Size Exclusion Chromatography In Proteins Biology Essay

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To purify the target protein, Glutathione S-Transferase (GST), from a PGEX-2T/E. coli lysate sample by use of size exclusion chromatography.

Principle of Methods

In order to study a specific protein, it first must be purified and isolated from its biological system. One way of doing this is through size exclusion chromatography (SEC). Proteins are very delicate structures so when you using purification techniques it is important to purify the protein in as few steps as possible because the more a protein is handled, the risk of denaturation increases exponentially (Lab Manual).

Most often, recombinant DNA technology using bacterial cells is used to prepare a source for a specific target protein. This means that the instructions to create a protein are inserted into a plasmid. This plasmid is a small circular piece of DNA that once in the bacterial cell can replicate and thus produce the target protein. To harvest this protein, the cells are must be lysed. Lysing of bacterial cells can be done by vibration against glass beads, nitrogen gas at high pressure, a French press, sonciation or enzymatic lysis. Once lysed and centrifuged, the formed supernatant is referred to as the lysate from which the target protein can be isolated (Lab Manual).

The principle of SEC is that is separated biomolecules based on their size. A long thin glass column contains microscopic porous beads of a predetermined size composed of cross-linked dextran. The pore size is determined based on what molecule is being purified; in this case, Sephadex G-75 beads are used because they allow for the inclusion proteins smaller than 65kDa, such as the target protein (GST). Also within this column is a buffer that continuously pumps through to help movement of the molecules as well as maintain pH and protect the proteins from denaturation. This column is also connected to a chromatography machine (Lab Manual).

Prior to running SEC, the lysate must be syringed through a filter to remove and miscellaneous things that could cause skewed data. The filtered lysate is then introduced to the column and the elution, or extraction, and the collection of fractions begins. This occurs by the principle that molecules which are larger than 65kDa with travel around the beads and exit the column first, and smaller molecules which become trapped in the pores of the beads and will elute out last. As the fractions are collected they pass by an optical unit that measure the absorbance of the sample at 280nm, which is the wavelength at which proteins absorb the most light. This information is then displayed on a chromatogram graph which displays absorbance versus time (Lab Manual).

These fractions are also indicated on the chromatogram to help identify which fractions occur during a peak on the graph. From this graph, students should choose fractions about every three samples during a peak to test for the presence of the target protein. In this case, CDNB was used to detect GST. Once the fractions have been tested by CDNB, the reaction velocity is calculated by determining the difference in change in absorbance over change in time multiplied by ml of the sample added (Lab Manual).

Materials/Reagents

pGEX-2T E. coli lysate

dH20

1.5 ml tubes

Fixed angle centrifuge

Syringe and filter

Biorad BiologicLP Chromatography machine with column containing 1-2 grams of Sephadex G-75 beads

50 mM Tris, pH 8.0

100 mM glutathione

10 mM CDNB

10X Reaction Buffer

Cuvettes

Spectrophotometer

Vortex

Parafilm

Results

Table 4-1: CDNB Assay Absorbance Value at 340nm, for SEC Fractions

Time (min)

Fraction 17

Fraction 19

Fraction21

Fraction23

Fraction25

Fraction 27

0

0.091

0.05

0.072

0.215

0.082

0.105

0.5

0.092

0.059

0.145

0.345

0.169

0.191

1

0.095

0.055

0.208

0.455

0.256

0.249

1.5

0.098

0.064

0.269

0.559

0.337

0.318

2

0.099

0.071

0.33

0.654

0.412

0.379

Reaction Velocity (units/ml)

0.8

3.2

24.4

39.4

31.2

26

Value 4-1: SEC Total Volume: appx. 5 ml

Value 4-2: SEC Final Reaction Velocity: 35.5 units/ml

Final Fractions Saved: #20-26

Discussion

The objective of this experiment was to purify the target protein, Glutathione S-Transferase (GST), from a PGEX-2T/E. coli lysate sample by use of size exclusion chromatography.

After running the SEC, students were able to determine where protein absorbance was highest using a CDNB Assay and from that information were able to calculate the reaction velocity of GST. This test showed that the highest reaction velocities were at the tail of the peak, between fractions 23 and 26. Fraction 23 had the highest reaction velocity of 39.4 units/ml. In order to obtain further purity, it was advised that the students save fractions 20 through 26. The average reaction velocity of these samples was then calculated to be 35.5 units/ml and the total volume came out to approximately 5 ml. This reaction velocity indicates that the students successfully isolated and purified Glutathione S-Transferase, which is further supported in an accurate chromatogram, with knowledge that GST is found towards the back end of the curve.

Kelly Pfeifler

Partner: Melissa Main

Section 11

Lab 5: Ion Exchange Chromatography

Objective

To further purify the target protein, Glutathione S-Transferase (GST), from a PGEX-2T/E. coli lysate sample by use of ion exchange chromatography

Principle of Methods

Ion exchange chromatography (IEX) is another way to purify and isolate a target protein; this process however separates based on charge, rather than size as seen in SEC. However, the charge of a protein is not constant and depends greatly on the pH of the solution that it is in. This means that when using IEX to separate proteins it is important to maintain a constant pH with your buffer to that the proteins maintain a constant charge (Lab Manual).

The columns used in IEX are much smaller than those in SEC, but also contain beads. These beads however are affixed with charged groups attached to them. Because opposite charges attract, beads with the opposite charge of the target protein is chosen. For example, a protein with a negative charge should be put through an IEX column with positively charged beads; this is also called anion exchange chromatography. Similarly, cationic exchange chromatography refers to a protein with a positive charge put through a negatively charged column. The type of chromatography therefore is named for what the beads will bind to, not the charge of the beads (Lab Manual).

When the sample is loaded into the column by movement of the buffer, proteins that contain like charges with beads will be repelled and fall out of the column first. Second, neutrally charged proteins will be next and will create an initial peak “flow throughâ€Â peak on the chromatogram. Now, proteins with opposite charge of the beads are now bound and are washed off by adding excess buffer. The elution of the bound proteins occurs because an increase in NaCl in the column competes with the bound proteins and will force the proteins off and the negative, or positive, ions will replace them. As the salt concentration increases, the more tightly bound proteins will begin to be eluted. This gradual increase in called the salt gradient (Lab Manual).

After elution, it is important that the salt concentration return to normal through a process called equilibration. If this does not occur, it will skew the data of subsequent testing as the IEX columns can be used several times over. For example, the following protein samples may not bind to the beads at all and flow directly through the column (Lab Manual).

In this lab, anion exchange chromatography was used. Which means that because GST has a pI of 5.0, a buffer of pH 8.0 is used to ensure that the protein will maintain a negative charge and will bind to the positively charged beads in the column. Additionally, when salting out the bound proteins, the negatively charged chloride ions from NaCl will exchange places and the bound proteins will be eluted (Lab Manual).

Materials/Reagents

pGEX-2T E. coli lysate

dH20

1.5 ml tubes

Fractions saved from Lab 4

AKTA Prime chromatography machine equipped with UNOsphere Q 1 ml cartridges, manufactured by Bio-Rad

50 mM Tris, pH 8.0

100 mM glutathione

100 mM CDNB

10X Reaction Buffer

Spectrophotometer

Cuvettes

Vortex

Parafilm

Results

Table 5-1: CDNB Assay Absorbance Value at 340nm, for IEX Fractions

Time (min)

Fraction 18

Fraction 20

Fraction 22

Fraction 24

Fraction 26

0

0.007

0.948

0.123

0.108

0.028

0.5

0.017

1.001

0.032

0.173

0.002

1

0.027

1.044

0.045

0.231

0.033

1.5

0.038

1.1

0.11

0.293

0.064

2

0.049

1.14

0.188

0.356

0.096

Reaction Velocity (units/ml)

2.2

9.6

14.3

12.5

6.3

**The chromatogram that was performed and emailed saved as .chd file and is unable to be opened and displayed as an image**

Value 5-1: SEC Total Volume: appx. .7 ml

Value 5-2: SEC Final Reaction Velocity: 14.3 units/ml

Discussion

The objective this lab was to further purify the target protein, Glutathione S-Transferase (GST), from a PGEX-2T/E. coli lysate sample by use of ion exchange chromatography.

Fractions were chosen, based on the chromatogram, surrounding the second peak. These fractions, 18-26, started slightly before, during and a little after the peak. These fractions were tested with a CDNB Assay and reaction velocities were calculated. Fraction 22 showed the highest reaction velocity with 14.3 units/ml. This reaction velocity shows a substantial decrease from that which was calculated in lab 4 using size exclusion chromatography. This could be explained by the decrease in protein concentration, because as previously stated, the more a protein is handled the risk of it denaturing increases. Therefore, the protein work comes with a tradeoff between sample purity and sample amount. Additionally, the lower reaction velocity also indicates that the IEX purification technique was perform effectively in that as the sample becomes more and more pure, as the protein has fewer things to react to. Following this method of purification it can be assumed that Fraction 22 contains the most pure sample of GST thus far.

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