Transcript 499Presentation_fina..

Affinity Chromatography:
Homemade
Microcystin-Sepharose Column
Cindy Lee
May 1, 2006
Affinity Chromatography
Molecule of Interest
Ligand
Matrix
Sepharose
Protein
Phosphatase-1
Microcystin-LR
Protein Phosphatase-1 (PP1)


Part of the Ser/Thr Phosphatase
Family
Important enzyme in the
regulation of many cellular
pathways
 Involved
Protein
Phosphatase-1
in reversible
phosphorylation of proteins
 Must counterbalance the activity
of several different protein
kinases
 Tightly regulated by regulatory
subunits
What is Microcystin?



Cyclic heptapeptide
Hepatotoxin found in
blue-green algae
(cyanobacteria)
Potent inhibitor of
protein phosphatase-1
(PP1)




Immediate binding
Covalent binding to
Cys-273 of PP1
>50 kinds
MC-LR and MC-LL
are the most common
Microcystin-LR Bound to PP1
Acidic
Groove
C-terminal
groove
Hydrophobic
groove


RVXF Motif
Binding Site
Binds to active site of PP1
RVXF motif binding site exposed
Goldberg et al. Nature 376 745-735 (1995)
Uses of Microcystin-Sepharose
Affinity Chromatography
Purify PP1
 Bind regulatory proteins of PP1 to PP1
that is bound to the column

 RVXF

motif binding site is exposed
Purify PP2A
 Part
of the same Protein Ser/Thr phosphatase
family as PP1
 Also inhibited by microcystin
 In the literature, there were problems with
eluting the PP2A
How to Create a MicrocystinSepharose Column
Step 1: Obtain Microcystin-LR (MCLR)
 Step 2: Add linker to MCLR

MCLR +

Step 3: React MCLR with the linker to
N-hydroxysuccinimide (NHS) activatedSepharose
+
Moorhead et. al. FEBS Letters 356 46-50 (1994)
Microcystin-LR Standard
HPLC:
Buffer A: 0.1%TFA/H2O
Buffer B: 0.1%TFA/Acetonitrile
Rate: 0.3% B/min over 3 hours
Absorbance (206nm)
0.300
121.04min
0.200
0.100
0.000
0
50
100
Retention Time (min)
150
Step 1: Obtain Microcystin-LR
Absorbance (206nm)
-fractions were collected and pooled from an HPLC purification of
microcystin from cyanobacteria from Little Beaver Lake in 1992
0.300
121.27 min
0.200
0.100
0.000
0
50
100 150
Retention Time (min)
Comparison: Standard vs Pooled Fractions
0.200
Absorbance (206nm)
121.04 min
Standard
0.100
0.000
121.27 min
Pooled Fractions
0.100
0.000
0
50
100 150
Retention Time (min)
Step 2: Add Linker to MCLR
Comparison: Before vs After Reaction with Linker
Absorbance (206nm)
0.200
121.27 min
Microcystin
pool
0.100
0.000
113.89;115.02 min
0.400
After Reaction
with Linker
0.200
0.000
0
50
100
150
Retention Time (min)
Step 3: React MCLR with Linker to
NHS-activated Sepharose
Comparison: Before vs After Reaction with Sepharose
Absorbance (206nm)
0.400
113.89;115.02 min
After reaction
with linker
0.200
0.000
Supernatant
after reaction
with NHS-activated
Sepharose
0.100
0.000
0
50
100
150
Retention Time (min)
Binding Experiments with PP1

Determine the Binding Capacity of the
Microcystin-Sepharose
 Add
increments of PP1 to the resin
 Supernatant was tested for activity and used to
determine the amount of PP1 that bound
Add
PP1
control
repeat
MicrcystinSepharose
Note: Tris-Sepharose resin was used as the “Control “ and ran in
parallel with Micrystin-Sepharose resin
Amount of PP1 Bound After Each Addition
of 17.5g of PP1 to 50L of MicrocystinSepharose Resin
Unbound
Amount of PP1
18
Bound
16
14
12
10
8
6
4
2
0
1
2
3
4
Addition #
Total PP1 Bound = 14.4+15.2+16.1+13.3 = 59g
50L of resin contains ~14.3g of MCLR:
How much PP1 can 1mg of MCLR bind?
59gPP1 * 1mgMCLR / 14.3gMCLR = 4.2mgPP1
Binding Experiments with PP1


Determine the Binding Capacity of the MicrocystinSepharose
Using Microcystin-Sepharose for purifying PP1

1) Bind PP1 to resin
 2) Wash resin (0.3M NaCl)
 3) Elute PP1 (3M NaSCN)
Add
PP1
control
MicrcystinSepharosel
Wash
And
remove
supernatant
Elute
PP1 Purification Experiment
PP1
MW
For
markers binding
MCCtrl
Seph
Resin
Resin
After incubation
with PP1
MCSeph
Ctrl
Resin Resin
After Elution with
3M NaSCN
Ctrl
Elution
(3M NaSCN)
(kDa)
75
50
37
25
20
15
10
MCSeph
Ctrl = control:Tris-Sepharose
MC-Seph = Microcystin Sepharose
PP2A Purification
Experiment
MCMCMW
markers
(kDA)
75
50
37
25
20
15
PP2A
For
binding
Ctrl
Resin
Seph
Resin
After incubation
with PP2A
Seph
Ctrl
Resin Resin
After Elution with
Okadaic Acid
MCSeph
Ctrl
Elution
(Okadiac Acid)
Future Work…

Repeat Experiments with PP2A
 Get

a more definite result
Try binding regulatory proteins to PP1 that
is bound to the column
Acknowledgements

Holmes Lab
 Especially
Marcia Craig