Lysozyme From Egg White Using Chromatographic Technique

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Protein purification is a process of obtaining a homogenous sample of protein of interest, retaining its original functional status. Chromatography is one of the most important techniques of biotechnology to purify therapeutic proteins from complex mixtures. (Shukla et al. 2007). Basically, there are two modes of chromatography: 1) bind/elute mode and 2) flow through mode. In bind/elute mode, target protein is bound and later eluted isocratically from the adsorbent. Whereas, in flow through mode, unwanted impurities bind to the adsorbent; and target species flow through the column. In bind/elute mode, numerous impurities may co-adsorb with the target product, and these impurities must be desorbed either by a wash step prior to product elution or allowed to remain adsorbed till the target species has been eluted. The similarity of the binding properties of the target product and impurities makes the process to chromatographic separation very challenging.

There are different forms of chromatographic techniques employed for protein purification. They all exploit the differences in the binding properties between the target product and the impurities. Out of these various techniques, ion -exchange chromatography is the method of separation of molecules according to the charge. Ion exchange resins like Carboxymethyl Sepharose (CM Sepharose) are cationic exchanger that traps positively charged molecules. Thus, positively charged proteins are bound to the CM sepahrose and are released by altering the ionic strength of the eluting buffer. (McCue, Engel and Thömmes 2009)

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Column chromatography can be carried out in gradient or displacement mode for protein purification (Yamamoto, Nomura and Sano 1987). Generally, this technique uses the increasing concentration of salts for elution. Alternatively, displacement column chromatography can be performed by subjecting sequential changes in inlet conditions of the column. For this, the column is first of all brought in equilibrium using a carrier of low ionic strength, which is later on introduced with a feed pulse under conditions of high retention. This is followed by a constant infusion of a displacer solution of higher dynamic affinity for stationary phase of the column. This higher affinity solution competes with the previously fed solutes for the adsorption sites on the stationary phase. Under optimum condition, the displacer solution induces the feed components to develop the adjacent "square wave" zones of concentrated and purified material, which passes out of the column before the displacer solution. (Barnthouse et al. 1998).

In this experiment, an attempt to purify lysozyme from egg white using CM Sepharose- based- ion-exchange chromatography has been carried out. Egg white consists of a complex mixture of varieties of proteins like ovalbumin, lysozyme, conalbumin, ovotransferrin , flavoprotein, G2 globulin and G3 globulin. Out of them, lysozyme is a low molecular weight protein (Molecular weight 14.3 kDa), which is a commercially important enzyme used as an effective antibacterial agent, a food additive and a precursor of the drug against ulcer and infections. (Yilmaz, Bayramoglu and ArIca 2005). This experiment has been aimed to demonstrate the practical application of CM Sepaharose resins on isolation of lysozyme from whole egg white. Here lysozyme has been selected as a basic protein and pH has been maintained at 9.2 using Tris buffer along with sodium chloride.

An end- point- colorimetric assay based on Beer and Lambert's law can be used to determine the concentration of protein sample by treating them with coomassie blue. Measuring the change on the absorbance of colour and comparing it with the colour produced by a standard protein solution of known concentration, the protein concentration of unknown sample is determined. Similarly, the lysozyme activity can be assayed by treating the sample with micrococcus suspension, and carrying out a fixed-time -kinetic assay. When lysozyme acts on micrococcus, it hydrolyses the peptidoglycan present on the cell wall of the bacteria. This process is monitored by clearing of the micrococcus suspension and gradual decrease on the absorbance of the reaction mixture.

SDS polyacrylamide gel electrophoresis (SDS-PAGE) is a technique to separate protein components on the basis of size, from a complex biological sample. The protein components can be visualised using a staining technique that will make the individual bands of protein more clearly visible. The aim of SDS-PAGE assay in this experiment is to confirm the purity of the lysozyme obtained by ion-exchange chromatography.

Aim

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To fractionate protein sample using column chromatograpy

To analyse protein samples using assays of activity and protein concentration

To compare protein samples using SDS-PAGE

Materials and Methods

In order to accomplish the target of this work, egg white was prepared at first. For this, an egg was cracked, and yolk part was discarded. The egg white was filtered in two layers of cheese cloth to get 5 ml of egg white which was mixed with 75 ml of buffer A, thus making 1: 16 dilution. This mixture was passed through a loose plug of glass wool to get clear filtrate called "egg white extract". It was labelled as Sample A.

In the second step of the experiment, Ion exchange column chromatography was carried out, using Carboxymethyl (CM) Sepharose resins at pH 9.2. For this, the column was, first of all, adjusted at the flow rate of 1ml/min and the buffer and elutant were checked to ensure that the required pH has been maintained. Concurremtly, 40 test tubes were taken and marked with a line for 2 ml capacity. Using a clean loading syringe, 5 ml of the egg white sample was transferred to the column and elutatant was collected, which was labelled as "column flow through".

Two buffers viz buffer A and buffer B were used in this experiment where buffer A was made up of the combination of 0.05 M Tris buffer with 0.05 M sodium chloride at pH 9.2. Similarly buffer B was the combination of 0.05 M Tris buffer with 1.00 M sodium chloride at pH 9.2. Before setting the gradient maker, the column was washed with 10 ml of buffer A. After this, the gradient maker was set by keeping 20 ml each of buffer A and B in two interconnected chambers and mixing them by a magnet stirrer. When the mixture of the buffer A and B was allowed to pass through the column, 2 ml fractions were collected on the tubes until the gradient ran out. During this experiment, 16 fractions were collected and their absorbance was measured at 280 nm. A graph was plotted keeping the all of these fractions against their absorbances (graph 1). Graph 1 showed that out of the 16 fractions, fractions 7, 8, 9 and 10 were seem to have distinctly higher protein concentration and they were used for the determination of protein concentration , specific lysozyme activity and SDS-PAGE assay.

For the determination of the protein concentration, 12 labelled eppendorf tubes were taken and loaded with standard protein (1mg/mL) and unknown proteins as mentioned on table 1.

Table 1: Protocol design for the determination of protein concentration of unknown samples, using standard protein

Contents

Tube Number

1

2

3

4

5

6

7

8

9

10

11

12

Standard Protein (1 mg/mL)(µL)

0

10

20

30

40

50

-

-

-

-

-

-

Unknown Protein(µL)

50

50

50

50

50

50

Water (µL)

50

40

30

20

10

0

-

-

-

-

-

-

(Note: Tube 7, 8, 9 and 10 contained fraction 7, 8, 9 and 10 respectively. Tubes 11 and 12 contained egg white and egg extract samples)

From each of the eppendorf tubes of table 1, 20µL of sample was taken and mixed with 1 ml of coomassie blue reagent. After mixing well, the preparation was incubated for 5 minute at room temperature and measured the absorbance at 595nm in colorimeter. The findings were plotted in graph and the protein concentration of unknown samples was calculated from the known standards. (as mentioned in graph 2)

After determining the protein concentration, lysozyme activity was assayed by mixing 40 µL of each of fractions 7, 8, 9, and 10 and egg white samples with 1 mL of micrococcus suspension and taking absorbance readings at 30 sec interval for 5 minutes, at 450 nm. Different absorbance readings obtained at different intervals for different fractions were plotted in graph against time. (Graph 3 to 12). The slope of the graph of individual sample was determined and lysozyme activity of individual sample was calculated as shown in table 3 and graphs 3 to 12.

Finally, 100µL of undiluted samples of fractions 7, 8, and 10 and 1:3 diluted sample of fraction 9 were taken and each of them were mixed with 100 µL of sample buffer. Similarly, 1:140 diluted egg white sample was mixed with 141 µL of sample buffer. These all preparations were boiled for 10 minute followed by centrifugation for 5 min at top speed. Using the protein concentration that have been already determined, it was calculated that 20µL of each of these samples (i.e. fraction 7,8, and 10 undiluted and fraction 9 and egg white diluted to 1:3 and 1: 140 respectively) are required to be loaded to retrieve 200ng of protein on each case. Thus, after loading 20 µL of each of these samples (along with marker) on gel, SDS-PAGE assay was carried out. The bands obtained were stained with dye and results were recorded.

Results

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Protein concentrations of egg white and fraction 7, 8, 9 and 10 were found to be 1.06mg/mL, 0.11mg/mL,0.08 mg/mL, 0.30 mg/mL and 0.11 mg/mL respectively. Similarly, lysozyme activity of these fractions was determined to be 8500 U/mL, 1287.5 U/mL, 2925 U/mL, 5750 U/mL and 3137.5 U/mL respectively.

Table 2: Protein concentration

Sample

Volume

(µL)

Dilution

Concentration of diluted sample(mg/mL)

Concentration before dilution (mg/mL)

Total amount of protein in sample (mg)

% of initial protein recovered

Egg white

5

1:1

1.06

1.06

5.30

100.00

Fraction 7

2

1:1

0.11

0.11

0.22

4.15

Fraction 8

2

1:1

0.08

0.08

0.16

3.02

Fraction 9

2

1:1

0.30

0.30

0.60

11.30

Fraction 10

2

1:1

0.11

0.11

0.22

4.15

Table 3: Lysozyme activity

Sample

Volume (mL)

Dilution

Activity of diluted sample(U/mL)

Activity of sample before dilution (U/mL)

Total activity (U)

% Initial activity recovered

Specific activity

(U/mg)

Egg white

5

1:1

8500.0

8500.0

42500.0

100

8019

Fraction 7

2

1:1

1287.5

1287.5

2575.0

6.0

11705

Fraction 8

2

1:1

2925.0

2925.0

5850.0

13.8

36563

Fraction 9

2

1:1

5750.0

5750.0

11500.0

27.1

19167

Fraction 10

2

1:1

3137.5

3137.5

6275.0

14.8

28523

Marker Egg white Fraction 7 Fraction 8 Fraction 9 Fraction 10

Fig.1: Output of SDS-PAGE showing the band of lysozyme against the protein marker (Chromatographic assay done at pH 9.2)

Discussion

Purification of lysozyme from egg white using ion exchange column chromatography and verification of the purity by biochemical and molecular technique was the sole aim of this experiment. Lysozyme, which is a positively charged protein, was made to bind with the cationic CM sepharose resins. It was followed by constant infusion of a displacer solution containing sodium chloride along with Tris buffer. The Na+ ions present on sodium chloride has higher dynamic affinity for the binding sites of CM sepahrose. Due to this higher affinity, Na+ competes with the previously fed lysozyme for the adsorption sites and causes the elution of lysozyme. Graph 1 shows that out of 16 fractions of the chromatographic assay, only fractions 7, 8, 9 and 10 shows potential lysozyme activity. Due to the potential similarities on the binding properties of lysozyme and its contaminant proteins, it was mandatory to determine specific lysozyme activity using micrococcus suspension. Lysozyme activity assay is based on the principle that when lysozyme acts on micrococcus, it hydrolyses the peptidoglycan present on the cell wall of the bacteria resulting in gradual decrease on the absorbance of the reaction mixture. This is the reason, why the decreasing curve of activity of enzyme has been seen on graphs 3 to 12. This assay indicated that major quantity of lysozyme was present on fraction 9 of chromatographic elutants. It means that the moment of elution of fraction 9 was the major "knock out" of lysozyme by the Na+ ions.

These results were further verified by SDS-PAGE assay. Figure 1 shows the distinct band of lysozyme of 14.2 kDa against the marker protein. Absence of any band above the level of 14.2 kDa indicates that the chromatographic purification assay was absolutely precise. Absence of lysozyme band and presence of few bands of impurities on the lane of fraction 10 (of Fig.1) imply that after the elution of fraction 9, the lysozyme content must have been absolutely cleared off the column. Consequently, the impurities that were eluted after fraction 9 were mixed in the fraction 10 of the elutant.

Conclusion

Lysozyme was successfully fractionallised and purified from egg white sample using CM sepharose based column chromatography at pH 9.2. The concentration and activity of the enzyme was determined to assess its purity, which was further confirmed, successfully, by SDS-PAGE based assay. These results indicated that the entire process of purification of lysozyme from egg white sample was quite efficient and productive.