Lysozyme From Egg White Using Chromatographic Technique

Protein purification is a procedure of obtaining a homogeneous sample of protein of involvement, retaining its original functional position. Chromatography is one of the most of import techniques of biotechnology to sublimate curative proteins from complex mixtures. ( Shukla et al. 2007 ) . Basically, there are two manners of chromatography: 1 ) bind/elute manner and 2 ) flow through manner. In bind/elute manner, mark protein is bound and subsequently eluted isocratically from the adsorbent. Whereas, in flow through manner, unwanted drosss bind to the adsorbent ; and aim species flow through the column. In bind/elute manner, legion drosss may co-adsorb with the mark merchandise, and these drosss must be desorbed either by a wash measure prior to merchandise elution or allowed to stay adsorbed till the mark species has been eluted. The similarity of the binding belongingss of the mark merchandise and drosss makes the procedure to chromatographic separation really ambitious.

There are different signifiers of chromatographic techniques employed for protein purification. They all exploit the differences in the binding belongingss between the mark merchandise and the drosss. Out of these assorted techniques, ion -exchange chromatography is the method of separation of molecules harmonizing to the charge. Ion exchange rosins like Carboxymethyl Sepharose ( CM Sepharose ) are cationic money changer that traps positively charged molecules. Therefore, positively charged proteins are bound to the CM sepahrose and are released by changing the ionic strength of the eluting buffer. ( McCue, Engel and Thommes 2009 )

Column chromatography can be carried out in gradient or supplanting manner for protein purification ( Yamamoto, Nomura and Sano 1987 ) . By and large, this technique uses the increasing concentration of salts for elution. Alternatively, displacement column chromatography can be performed by subjecting consecutive alterations in recess conditions of the column. For this, the column is foremost of all brought in equilibrium utilizing a bearer of low ionic strength, which is subsequently on introduced with a provender pulsation under conditions of high keeping. This is followed by a changeless extract of a displacer solution of higher dynamic affinity for stationary stage of the column. This higher affinity solution competes with the antecedently fed solutes for the surface assimilation sites on the stationary stage. Under optimal status, the displacer solution induces the provender constituents to develop the next “ square moving ridge ” zones of concentrated and purified stuff, which passes out of the column before the displacer solution. ( Barnthouse et al. 1998 ) .

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

An end- point- colorimetric check based on Beer and Lambert ‘s jurisprudence can be used to find the concentration of protein sample by handling them with coomassie blue. Measuring the alteration on the optical density of coloring material and comparing it with the coloring material produced by a standard protein solution of known concentration, the protein concentration of unknown sample is determined. Similarly, the muramidase activity can be assayed by handling the sample with micrococcus suspension, and transporting out a fixed-time -kinetic check. When lysozyme Acts of the Apostless on Micrococcus, it hydrolyses the peptidoglycan nowadays on the cell wall of the bacterium. This procedure is monitored by glade of the Micrococcus suspension and gradual lessening on the optical density of the reaction mixture.

SDS polyacrylamide gel cataphoresis ( SDS-PAGE ) is a technique to divide protein constituents on the footing of size, from a complex biological sample. The protein constituents can be visualised utilizing a staining technique that will do the single sets of protein more clearly seeable. The purpose of SDS-PAGE check in this experiment is to corroborate the pureness of the muramidase obtained by ion-exchange chromatography.

Purpose

To fractionate protein sample utilizing column chromatograpy

To analyze protein samples utilizing checks of activity and protein concentration

To compare protein samples utilizing SDS-PAGE

Materials and Methods

In order to carry through the mark of this work, egg white was prepared at first. For this, an egg was cracked, and yolk portion was discarded. The egg white was filtered in two beds of cheese fabrics to acquire 5 milliliter of egg white which was assorted with 75 milliliters of buffer A, therefore doing 1: 16 dilution. This mixture was passed through a loose stopper of glass wool to acquire clear filtrate called “ egg white infusion ” . It was labelled as Sample A.

In the 2nd measure of the experiment, Ion exchange column chromatography was carried out, utilizing 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 guarantee that the needed pH has been maintained. Concurremtly, 40 trial tubings were taken and marked with a line for 2 milliliter capacity. Using a clean burden syringe, 5 milliliter 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 Na chloride at pH 9.2. Similarly buffer B was the combination of 0.05 M Tris buffer with 1.00 M Na chloride at pH 9.2. Before puting the gradient shaper, the column was washed with 10 milliliters of buffer A. After this, the gradient shaper was set by maintaining 20 ml each of buffer A and B in two interrelated Chamberss and blending them by a magnet scaremonger. When the mixture of the buffer A and B was allowed to go through through the column, 2 milliliter fractions were collected on the tubings until the gradient ran out. During this experiment, 16 fractions were collected and their optical density was measured at 280 nanometer. A graph was plotted maintaining the all of these fractions against their optical densities ( chart 1 ) . Graph 1 showed that out of the 16 fractions, fractions 7, 8, 9 and 10 were seem to hold clearly higher protein concentration and they were used for the finding of protein concentration, specific muramidase activity and SDS-PAGE check.

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

Table 1: Protocol design for the finding of protein concentration of unknown samples, utilizing standard protein

Contentss

Tube Number

1

2

3

4

5

6

7

8

9

10

11

12

Standard Protein ( 1 mg/mL ) ( AµL )

0

10

20

30

40

50

Unknown Protein ( AµL )

50

50

50

50

50

50

Water ( AµL )

50

40

30

20

10

0

( Note: Tube 7, 8, 9 and 10 contained fraction 7, 8, 9 and 10 severally. Tube 11 and 12 contained egg white and egg infusion samples )

From each of the eppendorf tubings of table 1, 20AµL of sample was taken and assorted with 1 milliliters of coomassie bluish reagent. After blending good, the readying was incubated for 5 minute at room temperature and measured the optical density at 595nm in tintometer. The findings were plotted in graph and the protein concentration of unknown samples was calculated from the known criterions. ( as mentioned in graph 2 )

After finding the protein concentration, lysozyme activity was assayed by blending 40 AµL of each of fractions 7, 8, 9, and 10 and egg white samples with 1 milliliters of Micrococcus suspension and taking optical density readings at 30 sec interval for 5 proceedingss, at 450 nanometer. Different optical density readings obtained at different intervals for different fractions were plotted in graph against clip. ( Graph 3 to 12 ) . The incline of the graph of single sample was determined and lysozyme activity of single sample was calculated as shown in table 3 and graphs 3 to 12.

Finally, 100AµL of undiluted samples of fractions 7, 8, and 10 and 1:3 diluted sample of fraction 9 were taken and each of them were assorted with 100 AµL of sample buffer. Similarly, 1:140 diluted egg white sample was assorted with 141 AµL of sample buffer. These all readyings were boiled for 10 minute followed by centrifugation for 5 min at top velocity. Using the protein concentration that have been already determined, it was calculated that 20AµL 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 severally ) are required to be loaded to recover 200ng of protein on each instance. Thus, after lading 20 AµL of each of these samples ( along with marker ) on gel, SDS-PAGE check was carried out. The sets obtained were stained with dye and consequences were recorded.

Consequences

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 severally. 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 severally.

Table 2: Protein concentration

Sample

Volume

( AµL )

Dilution

Concentration of diluted sample ( mg/mL )

Concentration before dilution ( mg/mL )

Entire sum of protein in sample ( milligram )

% 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 ( milliliter )

Dilution

Activity of diluted sample ( U/mL )

Activity of sample before dilution ( U/mL )

Entire 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: End product of SDS-PAGE demoing the set of muramidase against the protein marker ( Chromatographic assay done at pH 9.2 )

Discussion

Purification of muramidase from egg white utilizing ion exchange column chromatography and confirmation of the pureness by biochemical and molecular technique was the exclusive purpose of this experiment. Lysozyme, which is a positively charged protein, was made to adhere with the cationic CM sepharose rosins. It was followed by changeless extract of a displacer solution incorporating Na chloride along with Tris buffer. The Na+ ions present on Na chloride has higher dynamic affinity for the binding sites of CM sepahrose. Due to this higher affinity, Na+ competes with the antecedently fed muramidase for the surface assimilation sites and causes the elution of muramidase. Graph 1 shows that out of 16 fractions of the chromatographic check, merely fractions 7, 8, 9 and 10 shows possible lysozyme activity. Due to the possible similarities on the binding belongingss of muramidase and its contamination proteins, it was compulsory to find specific muramidase activity utilizing micrococcus suspension. Lysozyme activity check is based on the rule that when lysozyme Acts of the Apostless on Micrococcus, it hydrolyses the peptidoglycan nowadays on the cell wall of the bacteriums ensuing in gradual lessening on the optical density of the reaction mixture. This is the ground, why the decreasing curve of activity of enzyme has been seen on graphs 3 to 12. This check indicated that major measure of muramidase was present on fraction 9 of chromatographic elutants. It means that the minute of elution of fraction 9 was the major “ knock out ” of muramidase by the Na+ ions.

These consequences were farther verified by SDS-PAGE check. Figure 1 shows the distinguishable set of muramidase of 14.2 kDa against the marker protein. Absence of any set above the degree of 14.2 kDa indicates that the chromatographic purification check was perfectly precise. Absence of lysozyme set and presence of few sets of drosss on the lane of fraction 10 ( of Fig.1 ) imply that after the elution of fraction 9, the muramidase content must hold been perfectly cleared off the column. Consequently, the drosss that were eluted after fraction 9 were mixed in the fraction 10 of the elutant.

Decision

Lysozyme was successfully fractionallised and purified from egg white sample utilizing CM sepharose based column chromatography at pH 9.2. The concentration and activity of the enzyme was determined to measure its pureness, which was further confirmed, successfully, by SDS-PAGE based check. These consequences indicated that the full procedure of purification of muramidase from egg white sample was rather efficient and productive.

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