讲解 BCMB20005 Purification of lysozyme using ion-exchange chromatography辅导数据结构语言

Purification of lysozyme using ion-exchange chromatography

Aims

The experiment aims to isolate the protein lysozyme from diluted hen egg white using cation-exchange chromatography and to evaluate the effectiveness of the purification through various analytical techniques. The purified fractions will be assessed for protein purity using SDS-PAGE, and the total protein concentration will be quantified using a colorimetric assay. These results will be used to construct a purification profile and to evaluate the efficiency of the isolation process.

Materials and Methods

Ion-Exchange Purification of Lysozyme

Hen egg white (fraction A) was subjected to cation-exchange chromatography using CM-Sepharose resin in 0.025 M sodium bicarbonate buffer, pH 9.0 (buffer 1) to achieve binding of positively charged proteins to the negatively charged resin on pages 197-200 in the BCMB LM. Proteins not bound to the resin were removed (Fraction B), and the resin was washed twice with Buffer 1 (producing Fractions C and D) prior to elution of bound protein in Buffer 2 (0.025 M sodium carbonate, pH 10.4, 0.35 M NaCl). Eluted proteins were subjected to centrifugal ultrafiltration using an Amicon Ultra-15 Centrifugal Filter Unit in Buffer 3 (0.025 M sodium acetate, pH 5.0) to achieve buffer exchange to stabilize lysozyme and reduce the volume of the purified protein (Fraction E).

Protein Crystallization

Vapour diffusion crystallization was used to crystallize purified lysozyme, following the procedure outlined on pages 201-202 in the BCMB LM. Two buffers were employed: Buffer A (1 M NaCl, 100 mM sodium acetate, pH 4.5) and Buffer B (0.97 M NaCl, 97 mM sodium acetate, pH 4.5 with blue dye). The protein droplet contained 0.12 M NaCl, 12.1 mM sodium acetate. For crystallization setup, 35 mL of protein sample was mixed with 5mL of Buffer B (7:1 ratio), and four conditions were tested using Fraction E and its 1/10 dilution, as well as commercial lysozyme (50 mg/mL) and its 1/10 dilution (5 mg/mL) as positive controls. After loading the droplets onto the Petri dish lid, 2.5 mL of Buffer A was added to the dish base, and the dish was sealed with paraffin film and stored at 4 for crystallization.

SDS-PAGE

Samples were applied to 4-20% (w/v) gradient polyacrylamide gel for separation by electrophoresis, as described on pages 206-207 in the BCMB LM. Gels were run at 150 V for 45-50 minutes in Tris-glycine SDS running buffer (25 mM Tris pH 8.4 192 mM glycine, 0.1% (w/v) SDS). Prior to loading, protein fractions were denatured in reducing denaturation buffer (60 mM Tris pH 7.5, 2% (w/v) SDS, 0.04% (w/v) bromophenol blue, 10% (v/v) glycerol, 100 mM DTT) by boiling for 5 minutes. Visualization of protein bands was achieved by overnight staining with 0.1% (w/v) Coomassie Brillant Blue R250 in 50% (v/v) ethanol and 7.5% (v/v) acetic acid, followed by destaining by in 7.5 (v/v) acetic acid.

Lowry Assay

Protein concentrations were determined using the Lowry method, as described on pages 215-219 in the BCMB LM. A six-point BSA standard curve (0-40 mg) was used. Each reaction contained 0.2 mL sample or standard, 1.0 mL of Lowry copper reagent and 0.1 mL Folin-Ciocalteu reagents, which with measurements taken at 750 nm after 10 minutes incubation. Absorbance was read using standard cuvettes and an Implen C40 spectrophotometer. Protein concentrations were calculated from a second-order polynomial standard curve.

Lysozyme Activity Assay

Lysozyme activity in Fraction A-D and 1/5 dilution of Fraction E was measured using turbidity assay, as described on pages 208-210 in the BCMB LM. Samples (50 mL for Fraction B-D. 20 mL for Fraction A and 1/5 diluted Fraction E) were added to 1.0 mL of bacterial suspension containing 0.2mg/mL Micrococcus luteus in 20 mM phosphate buffer (pH 6.2. 150 mM NaCl). Absorbance at 540 nm was recorded every 5 seconds for 1 minute using Sarstedt standard cuvettes and an Implen spectrophotometer. Water was used as a reagent blank. Lysozyme activity was quantified based on the rate of absorbance decrease, reflecting the lysis of bacterial cells by the enzyme.

Results

Crystallization of lysozyme

Crystallization of protein depended on purity of protein and presence of contaminant therefore it was a technique to examine of protein. The crystallization outcomes are shown in Figure 1.

Figure 1. Image of crystallization. Figure shows the crystallization outcome for four lysozyme samples: Droplet 1 (Commercial lysozyme, 50 mg/mL), droplet 2 (1/10 diluted commercial lysozyme, 5 mg/mL), droplet 3 (Fraction E) and droplet 4 (1/10 diluted fraction E). All sample were stained with E133 blue food coloring.

The appearance of crystals in each droplet varied significantly. Observations were made under a microscope and recorded based on crystal number, size, density and spatial distribution. A summary of these observations is presented in Table 1 below.

¹Droplet	²Crystal size	³Crystals quantity	Crystal density	²Spatial Distribution
Droplet 1 (Pure lysozyme, 50 mg/mL)	Medium	+++	Light blue	Evenly distributed throughout the droplet
Droplet 2 (1/10 diluted pure lysozyme, 5 mg/mL)	Small	+++++	Deep blue	Mostly concentrated toward the center
Droplet 3 (Fraction E)	Large	++	Deep blue	Clustered densely in the middle region
Droplet 4 (1/10 diluted fraction E)	/	/	/	Scattered near one side, uneven across the droplet

Table 1. Description of protein crystallization

¹Qualitative observations of lysozyme crystallization in four sample droplets. Samples included commercial lysozyme (50 mg/mL and 1/10 dilution) and purified lysozyme (Fraction E and 1/10 dilution).

²Crystal size and distribution were estimated visually, no precise measurements were taken

³ “+” = very few crystals (10), “++” = few (11-30), “+++” = moderate (31-60), “++++” = many (61-100), “+++++” = very many (>100)

SDS-PAGE

SDS-PAGE was used to monitor the purification of lysozyme from hen egg white across different fractions. Sample from each purification step, including crude extract, wash fractions, eluted protein (Fraction E) and commercial lysozyme were analyzed on a 4-20% (w/v) gradient polyacrylamide gel. Band intensity and position were used to evaluate purify, with conalbumin, ovalbumin and lysozyme as references. Lysozyme enrichment was observed in Fraction E and the commercial sample, as shown in Figure 2.

Figure 2. SDS-PAGE of Fraction A-E. 4-20% (w/v) gradient polyacrylamide gel in SDS-PAGE buffer (25 mM Tris pH 8.4, 192 mM glycine, 0.1% (w/v) SDS), stained with Coomassie blue R250 and destained with acetic acid. Lane 1: NEB protein standard Lane. Lane 2,8,13: 1/20 diluted fraction A. Lane 3,9,14: 1/20 diluted fraction B. Lane 4,10,15: fraction C. Lane 5,11: fraction D. Lane 6,12: 1/10 diluted fraction E

Activity Assay

Assay	Fraction	Volume (mL)	Reaction rate, ¹V₀(DA_{540 nm}min^-1)	²Enzyme activity (units/mL)	³Total units of activity in Fraction
1	D	50
2	C	50
3	B	50
4	A	50
5	1/5 diluted E	50

Table 2. Initial velocity rates and enzyme activity of activity assay

¹V₀ = change in Absorbance/change in time

² Enzyme activity = V₀/vol

³ Total activity unit = enzyme activity vol. of fraction from day 1

Lowry Assay

Protein concentrations in Fraction A-E were determined using the Lowry assay, with a six-point standard curve ranging from 0-40 mg. Absorbance at 750 nm was measured for each fraction in duplicate. Table 3 summaries the absorbance readings, dilution factors, and calculated protein quantities for all samples.

Table 3. 750 nm absorbance of BSA standards and duplication protein fraction A-E for the Lowry assay. Absorbance readings at 750 nm for BSA standards and calculated proteins amounts for BSA standards and protein purifications (A-E), based on the Lowry assay. Volumes loaded and dilution factors are indicated for each sample.

A standard curve was generated using unknown concentrations of BSA (0-40 mg) to establish a relationship between protein amount and absorbance at 750 nm. A second-order polynomial regression was fitted to the data, yielding a high degree of correlation (R² = 0.997). This curve was used to calculate the protein content of unknown samples.

Figure 3. Standard curve for Lowry assay using BSA (0-40 mg). Absorbance at 750 nm was measured for BSA standards (0-40 mg), and a second-order polynomial regression was fitted (y = -6E - 05x² + 0.0143x, R² = 0.9969) to interpolate unknown sample concentrations.

Fraction	Volume of fraction (mL)	Concentration (mg/mL)	Total protein amount (mg)	Specific activity (unit/mg)	Yield %	Purification
A	9.0	26.94	242.5
B	8.55	34.81	297.6
C	8.60	0.45	3.87
D	8.68	0.19	1.649
E	0.60	5.78	3.468		1.43	22.7

讲解 BCMB20005 Purification of lysozyme using ion-exchange chromatography辅导 数据结构语言

讲解 BCMB20005 Purification of lysozyme using ion-exchange chromatography辅导数据结构语言