties which induce intense ultrastructural changes in the cell features and, thereby, cause substantial cell damage in bacteria and fungi. It also acts synergistically with some antimicrobial agents (Wakabayashi et al., 2003) including other innate immunity proteins such as lysozyme and SLPI in destroying bacteria (Singh et al., 2000). Lf has been shown to boost the antimicrobial effects of some antibiotics such as doxycycline and tobramycin. The bactericidal effect of LFcin is due to its binding to the lipid A part of bacterial lipopolysaccharide, leading to an increase in membrane permeability. Other antibacterial activities of Lf include specific effects on the biofilm development, the bacterial adhesion and colonization, the intracellular invasion, the apoptosis of infected cells and the bactericidal activity of polymorphonuclear leukocytes, PMN (Orsi, 2004). Biofilm formation is a highly destructive mode of bacterial growth, biofilm bacteria being highly resistant to host killing and antibiotics. The inhibition of biofilm formation, crucial for host defense, is an extremely important property that is unique to lactoferrin. The anti-inflammatory, anti-viral, anti-lipopolysaccharide, anti-biofilm, antibacterial and anti-fungal properties of lactoferrin as observed in the respiratory tract are schematically shown in Fig. 3. The immunomodulatory function of lactoferrin is essentially due to its ability to bind unmethylated CpG motifs, which are bacterial DNA products capable of stimulating various inherent and acquired immune responses in human and murine models (Rogan et al., 2006). Fig.3. Action of lactoferrin released from neutrophils and respiratory tract epithelium as envisaged in the respiratory tract. (Modified from Rogan et al., 2006) LFcin acts against both enveloped and naked viruses such as rotavirus, enterovirus and adenovirus in the early phases of viral infection, through binding to the viral particle or to the cell receptors for virus. In its action against rotavirus, Lf from bovine milk (BLf) acting in a dual role, prevents virus attachment to intestinal cells by binding to viral particles, and, subsequently, inhibits a post adsorption step. In the case of poliovirus, BLf acts at an early infection step. However, it can also inhibit viral replication after the viral adsorption phase if the molecule is saturated with Zn2+ ions (Seganti et al., 2004). On the other hand, the anti-adenovirus action of BLf is directed against virus attachment to cell membranes which occurs through competition for common glycosaminoglycan receptors and a specific interaction with viral structural polypeptides. Thus the anti-viral action of Lf mainly occurs through deterrance of adsorption and internalization into cells caused by specific binding to cell receptors and/or viral particles (Seganti et al., 2004). BLf has also been recognized as a potent inhibitor of human herpetic viruses, such as cytomegalovirus and herpes simplex virus type 1 and 2 where it acts by binding to heparan sulphate containing proteoglycans that also act as cell receptors for herpetic viruses. Besides, BLf also inhibits the cell-to-cell spread of herpes simplex virus type 1 which makes it an excellent treatment choice for herpetic infections with the advantage of preventing viral infections caused by cell-associated virus. The binding of Lf to cell surface expressed heparan sulfate (HS) also explains its anti- Japanese encephalitis virus activity (Chien et al., 2008). Applications of lactoferricin Natural antibiotics are in great demand as components to supplement infant formulas and other speciality foods. This has stimulated much research into the production and effective utilization of lactoferrin (Smithers et al., 1996). In vivo synthesis of these peptides in substantial quantities could have important physiological significance specifically for breast-fed infants. The availability of recombinant human Lf has enabled intense research for commercial applications, but industrial-scale production and commercial exploitation of lactoferrin/lactoferricin is still limited for lack of suitable technologies (Korhonen and Pihlanto, 2007). Besides infant milk formula, the use of Lf in food preservation, fish farming, and oral hygiene are some of the applications being considered. The potential prophylactic and therapeutic applications of Lf for a wide spectrum of medical conditions are under scrutiny. The readily accessible body compartments for therapeutic Lf administration include skin, throat and small intestine while studies for possible medicinal applications in colon and systemic tissues are needed. Recently, López-Soto et al. (2010) have shown that lactoferrin-peptides, especially the chimera, have amoebicidal activity. Lactoferricin, therefore, promises be a useful therapeutic agent against amoebiasis which could also help curtail the use of the extremely toxic metronidazole. Lf being a natural product, it is expected that it should be highly biocompatible. However, potential hazards have been reported (Weinberg, 2003). Many studies have provided evidence to show that oral administration of lactoferrin exerts several beneficial effects on the health of humans and animals, e.g., antibiotic, anti-cancer, and anti-inflammatory effects, promoting the enhanced use of lactoferrin as a food additive. It has been used to supplement several foods and skin/oral care products such as mouth wash, mouth gel, toothpaste, chewing gum, infant formula, follow-up milk, skim milk, yogurt, nutritional enhancers and feed supplements (Wakabayashi et al., 2006). Recently, probiotic Lactobacillus casei has been employed to express and deliver human lactoferrin (hLF) to protect the host against bacterial infection. This could be a useful mode of application of lactoferrin for treatment of infections caused by clinically significant pathogens (Chen et al., 2010). Lactoferricin is an extremely attractive candidate as an anti-cancer drug. Studies have shown that bovine Lfcin induces cell death in tumor cells by targeting the plasma membrane and the mitochondria, following internalization (Eliassen, et al., 2006). Also, compared to conventional chemotherapy the cationic antimicrobial peptides including lactoferrin display a higher specificity for cancer cells versus normal cells. Furthermore, the peptides are able to kill cancer cells that have developed resistance to conventional chemotherapeutics. Purification of lactoferricin Worldwide annual production of bovine lactoferrin is approximately 20 to 30 tons from milk and milk products (Tomita et al., 2002). Considering the myriad potential applications of lactoferricin, obtaining it in the pure form has become absolutely necessary. However, the yield through natural resources such as bovine milk is limited. Production of recombinant lactoferricin is a promising, high-yielding alternative. Purification of Lf is needed in order to study the structure of the molecule which will be helpful for the preparation of the synthetic compound. Lactoferrin is a highly cationic protein (calculated pI > 8.5) being rich in arginine residues that give the molecule its highly cationic nature, and this property has been used to advantage in the process of isolation of the protein from whey. Also, most of the major and many of the minor, protein constituents of milk are acidic (pI < 7). Thus, cation-exchange column chromatography is the most commonly reported method for the extraction of lactoferrin, especially from whey (Smithers et al., 1996). The cationic lactoferrin gets readily adsorbed to a cation-exchange resin, from which it is recovered using salt solutions or by varying the pH of the resin (Tomita et al., 2002). The eluted crude LF is either desalted or pH neutralized depending on the nature of eluate. The treated eluate is concentrated using ultrafiltration and diafiltration techniques, and the LF concentrate is pasteurised to kill bacteria as well as viruses. Purified LF powder is obtained by freeze drying the pasteurised concentrate. Pasteurisation is done under acidic conditions since heat treatment of LF under neutral conditions causes denaturation of the protein (Tomita et al., 2002). Lactoferricin is produced by pepsin digestion of LF. The hydrolysate obtained is purified by cation-exchange chromatography as described earlier for LF. Reverse phase-HPLC is a suitable method using which lactoferrin has been purified (>90%) and quantitated at levels as low as 0.2 μg (Palmano and Elgar, 2002). Materials and Methods Digestion of Lactoferrin: 15g lactoferrin were dissolved in 300mL distilled water to obtain a 5% (w/v) solution. The solution was adjusted to pH 2.5 with 1M HCl, that being the optimum pH for enzyme (pepsin) activity (Saito et al., 1991). 0.45g of Sigma porcine pepsin was added to the mixture to get 3% (w/w substrate). The solution was incubated in a water bath at 37°C for four hours. The reaction was stopped at the end of four hours by heating the protein mixture to 80°C for 15 minutes. The solution was next neutralized (pH 7.0) with dropwise addition of 1M NaOH. The resultant mixture was centrifuged for 10 min at 12000rpm, the supernatant was collected and stored frozen. 5mL of the sample was filtered through sterile 0.45µm cellulose acetate filter. Also, another lactoferrin sample was prepared by adding 5g lactoferrin to 100ml water and stored at -80°C for the plate assay. Ion-exchange Chromatography: The stationary phase of the ion exchange column used was Macro-prep CM, a weak cationic exchange support. The column (28mm X 230mm) was initially washed with de-gassed water at the rate of 30mL/h for 2.5 hours, pump speed setting at 7 to clear all protein residues from earlier experiments. 500mL each of 25mM ammonium acetate, 20% ethanol, 10% acetic acid were prepared and de-gassed along with 500 mL of distilled water to minimise air interference with the chart reading. The column was washed for approximately 20 min using the de-gassed ammonium acetate solution to stabilise the chart baseline. Immediately after the ammonium acetate solution had disappeared into the column bed, 5mL of the digested LF was added to the top of the column bed using a clean pipette and allowed to run by gravity. Before elution, the column was washed with ammonium acetate for 3 hours using the pump, until the chart baseline was fairly steady. By the end of this step, all the negatively charged material in the sample would have been washed out of the column, leaving the cationic molecules of the sample behind. The cationic matter bound to the column was next eluted from the column with the help of 10% acetic acid for a period of 2 hours. The fraction collector speed was set at 10min per tube. The chart recorded the data at a sensitivity of 0.2, and chart speed of 30mm/hr. The column was regenerated using 1M NaCl, the volume added being 2-4 times the column volume (28mm diameter and 230mm length), followed by a similar volume of distilled water. The final washing consisted of 20% ethanol passed for 1.5 hours, to disinfect the column bed. The test tubes corresponding to each peak on the chart namely, fraction1 (F1) and fraction2 (F2) were collected. Acetic acid in sample F2 was removed by rotary evaporation of the sample for 15min in a water bath at 40o C, to ensure the safety of the freeze drying machine. Next, both the samples were transferred to freeze drying flasks, frozen at -80o C, and subjected subsequently to freeze drying overnight. Plate Assay1: To investigate the antibacterial activities of lactoferrin and its active peptides, Escherichia coli 0111, a pathogenic strain of bacteria, was cultured in Brain Heart Infusion (BHI) agar overnight. It was next subcultured and grown in BHI broth at 37º C for 6 hours. Polymyxin B solution (1mg/ml) was prepared as a positive control. Soft agar for plate media was prepared by dissolving in 500ml distilled water, 5g peptone (1%) and 4g agar (0.8%) by gentle heating. The agar medium was autoclaved at 120º C for 20min. Into a series of previously autoclaved 100 mL bottles containing 40mL of the soft agar plate media, 25µL, 40µL, and 50µL of E.coli cells in the log phase of growth were added, mixed well and immediately poured aseptically into sterile petri plates. After the media had solidified, holes were made in the agar with sterile toothpick under UV light. The excess liquid agar in the holes was sucked using a pipette. 50µL samples of lactoferrin solution, digested lactoferrin solution and the positive control, Polymyxin B solution were pipetted into marked wells, while the negative control well remained empty. The plates were left at 4º C for 2-3 hours to allow the sample to diffuse in to the agar. The plates were transferred to a room at 37º C and left there overnight. Plate Assay2 : The freeze dried samples were dissolved in a minimum volume of 0.1% acetic acid. Samples F1, F2 , LF, digested LF, positive control Polymyxin B and negative control 0.1% acetic acid were plated as described earlier. The samples were placed in the wells of three replicate petri dishes containing 25μL log phase cells, stored in a refrigerator at 4º C for 2 hours, and next in a 37º C room overnight. Results The originally orange coloured lactoferrin solution was observed to turn yellow upon peptic digestion. The cow’s milk lactoferrin hydrolysate was fractionated by Macro-prep CM column chromatography. Upon elution with 10% acetic acid, two peaks were obtained (F1 and F2 in Fig. 4). The fractions F1 and F2 were concentrated by freeze drying and subjected to Plate assay against E.coli 0111. Compared to plates containing 40µL and 50µL log cells, the results with 25µL log cell plates had much better clarity. The results of the plate assay with 25µL log cell and using Lf, pepsin digested Lf, protein fractions F1 and F2, positive control Polymixin B and negative control are shown in Fig.5. Fig. 4. Cation exchange chromatogram of pepsin-digested lactoferrin from cow’s milk conducted on matrix Macro-prep CM. Protein fractions F1 and F2 were freeze-dried and analysed by Plate assay. Fig. 5. Plate assay of lactoferrin (Lf), pepsin digested lactoferrin (Lf PD), freeze-dried protein fractions F1 and F2, positive control (Polymyxin B) and negative control (0.1% acetic acid) against E.coli 0111. Discussion As expected, only after digestion did lactoferrin show antibacterial activity as seen in Fig. 5, spot LF PD, while the undigested lactoferrin (LF, Fig. 5) did not show any activity. Both the peaks of the bovine lactoferrin hydrolysate obtained by ion exchange chromatography were tested for antibacterial activity plate assay. Only fraction F2 showed activity to inhibit the growth of E.coli, indicating that fraction F2 contains the active peptide moiety, lactoferricin. Purification of lactoferricin, a cationic antimicrobial peptide, was accomplished by peptic digestion and fractionation by ion exchange chromatography. The plate assay results had the best clarity when conducted with 25µL log cell plates indicating that the concentration of the peptide in the test solution was not adequate to inhibit the growth of a higher biomass of bacterial cells present in plates containing 40µL and 50µL of log phase cells. Thus, the experiments have shown that cow’s milk contains biochemical factors with potent antibacterial activity. However, the activity becomes apparent only after proteolytic digestion of the milk protein, lactoferrin. Future analysis: Ion exchange chromatography actually yielded 3 peaks as seen in Fig. 4, although the last two peaks were not separated well. For analysis, therefore, peaks 2 and 3 were combined to form fraction 2 (that is, F2). Reducing the rate of elution would probably lead to better separation of the peaks. Future work should examine all three peaks individually for antimicrobial properties. Also, subjecting the digested sample to reverse phase-HPLC fractionation could lead to better resolution of the peptide fractions. Such an experiment would also indicate the presence of isoforms of lactoferricin. The Plate assay was qualitative in nature. A quantitative assay would provide more interesting and useful information. Future studies should also evaluate activity against other species of pathogenic bacteria, besides anti-viral, anti-fungal, and, more importantly, anti-tumourigenic activity of the lactoferricin containing fractions. Molecular weight estimations should be done of the active compounds. Conclusion: Cow’s milk was shown to contain biochemical factors with potent antibacterial activity. The undigested lactoferrin from cow’s milk was devoid of inhibitory activity against E.coli but after peptic digestion, the protein showed significant activity. Purification of lactoferricin was accomplished by peptic digestion and fractionation by ion exchange chromatography. References Baker, E.N., & Baker, H.M., 2005. Molecular structure, binding properties and dynamics of lactoferrin. Cellular & Molecular Life Sciences, 62(22):2531-2539. Beddek, A.J., & Schryvers, A.B., 2010. The lactoferrin receptor complex in gram negative bacteria. Biometals, 23(3): 377-386. 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