As shown in figures 5.10 and 5.11, G48V “4833” protein sample was applied on a DEAE- Cellulose column and collected sample fractions were analysed by SDS-PAGE in 20% gel concentration and polyacrylamide gel stained with Coomassie blue.
Fig. 5.10: Analysis of eluted G48V “4833” protein samples by DEAE-Cellulose chromatography on SDS-PAGE (pH 5.6).
KDa 1 2 3 4 5 6 7
Lane 1 : Biorad blue™ marker (prestained protein standards) Lane 2: Sample protein fraction eluted with LSB
Lane 3: Sample protein fraction eluted with 0.2 M NaCl. Lane 4: Sample protein fraction eluted with 0.4 M NaCl Lane 5: Sample protein fraction eluted with 0.9 M NaCl.
Lane 6: Protein sample o f pepstatin affinity purified G48V “4833” Lane 7: Kaleidoscope marker
Fig. 5.11: Elution profile of G48V “4833” by DEAE-Cellulose ion exchange chrom atography O CO N (D - e
o
4
3
2
1
0
H--- 1--- 1--- 1--- 1--- 1-1 - — I— --- 10
2
4
6
8
10
12 14
16
18 20
22
N u m b e r o f f r a c t i o n sFigure 5.11 shows the optical density (O.D) 280 nm trace from the DEAE-Cellulose ion exchange chromatography.
A number o f fractions were collected. Results o f figure 5.10 showed that the 15 kDa protein was hydrolysed into 7 kDa or smaller proteins and eluted in the presence o f 0.2 M NaCl. These were impossible to separate from the 11 kDa protein (22 kDa dimer) by acetone precipitation or concentrating using a 10 kDa cutoff Centriprep™ . The 7 kDa protein was only present in the fractions which eluted with low salt.
Fig. 5.12: SDS-PAGE analysis of G48V “4833” protein sample by DEAE-Cellulose ion exchange chrom atography.
49 ^
Lane 1 : Biorad blue™ m arker (prestained protein standards)
Lane 2: Protein sample G48V “4833” during initial purification at pH 5.6 before being applied on DEAE-Cellulose column.
Lane 3: Protein sample during initial purification at pH 5.6; unbound proteins after being applied on DEAE-Cellulose column.
Fig. 5.13: Centriprep*'^ concentration of G48V “4833” protein fraction from DEAE- Cellulose chromatography. Samples analysed by SDS-PAGE in 20% gel concentration and polyacrylamide gel stained with Coomassie blue.
KDa 49
>
33>
21>
8
>
Lane 1 : Biorad blue™ marker (prestained protein standards)
Lane 2: G48V “4833” protein sample in 0.2 M NaCl obtained from DEAE-Cellulose column before concentrating using a Centripre™.
Lane 3: G48V “4833” protein sample in 0.2 M NaCl obtained from DEAE-Cellulose column after concentrating using a Centriprep™.
In figure 5.12, the result indicates that only a very small percentage o f different molecular weight
proteins bound to the resin and only 1 proteinase species eluted. In addition, it seems that protein
samples equilibriated at pH 5.6 causes some of the proteins break down into small molecular weight
to proteins below 11 kDa.
On SDS-PAGE analysis (fig. 5.13), it was clear that the 11 kDa protein remained the same size and
did not cleave to yield further fragments but the 15 kDa protein was no longer present. Since the 11
and 15 kDa peptides behaved differently, it would be important to know the sequence o f the 7 kDa
protein to determine the point o f cleavage. To separate the 7 kDa fragment from the 11 kDa protein,
the sample was precipitated with acetone since the 7 kD a protein maybe soluble in acetone. As an
alternative approach, a (Centriprep™ Amicon) concentrator was used. In theory, the 7 kDa protein
would pass through the 10 kDa membrane, if it were not present as a dimer. Separation of 7 kDa
protein from 11 kDa protein was not achieved by acetone precipitation or concentration using a
Centriprep™ concentrator. This could be due to the fact that the 7 kDa protein is either multimeric
/ aggregated or acts as a sticky protein. It appeared that pH 5.6 facilitated cleavage o f the 15 kDa
protein, so perhaps this was not the appropriate pH for complete cleavage.
Accordingly an experiment was then carried out with the protein sample fraction o f0 4 8 V “4833”
which was eluted with 0.1 M NaCl obtained from DEAE-Cellulose column at pH 7.0 rather than
pH 5.6. The protein sample was analysed on a DEAE-Cellulose column, equilibriated with pH 7.0
buffer.
Fig 5.14: Analysis of G48V “4833” protein sample fraction on DEAE-Cellulose chrom atography by SDS-PAGE.
1 2 3 4 5 6
w
Lane 1: Biorad blue™ marker (prestained protein standards)
Lane 2: G48V “4833” protein sample fraction in 0.1 M NaCl pH 7.0 before application to a DEAE-Cellulose column.
Lane 3: G48V “4833” protein sample fraction in 0.1 M NaCl pH 7.0 after application to a DEAE- Cellulose column.
Lane 4: Eluted protein sample with 0.2 M NaCl. Lane 5: Eluted protein sample with 0.4 M NaCl. Lane 6: Eluted protein sample with 0.6 M NaCl.
As shown in figure 5.14, the result obtained were similar to those seen at pH 5.6 and the 11 and 15 kDa proteins behaved as sticky proteins as before. Carrying out this experim ent at pH 8.0, increased the proteinase activity slightly, but did not separate the two proteins.
There was no success in separating the 11 and 15 kDa proteins on a DEAE-Cellulose column at different pHs. Therefore, it was decided to analyse the sample on an SP-sepharose ion exchange column. The protein sample was dialysed at pH 5.6 and the column equilibriated with pH 5.6 buffer. A number o f 14mls fractions were collected and analysed on SDS-PAGE.
Fig. 5.15: Analysis of crude G48V ‘‘4833” protein sam ple at pH 6.0 on SP-sepharose chrom atography by SDS-PAGE. Samples were analysed on 20% gel concentration and polymerised gel was stained with silver.
Lane 1 : Biorad blue™ marker (prestained protein standards)
Lane 2: Crude G48V “4833” protein sample at pH 5.6; before been applied on SP-sepharose ion exchange chromatography.
Lane 3: Crude G48V “4833” protein sample at pH 5.6; after been applied on SP-sepharose ion exchange chromatography.
Lane 4: Eluted material obtained from column wash with LSB after applying protein sample on column.
Lane 5: Protein sample eluted with 0.4 M NaCl. Lane 6: Protein sample eluted with 0.5 M NaCl. Lane 7; protein sample eluted with 0.5 M NaCl. Lane 8: Protein sample eluted with 0.9 M NaCl.
SDS-PAGE analysis (fig. 5.15) revealed that there was no longer a band noted for the 15 kDa protein, but the 11 kDa and smaller sized proteins were observed. Since the sample loaded had both 11 and 15 kDa bands, there was no separation o f these tw o proteins on ion exchange columns. It appears that when the proteinases are analysed on the ion exchange at pH 5.6, they no longer behave as 11 and 15 kDa protein. The 11 kDa protein is detected but the 15 kDa becomes proteolysed to smaller peptides such as the 7 kDa protein. In all cases the 11 and 7 kDa proteins eluted o ff the column together. Hence, a single peak is seen, which on SDS-PAGE is fractionated into two bands o f 11 and 7 kDa .
Ion exchange separates proteins by charge not size. The 11 and 15 kDa must both have similar
charge to behave in the same way on the ion exchange chromatograpy (fig. 5.9). Subsequent SDS-
PAGE analysis of the ion exchange fractions revealed peptides o f 11 and 7 kDa, although the sample
load had 11 and 15 k D a . This suggests that the 15kDa has been hydrolysed during the experiment
to give the 7 kDa peptide.