Size Exclusive

Chromatography

CHE 447-01
By Grant Akalonu
Abstract
In this experiment, green fluorescent protein (GFP) and blue fluorescent protein (BFP)
from bacterial samples were isolated using size exclusion chromatography. GFP and BFP were
successfully extracted from the bacterial samples. After the experiment, the molecular weight of
GFP and BFP was estimated to be about 36kDa, which is within 10% of the accepted value of
40kDa.

Introduction

The two proteins of interest in this experiment, green fluorescent protein (GFP) and blue
fluorescent protein (BFP) belong to a family of luminescent proteins found in marine
microorganisms and jellyfish. GFP has 238 amino acid residues and has a molecular weight of
approximately 40kDa. Most of the intact protein is required for maintaining fluorescence and
only small deletions of a few amino acids are allowed without compromising the integrity of the
protein structure. The chromophore responsible for light emission is the primary structure of the
protein and consists of a tripeptide at positions 65 to 67 which is cyclic and is composed of a
sequence of Ser-Tyr-Gly residues. The importance of protein folding is clearly demonstrated
with where the protein is fluorescent only upon proper conformational folding. The blue
fluorescent protein (BFP) is a derivative variant of the GFP. Their differecnes include a His-66
sub- stitution at the Tyr-66 position and a second substitution from Tyr-145 to Phe-145. The
initial BFP known as P-4 had only the His-66 substitution and was not as bright as the mutant
with a double substitution.

Size exclusion chromatography can purify a solution containing proteins or other

macromolecules such a carbohydrates and nucleic acids based on their size and shape. The
macromolecules of interest are separated by their size through a gel which contains a solid phase
consisting of a network of beads. Larger proteins will evade the pores between the beads and
elute to the bottom of the chromatography chamber first, while smaller ones will travel through
the pores and take longer to elute through the chamber. The purified GFP and BFP samples will
have their absorbance measured at 280 nm, and studied using denaturing polyacrylamide gel
electrophoresis.

Materials/Methods

The first step is to prepare the chromatography column. The elution buffer was prepared
by mixing 450 mL of distilled water with 50 mL 10X column elution buffer. Then 50 mL of 1X
elution buffer was used to swell the dry matrix. The slurry gel was stirred for 30 minutes. The
column was then wet and then the slurry mixture was added to it. Precaution was taken to make
sure that the gel was well packed without air bubbles. Then, 2mL of elution buffer was used to
equibriate the column after packing several times to prevent the column from drying. Then 300
L of GFP was added to the the sample column in circular motion, and the displacement emerging
from the column was collected immediately. The 1X elution buffer was added continuously until
7 fractions of of protein were collected. This same process was carried out for BFP.

The SDS-Page electrophoresis gel to study the green and blue fluorescent proteins was
prepared. First, the 1X Tris-Glycine-SDS electrophoresis buffer was prepared from dilution to a
1x electrophoresis buffer. The BFP and GFP fractions with the best absorbance reading chosen to
run on the SDS PAGE gel. Twenty-five microliters of each fraction were pipetted into centrifuge
tubes with 20 L of sample loading buffer. The samples were then incubated for 5 minutes at
95C, and the sample was quick spun afterwards. Then the samples were loaded into the gel. A
potential of 180V was applied to the gel for about 40 minutes. Then the gel was removed from the
plastic case and stained over night with Coomassie staining solution.

Results

GFP/BFP Purification

Fig.1 GFP fractions visualized with UV light.

 Blue Flourescent Protein
BFP Absorbance
1.8 Fraction
1.6 1 0.137
1.4 2 1.545
1.2 3 1.326
Absorbance

1 4 0.776
5 0.385
0.8
6 0.289
0.6
7 0.128
0.4
Table 1. BFP absorbance at 280 nm.
0.2
0
0 2 4 6 8
BFP Fraction

Fig,2. Graph of BFP fraction vs absorbance.

 0.9 Green Flourescent Protein
0.8

0.7
GFP Absorbance
0.6 Fraction
1 0.125
Absorbance

0.5
2 0.772
0.4 3 0.376
0.3 4 0.572
5 0.307
0.2
6 0.223
0.1 7 0.07
0 Table 2. GFP absorbance at 280 nm.
0 2 4 6 8
GFP Fraction

Fig.3. Graph of GFP fraction vs absorbance

Lane Fraction

1 Molecular Marker

2 Total GFP extract

3 GFP Fraction 2

4 GFP Fraction 3

5 GFP Fraction 4

6 Total BFP

7 BFP Fraction 2

8 BFP Fraction 3

9 BFP Fraction 4

Table 3: The contents for each well in

the SDS-page gel from left to right,
Fig.4. The SDS-Page Gel of GFP-BFP proteins after
destaining
 Lane Fraction Distance Traveled (cm)
2 Total GFP extract 18
3 GFP Fraction 2 19
4 GFP Fraction 3 16
5 GFP Fraction 4 2
6 Total BFP 3
7 BFP Fraction 2 17
8 BFP Fraction 3 17
9 BFP Fraction 4 18
Table 4. The distance that each protein fraction traveled in each well.

Discussion & Conclusion

In this experiment, green fluorescent protein and blue fluorescent proteins were successfully
isolated from a bacterial mixture. From the graph showing the absorbance change with the fraction
of blue fluorescent protein, it appears that fractions 2 and 3 had the maximum concentration of the
protein, and this result is supported by the SDS-PAGE result in Figure 4. Also, the green
fluorescent protein graph shows two maximums for fraction two and for fraction 4. This result
does not seem to be supported by SDS-PAGE however. It is likely that there was some form of
error introduced into transferring the fraction sample to the well of the gel before SDS-PAGE.
Most of the fractions traveled a similar distance for the origin of the gel. Based on the legend
provided by the molecular marker, the molecular weight of both GFP and BFP can be estimated
to be 36kDa.
References

[1] Edvotek kit #255: Purification & Size Determination of Green (GFP) & Blue (BFP)
Fluorescent Proteins.