Studies on Recovery of Lactoferrin from Bovine ... - Springer Link

Biotechnology and Bioprocess Engineering 20: 148-156 (2015) DOI 10.1007/s12257-014-0408-7

RESEARCH PAPER

Studies on Recovery of Lactoferrin from Bovine Colostrum Whey Using Mercapto Ethyl Pyridine and Phenyl Propyl Amine HyperCel™ Mixed Mode Sorbents R. Ravichandran, Venkatesh Padmanabhan, M. A. Vijayalakhsmi, and N. S. Jayaprakash

Received: 7 June 2014 / Revised: 8 August 2014 / Accepted: 17 September 2014 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract Lactoferrin is one of the important nutraceutical with several physiological and biological functions. Bovine colostrum contains higher concentrations of lactoferrin than in the milk. The present work describes the use of two mixed mode sorbents, mercapto ethyl pyridine (MEP HyperCel™) and phenyl propyl amine (PPA HyperCel™), for the recovery of lactoferrin from bovine colostrum whey. The dynamic binding capacity study showed that, in MEP the mode of interaction of lactoferrin is similar to IgG and had a more or less same binding capacity of ~20 mg/mL of sorbent. In PPA, the interaction of lactoferrin was hydrophobic and was influenced by the addition of salt. The binding capacity of lactoferrin with PPA in absence and presence of salt was found to be ~17 and ~18 mg/mL sorbent respectively. The binding buffers used for the chromatographic experiments were 0.05 M sodium phosphate buffer, pH 7.4, with or without sodium chloride (0.15 M). A decreasing step pH gradient of pH 6.0, 5.5 and 4.0 was performed for elution. Both lactoferrin and immunoglobulin were obtained with high homogeneity. The recovered lactoferrin was confirmed by immunoblot analysis. The chromatographic elution fractions obtained from MEP Hypercel did not exhibit lactoperoxidase activity. The highest recovery of lactoferrin (~91%) with 2.9 fold rise in purity was obtained when the MEP resin column was used with the binding buffer without sodium chloride. Thus, MEP HyperCel™ could be a potential alternative to existing systems for separation of lactoferrin from bovine colostrum whey. R. Ravichandran, Venkatesh Padmanabhan, M. A. Vijayalakhsmi, N. S. Jayaprakash* Centre for Bioseparation Technology, VIT University, Vellore 632-014, Tamil Nadu, India Tel: +91-416-2202377; Fax: +91-416-2243092 E-mail: [email protected]

Keywords: mixed mode sorbent, MEP, PPA, lactoferrin, bovine colostrum

1. Introduction Bovine colostrum is rich in nutrients and contains bioactive molecules that nourishes and protects the calves from the diseases. The concentration of lactoferrin present in the colostrum is tenfold higher than in normal milk [1]. Lactoferrin molecule is an 80 kDa glycosylated protein with iron-chelating property [2]. Lactoferrin acts as a multifunctional biomolecule and have been used in the treatment of bacterial and viral diseases [3-5]. Lactoferrin is known to interact with a variety of bio-molecules like DNA, RNA, polysaccharides and heparin [6-8]. The increase in demand for lactoferrin and other whey proteins in food industry as food supplements necessitate the large-scale downstream processing with high recovery at low cost and process time [9]. Several approaches have been applied over the years for the recovery of protein from whey. Generally, ion-exchange methods like anion/cation exchange systems are widely used in the whey protein fractionation. However, this procedure requires high saltbuffer systems for the fractionation of biomolecules along with desalting/ultra filtration systems for the concentration of bio-products. Presently, the membrane ultrafiltration is used on an industrial scale for the production of lactoferrin. But, the above process has drawbacks such as product recovery and purity [10]. There are a few other reports on lactoferrin purification using immobilized peptides and dyes as affinity ligands, but they have to be further optimized for industrial operation [10-13]. Sulkowski [14] and Hutchens [15] have studied the conditions for adsorption/desorption

Studies on Recovery of Lactoferrin from Bovine Colostrum Whey Using Mercapto Ethyl Pyridine …

of lactoferrin and transferrin with immobilized metal ion affinity chromatography (IMAC) performed with metal ions such as Cu2+ and Fe3+. In the present study, we evaluated MEP HyperCel™ and PPA HyperCel™ mixed mode sorbents for the recovery of lactoferrin from bovine colostrum whey. These are newly designed industrially scalable chromatographic sorbents introduced to market by Pall Life Sciences. Previously, it has been studied in our laboratory for the purification of antibodies and its mechanism of interaction [16]. The objective of this work is to understand the molecular interaction of bovine lactoferrin and other whey proteins with MEP HyperCel and PPA HyperCel, in presence or absence of salt. For this study, a simple and economical screening protocol for identifying optimal buffer conditions for purification and recovery of lactoferrin was established with micro volume of the sorbent in batch mode. To the best of our knowledge, application of these sorbents for recovery of lactoferrin is not reported before. In this communication, the mechanism of interaction of lactoferrin with the mixed mode sorbents and the effects of salt in buffer system has been discussed.

2. Materials and Methods 2.1. Materials The chromatographic sorbents (MEP HyperCel™ and PPA HyperCel™) were obtained from Pall life sciences, Sergy, France. All chemicals and reagents were of analytical grade and were obtained from Sigma (St Louis, MO, USA). All buffers and reagents were prepared with Milli–Q® water (Millipore, Bedford, MO, USA) and were degassed prior to use. 2.2. Screening for optimal buffer conditions Bovine lactoferrin, human IgG, BSA, and beta lactoglobulin were chosen as model proteins. Five milligrams of each of the model proteins were tested. We initiated screening experiments with MEP and PPA sorbents using two different adsorption buffers, (A) sodium phosphate buffer, 0.05 M, pH 7.4, (B) sodium phosphate buffer, 0.05 M, pH 7.4 with 0.15 M NaCl and, elution with 0.05M sodium acetate, pH 4.0. 2.3. Determination of dynamic binding capacity of lactoferrin Determination of dynamic binding capacity (DBC) was done using lactoferrin. The protein feedstock was prepared with sodium phosphate buffer with or without 0.15 M NaCl containing 25 mg of commercial lactoferrin and was pumped into MEP and PPA column at a flow rate of

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0.25 mL/min. Protein was injected until a breakthrough curve was achieved. The bound proteins were eluted using 50 mM sodium acetate buffer, pH 4.0. After each run, the column was regenerated with 1 M NaOH followed by water wash. 2.4. Preparation of whey Bovine milk colostrum was obtained from a local cattle farmer in a sterile storage container. It was acid stripped (sodium acetate, 0.1 M, pH 4.0) at 4ºC to precipitate the casein and then ultracentrifuged (Beckman coulter) at 30,000 g for 30 min at 4ºC. The soluble supernatant was carefully removed without disturbing the pellet using a Pasteur pipette and was adjusted to pH 7.0 with 1 M NaOH. The whey was dialysed against 0.05 M sodium phosphate buffer (pH 7.0) for a period of 12 h at 4ºC with three changes of buffer and then lyophilized. The prepared powdered protein samples were rehydrated with the equilibration buffer and sterile filtered using 0.22 µ PVDF membrane (Millipore, USA). 2.5. Column preparation and chromatographic experiments Chromatographic experiments were done using a medium pressure pump (Econo™ pump, Bio-Rad, Hercules, CA, USA) with an UV detector set at 280 nm. The chromatographic sorbents MEP HyperCel™ and PPA HyperCel™ were supplied as slurry in 1 M NaCl containing 20% (v/v) ethanol. The columns were packed tightly without any air bubble with an adjustable plunger. The column had a dimension of 1.1 cm I.D. × 4.5 cm with a bed volume of approximately 5 mL. All the experiments were carried out with a constant volumetric flow rate of 2 mL/min (126 cm/h). The dialysed bovine colostrum whey sample was fed into the column. The binding buffers used were phosphate buffer (0.05 M, pH 7.4) without sodium chloride (binding buffer A) and with 0.15 M sodium chloride (binding buffer B). Elution was done by decreasing step pH gradient with buffers, sodium phosphate (0.05 M, pH 6.0); sodium acetate (0.05 M, pH 5.5) and sodium acetate (0.05 M, pH 4.0). The columns were washed thoroughly and equilibrated with the respective buffers, before and after each chromatographic experiment. 2.6. Total protein quantification The total protein concentration of the samples was determined by Bradford method [17], using bovine serum albumin (BSA) as a reference protein. 2.7. Lactoperoxidase assay The concentrated chromatographic fractions were checked for lactoperoxidase activity using TMB/H2O2 as the

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substrate [10]. Briefly, the samples were incubated for 15 min and the reaction was terminated using 0.5 M H2SO4. The end point titer values were determined by measuring the absorbance at 450 nm. Phosphate buffer was used as a blank against each of the test samples. 2.8. SDS-PAGE analysis Chromatographic fractions were analysed by SDS-PAGE (12% acrylamide gels) under non-reducing conditions [18] in a mini PROTEAN system (Bio-rad, USA). The gels were stained with silver nitrate [19]. 2.9. Quantification of lactoferrin and immunoglobulin by ELISA The lactoferrin concentration was determined by sandwich ELISA. Mouse anti-bovine lactoferrin polyclonal antibodies (capture antibody) prepared in 50 mM bicarbonate buffer, pH 9.6 was coated at the concentration of 1 µg/100 µL/well and incubated overnight at room temperature. The plates were washed thrice with wash buffer (0.05 M Tris-base, 0.14 M NaCl, 0.1% Tween20, pH 7.4) and then blocked with the same buffer for 30 min. Bovine lactoferrin (Sigma, USA) was used as a standard protein. Mouse antibovine lactoferrin polyclonal antibody was added to each well, and the mixture was incubated at 37ºC for 1 h. The plates were then washed and secondary goat anti-mouse IgG horseradish peroxidase conjugate (1:3,000) was added to each well. After 1 h of incubation, the plates were washed and tetramethylbenzidine (TMB) /H2O2 substrate solution (Genei, Bangalore, India) was added. After 10 min of incubation, the enzymatic reaction was stopped by the addition of 50 µL of 2M sulphuric acid. The absorbance was measured at 450 nm using a microplate reader (FLUOstar Optima, BMG, Germany). Bovine IgG concentration was determined by competitive ELISA. Protein G affinity purified bovine IgG was quantified by Bradford method and coated overnight on to the ELISA plate with a fixed concentration of 1 µg/100 µL/well. The plates were washed thrice using wash buffer (0.05 M Trisbase, 0.14 M NaCl, 0.1% Tween20, pH 7.4) and then blocked using the same buffer for 30 min. The standard graph was obtained by serially diluting (double dilution) the known concentration of bovine IgG starting with 10 µg, and incubated with the fixed concentration of HRP conjugated secondary anti-bovine IgG as the detection antibody. Then the plates were incubated for 45 min. The plates were washed and TMB/ H2O2 substrate solution was added. After 10 min of incubation, the enzymatic reaction was stopped by the addition of 50 µL of 2M sulphuric acid. The absorbance was measured at 450 nm using a microplate reader (FLUOstar Optima, BMG, Germany). The decrease in the ELISA end point titer indicates higher concentration

Biotechnology and Bioprocess Engineering 20: 148-156 (2015)

of bovine IgG in the sample. The unknown concentration of bovine IgG from each of the chromatographic fraction was determined from the standard graph. 2.10. Immunoblotting The chromatographic fractions were electrophoresed in SDS-PAGE and the proteins were electrophoretically transferred to a nitrocellulose membrane using trans-blot apparatus (Bio-Rad, Hercules, CA, USA). The membrane was blocked with wash buffer (PBS, 0.1% Tween 20) and incubated with mouse anti-lactoferrin polyclonal antibody for 1 h. After extensive washing with PBS-Tween, alkaline phosphatase conjuagated anti-mouse IgG was added and incubated for 1 h. The membrane was washed thoroughly and developed with NBT/BCIP substrate solution prepared in 0.1 M Tris buffer, 0.1 M NaCl, 5 mM MgCl2. 2.11. Computational analysis on mechanism of interactions of 4-mercaptoethyl pyridine The mechanism of interactions between MEP and the surface domains of bovine lactoferrin was evaluated by molecular docking using Auto dock vina [20]. The molecular structure of bovine lactoferrin was retrieved from protein data bank (PDB) with ID number 3RGY. The two ligands, N-acetyl-D-glucosamine (NAG) and lipopolysaccharide (LPS) which were already known to interact with a few surface exposed amino acid residues of bovine lactoferrin in the C-terminal regions were chosen for the docking experiment.

3. Results and Discussion MEP HyperCel™ and PPA HyperCel™ are newly designed industrially scalable chromatographic sorbents having dual behaviour of hydrophobic as well as ionic interaction and are predominantly used in antibody purifications [21]. MEP is a kind of biomimetic hydrophobic ligand with an ionisable head group with an aromatic tail and a thiol group contributing to the selective binding of immunoglobulin species [22]. PPA is an aromatic ligand with an amine group favouring both hydrophobic and electrostatic interaction. The important features of these sorbents are its capacity to adsorb bio molecules at near neutral pH and to desorb it by reducing the pH conditions of mobile phase through electrostatic repulsion. Thus, the mixed mode sorbents interact in different ways like hydrophobic, aromatic, electrostatic and hydrogen bonding based on the affinity domains available on the surface of the proteins. Moreover, mixed mode sorbents are reported to have very high dynamic binding capacity for human immunoglobulin [16].


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Table 1. Comparative table showing the interaction of model whey proteins with MEP and PPA HyperCel in presence or absence of salt in buffer system Buffer system

Bovine lactoferrin (mg) FT

Human IgG (mg)

E

FT

E

BSA (mg) FT

Beta lactoglobulin (mg) E

FT

E

3.8 ± 0.01 0.54 ± 0.04

3.2 ± 0.04

1.5 ± 0.06

4.7 ± 0.03 4.32 ± 0.03 1.05 ± 0.01

3.7 ± 0.03

1.2 ± 0.03

4.2 ± 0.04

1.8 ± 0.03

2.4 ± 0.04

4.5 ± 0.04 0.52 ± 0.03 4.48 ± 0.01

1.4 ± 0.03

2.4 ± 0.04

MEP HyperCel Buffer A 0.05 M Sodium phosphate without 0.15 M NaCl, pH 7.4 Buffer B 0.05 M Sodium phosphate with 0.15 M NaCl) pH 7.4

0.7 ± 0.05 4.14 ± 0.03 0.72 ± 0.01 4.07 ± 0.03

0.84 ± 0.05 4.18 ± 0.04 0.45 ± 0.01

PPA HyperCel Buffer A 0.05 M Sodium phosphate without 0.15 M NaCl, pH 7.4 Buffer B 0.05M Sodium phosphate with 0.15 M NaCl) pH 7.4

1.2 ± 0.02 3.34 ± 0.03 0.24 ± 0.03 4.71 ± 0.04 0.24 ± 0.01

1.74 ± 0.05

3.1 ± 0.04

0.6 ± 0.02

FT: Flowthrough; E: Elution with 0.05 M sodium acetate pH 4.0.

Initially, we carried out the screening experiments with model whey proteins using phosphate buffer (0.05 M, pH 7.4) without sodium chloride (binding buffer A) and with sodium chloride (binding buffer B). The results showed that the lactoferrin was very selective with MEP when compared to PPA (Table 1). Moreover, bovine serum albumin was poorly retained with MEP even in the presence of salt. This corroborates with the study done by Wang et al., [23] where they also show that bovine serum albumin is not retained in MEP. It was observed that the retention of bovine serum albumin with PPA was higher and comparable with human IgG. Also, it was found that the retention of beta lactoglobulin was better with PPA than MEP. The dynamic binding capacity of lactoferrin was studied with MEP and PPA sorbents (data not shown). We found that the MEP resin had better binding capacity for lactoferrin and showed a binding capacity of 20 mg/mL of the sorbent with both buffers A and B. The PPA ligand showed a binding capacity of 17 mg/mL of the sorbent with binding buffer A and 18 mg/mL with binding buffer B. Further, we studied the adsorption behaviour and recovery of lactoferrin with the above mentioned mixed sorbents using the binding buffer A and binding buffer B. The pH was adjusted to 7.4 in both equilibration buffers. Prior to the chromatography, the bovine colostrum whey was prepared by acid precipitation, followed by buffer exchange and repeated dialysis. The colostrum whey sample was adsorbed onto the column using the binding buffers A or B. The proteins were desorbed by step pH gradient (pH 6.0, 5.5, and 4.0).

The adsorption behaviour of whey proteins with the MEP resin differed with respect to binding buffers A and B as shown in the chromatogram (Figs. 1A and 1B). It was clear that with binding buffer A, both lactoferrin and bovine immunoglobulin was retained on the column with its native hydrophobicity whereas other whey proteins, especially bovine serum albumin and lactoglobulin were seen in the non retained fractions (Fig. 1A, Lane 1). With binding buffer A, the MEP resin at pH 5.5 step gradient elution showed only the low molecular weight whey proteins like alpha and beta-lactoglobulins (Fig. 1C, Lane 2). However, with the binding buffer B, the MEP resin at the same pH 5.5 elution showed coelution of lactoferrin and IgG (Fig. 1B, Lane 2). It is also evident that, at pH 4 step gradient elution, most of the strongly retained contaminants came along with the lactoferrin and bovine IgG (Fig. 1B, lane 3). This suggests that the addition of salt increased the hydrophobic complementarities with the ligand and other whey proteins. Similarly, the selectivity of the lactoferrin with the PPA resin was studied with binding buffer A and B. It was found that both the buffers induced predominantly hydrophobic interaction. Figs. 1C and 1D showed that most of the whey proteins were retained on PPA resin. With binding buffer B, the bovine serum albumin was retained more tightly and it was seen in the subsequent NaOH wash along with other whey proteins (Fig. 1D, Lane 4). Hence, the presence of the salt in the buffer made the bovine serum albumin to bind stronger than the lactoferrin and IgG. When compared to the PPA, the recovery of lactoferrin

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Fig. 1. (A) and (B) Chromatogram of separation of bovine lactoferrin and bovine IgG from bovine colostrum whey with MEP HyperCel using two binding buffers A (0.05 M sodium phosphate dibasic) and B (0.05 M sodium phosphate dibasic + 0.15 M NaCl). Experimental conditions: protein loaded ~30 mg; flow rate 2 mL/min; binding buffers A (0.05 M sodium phosphate dibasic, pH 7.4) and B (0.05 M sodium phosphate dibasic + 0.15 M NaCl, pH 7.4); elution is done with decreasing pH step gradient, pH 6.0 (0.05 M sodium phosphate), pH 5.5 (0.05 M sodium acetate), pH 4.0 (0.05 M sodium acetate); column regeneration with 1M NaOH. (C) and (D) Chromatogram of separation of bovine lactoferrin and bovine IgG from bovine colostrum whey with PPA HyperCel™. Experimental conditions: protein loaded ~30 mg; flow rate 2 mL/min; binding buffers A (0.05 M sodium phosphate dibasic, pH 7.4) and B (0.05 M sodium phosphate dibasic + 0.15 M NaCl, pH 7.4); elution is done with decreasing pH step gradient, pH 6.0 (0.05 M sodium phosphate); pH 5.5 (0.05 M sodium acetate); pH 4.0 (0.05 M sodium acetate); column regeneration with 1 M NaOH. (E) Immunoblot of bovine lactoferrin obtained from the chromatographic fractions using MEP Hypercel with binding buffer A.

MEP Hypercel - In the absence of salt Fractions Load Non-retained pH 6 pH 5.5 pH 4 NaOH wash

Total proteina

Lactoferrin (mg)b

Lactoferrin (µg) /total protein (mg)

% Recoveryc

Purification foldd

Bovine IgG (mg)b’

30.9 ± 0.1

2.4 ± 0.2

77.92 ± 0.45

100

1

10.08 ± 0.27

13.04 ± 0.14

0.12 ± 0.01

8.75 ± 0.31

0.0275 ± 0.003

0

0 7 ± 0.7

5 ± 0.46 0

5.9 ± 0.2

0.0425 ± 0.005

8.65 ± 0.25

2.195 ± 0.015

229.5 ± 1.06

1.2 ± 0.1

0.0149 ± 0.002

0.0148 ± 0.016

0.62 ± 0.13 100

0.112 ± 0.007 0

1.72 ± 0.017 0

1.765 ± 0.215

0.089 ± 0.012

0.14 ± 0.114

91.45 ± 0.135

2.94 ± 0.04

7.62 ± 0.072

0.015 ± 0.003

Bovine IgG (µg) % Recoveryc’ /total protein (mg) 326.34 ± 0.37 131.94 ± 0.12 0 23.72 ± 0.058

100 17.087 ± 0.11 0 1.4

Purification foldd’ 1 0.405 ± 0.008 0 0.07 ± 0.01

881.14 ± 0.2

76 ± 0.044

2.7 ± 0.07

0.317 ± 0.048

265.54 ± 0.035

3.1 ± 0.07

9.81 ± 0.15

317.5 ± 0.044

100

1.13 ± 0.068

74.38 ± 0.008

11.5 ± 0.154

0.23 ± 0.028

0.14 ± 0.005

528.3 ± 0.21

1.7 ± 0.078

1.66 ± 0.22

0.83 ± 0.2

MEP Hypercel - In the presence of salt Load Non-retained

31.05 ± 0.35

2.595 ± 0.065

83.56 ± 0.81

15.2 ± 0.4

0.081 ± 0.004

5.3 ± 0.07

pH 6

0.265 ± 0.065

pH 5.5

6.095 ± 0.205

pH 4 NaOH wash

0

7.95 1.4 ± 0.3

0

3.12 ± 0.075 0

1 0.063 ± 0.001 0

0.89 ± 0.02

146.24 ± 5.83

34.29 ± 1.62

1.75 ± 0.125

2.76 ± 0.038

454.84 ± 0.035

1.595 ± 0.015

200.73 ± 4.01

61.46 ± 0.96

2.406 ± 0.0355

5.44 ± 0.18

684.76 ± 0.014

0.13 ± 0.001

0.29 ± 0.024

208.31 ± 0.04

0.135 ± 0.005

11 ±.141

5.2 ± 0.325

2.475 ± 0.045

78.91 ± 0.31

100

28.2 55.45 ± 0.082 2.9± 0.14

1

1.43 ± 0.052 2.17 ± 0.07 0.65 ± 0.31

PPA Hypercel – In the absence of salt Load Non-retained pH 6 pH 5.5 pH 4 NaOH wash

30.6 ± 0.4 9 ± 0.1

19.87 ± 1.31

1

0.48 ± 0.04

3.885 ± 0.18

0.64 ± 0.06

0.0365 ± 0.002

5.75 ± 0.67

10.25 ± 0.25

1.145 ± 0.025

111.8 ± 3.67

47.41 ± 0.15

8.95 ± 0.15

0.765 ± 0.045

85.4 ± 2.54

31.61 ± 1.27

0.915 ± 0.135

0.018 ± 0.003

16.24 ± 0.6

0.62 ± 0.11

0.205 ± 0.01

2.232 ± 0.048

73.084 ± 0.67

1.51 ± 0.075

0.049 ± 0.003

10.77 ± 0.085

100

1

177.3 ± 0.22

14.61 ± 0.022

0.072 ± 0.04

113.58 ± 0.31

6.7 ± 0.15

0.34 ± 0.2

1.416 ± 0.06

3.78 ± 0.21

366.72 ± 0.052

36.02 ± 0.035

1.039 ± 0.044

1.082 ± 0.035

5.81 ± 0.031

652.3 ± 0.028

54.11 ± 0.092

1.82 ± 0.31

0.34 ± 0.014

372.3 ± 0.17

0.073 ± 0.011

1.57 ± 0.034

352 ± 0.076

3.1 ± 0.164

0.5 ± 0.14

1.056 ± 0.024

PPA Hypercel – In presence of salt Load

30.54 ± 0.26

Non-retained

0.73 ± 0.008

pH 6

0.38 ± 0.006

pH 5.5 pH 4 NaOH wash

0.24 ± 0.025 0

11.23 ± 2.008 0

100 10.75 ± 1.10 0 76.61 ± 0.3

11.17 ± 0.062

367.52 ± 0.08

100

0.153 ± 0.019

1

0.37 ± 0.017

506.44 ± 0.28

3.35 ± 0.018

0.1 ± 0.4

0.018 ± 0.024

91.27 ± 0.064

0.164 ± 0.014

1 1.37 ± 0.0162 0.261 ± 0.1

10.98 ± 0.15

1.71 ± 0.03

156.1 ± 0.42

2.13 ± 0.023

2.67 ± 0.16

245.87 ± 0.17

23.94 ± 0.054

0.68 ± 0.12

8.55 ± 0.35

0.207 ± 0.012

24.21 ± 2.67

9.27 ± 0.74

0.33 ± 0.032

7.75 ± 0.21

906.43 ± 0.072

70.04 ± 0.11

2.46 ± 0.04

8.9 ± 0.2

0.049 ± 0.007

5.5 ± .45

2.19 ± 0.36

0.0752 ± 0.010

0.145 ± 0.32

66.84 ± 0.038

1.34 ± 0.036


Table 2. Performance of MEP Hypercel and PPA Hypercel in presence of different buffer (phosphate buffer saline and phosphate buffer)

0.19 ± 0.011

a

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Determined by Bradford assay. b, b’ Determined by ELISA. c The yield is determined as a ratio of lactoferrin present in the eluates to the total lactoferrin present in the crude. c’ The yield is determined as a ratio of bovine IgG present in the eluates to the total bovine IgG present in the crude. d Lactoferrin to protein mass ratio after purification/ lactoferrin to protein mass ratio in crude. d’ Bovine IgG to protein mass ratio after purification/ bovine IgG to protein mass ratio in crude.

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using MEP resin with buffer A was higher (91.45 ± 0.135%) with a purification fold of 2.94 ± 0.04. A recovery of 76 ± 0.044% of bovine IgG was obtained with MEP resin using binding buffer A. However, a recovery of only 76.61 ± 0.3 of lactoferrin and 70.04 ± 0.11% of bovine IgG was obtained with PPA resin with binding buffer B (Table 2). A recent study by Du et al., [24,25] described linear salt gradient elution method for the recovery of lactoferrin and IgG in a single step using a sequential expanded bed packed with cationic exchanger (Fastline SP) and mixed mode resin (Streamline Direct CST-1). Similarly, a study on the fractionation of ideal whey proteins with three mixed mode sorbents (MEP, PPA, HEA HyperCel) showed the separation of β-lactoglobulin from α-lactoglobulin. However, the separation of lactoferrin and IgG was not reported in their study [26]. Here in our study, we have optimized a pH step gradient elution method for the recovery of lactoferrin and IgG in a single step using MEP HyperCel™. MEP resins are economically feasible for downstream purification of nutraceuticals (lactoferrin, IgG) as utilization of the salt


free systems are more advantageous over the ion exchange/ hydrophobic chromatography based purification system where the requirement of salts in the buffer is higher [27,28]. The recovery of lactoferrin along with bovine immunoglobulin with high purity gives an added advantage and could attract its application as a nutraceutical [29,30]. We confirmed the recovered protein as lactoferrin molecule by immunoblot analysis with mouse anti-lactoferrin antibody. A band at 80kD corresponding to lactoferrin was observed (Fig. 1E, Lanes L and 3). The result of this immunological assay has confirmed that the chromatographic conditions that were employed in this study ensured the recovery of the intact lactoferrin molecule. The chromatographic fractions were analyzed for lactoperoxidase activity since the molecular weight of lactoperoxidase is close to lactoferrin. The analysis of chromatographic fractions showed that the MEP ligand did not favour the adsorption of lactoperoxidase in both binding buffer A and B, whereas PPA ligand showed better adsorption and the lactoperoxidase retained even stronger

Fig. 2. The binding modes of MEP ligand on cavity 1 and 2 are shown in Figs. 2A and 2C. The MEP ligands are shown in thick line and surrounding amino acid residues are shown in thick stick. Here the surface of the protein is displayed in a transparent mode. The two dimension planar mode of the ligand (MEP) interacting with the cavity 1, 2 are displayed using Ligplot as shown Figs. 2B and 2D.


in the presence of salts in the binding buffer (data not shown). In our MEP-lactoferrin docking analysis, we docked ligand MEP with two surface cavities seen on the C-terminal region of lactoferrin, which is already known to interact with lipopolysaccharide (LPS) and N-acetyl galactosamine (NAG). We found that the ligand MEP interacted with few amino acid residues on cavities present in LPS and NAG. MEP interacted with lactoferrin in the LPS cavity region with the amino acids, Pro 409, Val 410, Leu 651, Arg 654, and Pro 655, and in the NAG cavity region with Gln 615, Ala 616, Gly 619, Gly 622, Lys 620, Asn 621, Glu 646 and Cys 647. All of the interacting residues with the ligand showed hydrophobic interactions and among them, one residue Glu 646 in the NAG cavity showed hydrogen bonding with the nitrogen atom of the ligand with bond length of 2.73Å. The ligand (MEP) showed a binding energy value of -5.14 and -4.64 kcal/mol with the two surface cavities selected from the C-terminal lobe of lactoferrin. The three dimensional interaction of MEP with the amino acids seen on the surface cavities of LPS and NAG are shown in Figs. 2A and 2C. The two dimensional interaction of the ligand with the cavities of LPS and NAG are shown in the Lig plot in Figs. 2B and 2D.

4. Conclusion The sodium phosphate buffer without salt gave higher purity and recovery of lactoferrin with the experiments carried out with MEP resin. The band corresponding to the bovine lactoferrin showed specific reactivity to mouse antilactoferrin antibody by immunoblotting. The molecular docking study revealed that the interaction of MEP ligand with bovine lactoferrin is mainly due to non-covalent interaction involving hydrophobic and hydrogen bonding. Further studies are ongoing in molecular mechanism of interaction of lactoferrin on to the synthetic ligands like MEP to understand the purification process and to develop newer process analytical techniques for large scale purification. Thus, mixed mode sorbents like MEP HyperCel™ could be used as a new alternative purification system in the milk processing and pharmaceutical industry towards the existing methods like ion-exchange, affinity and membrane based systems.

Acknowledgements The first author thanks the council of scientific and industrial research (CSIR) for providing the senior research fellowship. The authors thank the Department of Science

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and Technology, Government of India (DST), for funding. The authors also thank Mr. Bavani Prasad Vipperla, VIT University, Vellore, for helping us in docking studies.

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