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CXCR4 Introduction
• G protein coupled receptor (GPCR)
• Activated by SDF-1, a chemokine
• Thought to be important to two major diseases
• HIV- Acts as co-receptor for M Tropic
• Possible cure for M Tropic infections
• Cancer- Expressed more in breast and other cancers
• Aids in metastasis of cancer to lungs and bones
•Stop CXCR4 from working, metastasis would stop to lungs and
bones
Project
• The purpose of the project was to mutate the CXCR4 Constitutively
Active Mutant (CAM) receptor, do experiments in order to determine
whether or not the receptor is active given the random mutations,
and to find out exactly where and what the mutations are.
• Experiments:
1) Filter Lift Assay
2) His Growth Assay
3) Plasmid Manipulation
4) Restriction Digest
5) Fluorescent Lac Z
6) PCR
7) Western Blot
8) Sequencing
9) Mutagenesis
10) 3-D Modeling
Filter Lift Assay
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•
•
•
Quickest and easiest method to mass screen for receptor activity
Yeast is grown on an agar plate with a grid in order to identify the
colonies. The colonies are the lifted with a piece of filter paper
that is pre-soaked in X Gal and Z Buffer.
The yeast are permeabilised and then incubated over 2-3 hours
and the active colonies turned blue.
Yeast with an active receptor synthesize B-Galactosidase via
FUS-1 activation. X Gal is a substrate for B-Gal. Complete X Gal
is colorless; cleaved X Gal is blue.
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His Growth Assay
•
Yeast is grown in a medium that
lacks a necessary amino acidHistidine.
This test is used to confirm the
filter lift. If the yeast grows then it
is active.
His Growth Assay of Single
Mutants
0.75
OD 600
•
0.50
0.25
0.00
0
10
20
Time (Hours)
30
wt
n119s
M63L1
V82G2
V82G1
V82G3
F248I1
F248I2
F248I3
Plasmid Manipulation
• The plasmid from the revert mutants is extracted by minipreps.
• The plasmid is then transformed into Nova Blue competent cells
• The DNA is extracted from the competent cells and used for
downstream experiments and sequencing.
Selection Procedure
• Different mediums select different plasmids i.e.:
CP4258 –Leu
CP1584 –Trp
• Different antibiotics select different plasmids in bacteria i.e.:
Cp4258 & CP1584 are grown in Ampicillin
Restriction Digest
•
•
Cuts plasmid and shows whether
or not CXCR4 is present
The plasmid is cut with restriction
enzymes ( XbaI and NcoI)
NcoI- CCATGG
XbaI- TCTAGA
PCR
•
•
•
•
A polymerase is added to DNA and replicates the desired DNA.
The primers have complementary sequences that line up with the desired
segment of the DNA.
Taq polymerase adds nucleotides to make up the DNA.
Used to make sure CXCR4 is present.
Fluorescent Lac Z
Same principle as filter lift.
FDG is used as substrate in place of X Gal.
When cleaved, FDG fluoresces.
Fluorescent Lac Z Assay of Single Mutants
75000
Fluorescence
•
•
•
50000
25000
0
wt
N119S
M63L1
M63L2
M63L3
Mutants
V82G1
V82G2
V82G3
F248I1
Western Blot
•
•
•
•
Shows if protein is present in
sample.
9E10 is the antibody that binds to
Myc tagged CXCR4.
Used to see the size of protein
present.
Results came out blank.
Sequencing
• DNA is sent to be sequenced.
• Results where mutations, if any, are present.
• Mutations that we found:
Revert 2-13: V82G
Revert 2-16: V82G
Revert 2-21: M63L, S312P
Revert 2-26: S123I, E288G
Revert 2-32: F29S, V59A, F248I, V320E
Revert 2-49: F248I
Overall Mutation Rate:
Mutation Rate:
1/1089
1/1089
2/1089
4/1089
4/1089
2/1089
14/6532= .21%
Mutagenesis
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•
•
•
Site directed mutagenesis of CXCR4 CAM
Separates multiple mutations
Mutate into pcDNA3.1zeo(+) for transfection into Mammalian Cells
Mutations:
M63L
V82G
F248I
E288G
3-D Modeling
• Displayed mutations on CXCR4
• Saw possible interactions with other residues
• Possible important hydrophobic interactions:
S123: W252, N119
L244: L127, S123, Y76, I126
L246: H228, L301
F248: Y256, L253, I215, L127
PLATE
1
PLATE
2
PLATE
3
PLATE
4
PLATE
5
PLATE
6
PLATE
7
PLATE
8
PLATE
9
TOTAL
Filter Lift
51
44
47
45
45
48
49
47
48
424
His Growth
0
36
47
38
44
41
0
40
0
246
Yeast Miniprep
0
33
43
21
43
31
0
37
0
208
Bact Transformation
0
6
5
0
0
11
Bact MinipreP
0
6
5
0
0
11
Sal 1 and bact transf. Miniprep
0
6
5
0
0
11
Yeast TRAnsformation (lac Z)
0
6
0
0
6
Fluorescent lac Z
0
6
0
0
6
Sequenced
0
6
0
0
6
Site Direct Mutagenesis (single
mutations)
0
4
0
0
4
Yeast Transformation (His, LacZ, FUI)
0
0
0
0
His Growth
0
0
0
0
FUI
0
0
0
0
Fluorescent lac Z
0
0
0
0