Engineering the growth factor,
CXCL12α, for heart tissue
regeneration
Emily Buck
Whitaker Conference
April 30, 2015
Cardiovascular diseases account for the highest number of
deaths worldwide1.
Blockage of coronary arteries causes a reduction in
blood supply to heart tissue
CORONARY (ISCHEMIC) HEART DISEASE
Heart tissue is damaged when blood supply is
totally cut off from heart tissue
HEART ATTACK (MYOCARDIAL INFARCTION)
MAJOR CONSEQUENCES:
Loss in cardiac function
Possibility of complications
1.
World Health Organization, “Global status report on noncommunicable diseases,” p. 9, 2011
-2-
What is damaged by reduced
blood supply?
-3-
Ischemic tissue transiently expresses growth factors, such
as VEGF and CXCL12α (SDF-1α), to initiate mechanisms
2
for revascularization in the damaged area
CXCL12α promotes2…
• expression of its
receptor, CXCR4
• migration of progenitor
and endothelial cells to
ischemic area
After myocardial infarction,
overexpression of CXCL12α
has been shown to
• Reduce cardiac
myocyte apoptosis3
• Promote rebuilding of
capillaries and small
arterioles3
PROBLEM: Short half-life and
easy diffusion!
2. C. Cencioni et al, Cardiovascular Research, 94: 400-407, 2012
3. M. Zhang et al, The FASEB Journal, 21: 3197-3207, 2007
-4-
How can we improve the regenerative capacity of
CXCL12α?
Previous studies in Dr. Hubbell’s lab4…
(1) Identification of a domain of platelet-derived growth factor 2 (PlGF-2123-144)
binds with high affinity to many ECM proteins
(2) Fusion of this PlGF-2 domain to growth factors (BMP-2, PDGF-BB, and VEGF-A)
enhanced their affinities for the ECM
(3) Engineering the growth factors by fusion of the PlGF-2 domain improved their
capacity for wound healing and lowered the dose required for efficacy
Let’s try with CXCL-12α!
4. M. Martino et al, Science, 343: 885-888, 2014
-5-
My lab in Lausanne
Dr. Jeffrey Hubbell
Laboratory for Regenerative Medicine
and Pharmacobiology (LMRP)
Priscilla Briquez
-6-
How will we engineer CXCL12α?
1. Design and Clone
DESIGN:
Establish sequence with the
PlGF-2 domain fused to
N- OR C-terminus of CXCL12α
AND Fc tag to produce wild-type5
2. Produce and
Purify
PRODUCE:
Transient gene expression of
CXCL12α variants in mammalian
cells
SDS-Page, Western blotting
CLONE:
PCR, agarose gel electrophoresis
Restriction, ligation and
transformation into plasmid
Sequence verification
Amplify plasmid for production
5. D. Czajkowsky et al, EMBO Mol Med, 4: 1015-1028, 2012.
3. Characterize
ECM-binding
affinity and
bioactivity
CHARACTERIZE:
Binding affinity for ECM
components through…
ELISA
Release from fibrin hydrogels
PURIFY:
Separate CXCL12α from
contaminants through
Bioactivity testing through…
His-tag affinity chromatography
MSC migration assay
Size exclusion chromatography
Phosphorylation assay
-7-
I. DESIGN AND CLONE
wtCXCL12α
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK
68 AA
RRRPKGRGKRRREKQRPTDCHL
PlGF-2123-144
22 AA
SHL-CXCL12α
RRRPKGRGKRRREKQRPTDSHLKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLK
NNNRQVCIDPKLKWIQEYLEKALNK
90 AA
CXCL12α-SHL
90 AA
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN
KRRRPKGRGKRRREKQRPTDSHL
SHL-CXCL12α CXCL12α-SHL
(288 bp)
(288 bp)
AgeI
BamHI
Ligate
300
200
300
200
in plasmid
by restriction
enzymes
Transform
into DH5α
competent
bacteria
PLASMID:
pXLG*Chis
Verify DNA
sequence
-8-
I. DESIGN AND CLONE
wtCXCL12α
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK
68 AA
RRRPKGRGKRRREKQRPTDCHL
PlGF-2123-144
22 AA
SHL-CXCL12α
RRRPKGRGKRRREKQRPTDSHLKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLK
NNNRQVCIDPKLKWIQEYLEKALNK
90 AA
CXCL12α-SHL
90 AA
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN
KRRRPKGRGKRRREKQRPTDSHL
SHL-CXCL12α CXCL12α-SHL
(288 bp)
(288 bp)
AgeI
BamHI
Ligate
300
200
300
200
in plasmid
by restriction
enzymes
Transform
into DH5α
competent
bacteria
PLASMID:
pXLG*Chis
Verify DNA
sequence
-9-
I. DESIGN AND CLONE
wtCXCL12α
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK
68 AA
Fc domain6
227 AA
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK + FLAG cleavage site
CXCL12α
(204 bp)
CXCL12α-Fc
(1056 bp)
Fc Tag
(786 bp)
800
700
300
200
CXCL12α-Fc
(984 bp)
Ligate
Amplify
two fragments
by restriction
enzymes
band of
correct size
by PCR
AgeI
Ligate
in plasmid
by restriction
enzymes
6. J.W. Murphy et al, J Biol Chem, 282: 10018-10027, 2007
Transform
PLASMID:
pXLG*Chis
HindIII
into DH5α
competent
bacteria
Verify DNA
sequence
-10-
1. Supernatant + 1. NaCl wash +
2. Supernatant – 2. NaCl wash –
3. PBS wash +
3. Pellet +
4. PBS wash –
4. Pellet -
II. PRODUCE AND PURIFY
HEK ± VPA
CHO ± DMSO
CHO
HEK
wt CXCL12α
CXCL12α-Fc
CXCL12α-SHL
SHL-CXCL12α
9.2 kDa
36.6 kDa
12.1 kDa
12.1 kDa
50 kDa
15 kDa SDS-page for Day 7 supernatant, PBS wash, NaCl wash, and pellet
10 kDa
Need to add Fc for PlGF-2 variants
-11-
II. PRODUCE AND PURIFY
Fc tag for PlGF-2 variants
Ligate Fc tag in PlGF-2 variant
plasmids with restriction
enzymes
AgeI
PLASMID:
pXLG*Chis
1. CXCL12α-SHL-Fc (39 kDa)
2. SHL-CXCL12α-Fc (39 kDa)
1
1*
2
2*
BamHI
Fc
Tag
Transform
into DH5α competent
bacteria
BgI II
Verify DNA sequence
HEK + VPA
50 kDa
15 kDa
10 kDa
-12-
II. PRODUCE AND PURIFY
His-affinity for CXCL12α-Fc
2.
1.
Elution buffer
Supernatant with
CXCL12α-Fc
1.
Contaminants
2.
As-received
Day 7
supernatant
Protein in elution buffer
His purification fractions
(left to right) A6, A6*, A7, A11, A13, B15,
B15*, B13, B11, B5, B3, B1, B1*, and C3
-13-
II. PRODUCE AND PURIFY
Cleave Fc tag from CXCL12α-Fc
4
37
kDa
CXCL12α
0.01% EK to CXCL12α-Fc
0.0001% 0.00064%
8
16
4
8
16*
4
8
16
Fc Tag
FLAG
50
+ Enterokinase (31 kDa)
Incubate
RT
CXCL12α
9 kDa
+
0
25
Fc Tag
FLAG
28 kDa
VARIABLE 1:
10
VARIABLE 2:
Enterokinase (%) per weight CXCL12α-Fc
Incubation time at room temperature
1. 0.0001%
2. 0.00064% (1 unit)
3. 0.01%
1. 4h
2. 8h
3. 16h
-14-
II. PRODUCE AND PURIFY
Separate Fc tag from CXCL12α
Size exclusion chromatography
Western blot
To separate cleaved Fc tag from CXCL12α
To confirm presence of CXCL12α after cleavage
1 1* 2 2* 3 4 4* 5 5* 6 6* 7 8
12 12* 13* 14 14* 29 30 30* 31 32 33 34 35 36
50
25
10
3. Western blot anti-his
Fc tag + EK
CXCL12α
1.
2.
3.
4.
Before his purification
After his purification
Dialysis in 1X PBS
After EK cleavage
1. Size exclusion 12-16
2. Size exclusion 29-31
3. Size exclusion 32-35
-15-
NEXT STEPS…
II. PRODUCE AND PURIFY
1. CXCL12α-SHL-Fc
Optimize cleavage of Fc tag from CXCL12α-SHL
Separate CXCL12α-SHL from tag and enzymes
Perform western blot to confirm presence of CXCL12α
2. CXCL12α
Mass spectrometry and amino acid sequencing
3. SHL-CXCL12α-Fc
Optimize production
III. CHARACTERIZE
Binding to ECM components
Study bioactivity of CXCL12α
Compare binding and bioactivity of CXCL12α-SHL
with
that of CXCL12γ
-16-
In-vivo model
1. Induce infarction by coronary
artery ligation and reperfuse
2. Inject saline, wtCXCL12α,
wtCXCL12γ, OR CXCL12αPlGF2 variant into infarcted
area after surgery
3. Study regeneration of
myocardium through
H&E staining
Immunohistochemistry
Flow cytometry
-17-
Acknowledgements
Dr. Jeffrey Hubbell
Priscilla Briquez
Jean-Phillipe Gaudry
Protein Expression Core Facility
Fellow members of LMRP
ERC Cytrix Grant
-18-
Thank you for your attention
QUESTIONS?
-19-
S1. Previous studies by LMRP
GF binding to ECM proteins, measured by ELISA4
Representative histology at 15 days for the topical
groups (hematoxylin and eosin staining). Black
arrows indicate wound edges; red arrows indicate tips
of the healing epithelium tongue. Scale bar, 1 mm. 4
Db/db mice with skin wound
4. M. Martino et al, Science, 343: 885-888, 2014
-20-
S2. Primers
CXCL12-SHL AAG CCC GTC AGC CTG AGC TAC AGA TGC CCA TGC CGA TTC TTC GAA AGC CAT GTT GCC AGA GCC AAC GTC AAG CAT
CTC AAA ATT CTC AAC ACT CCA AAC TGT GCC CTT CAG ATT GTA GCC CGG CTG AAG AAC AAC AAC AGA CAA GTG TGC
ATT GAC CCG AAG CTA AAG TGG ATT CAG GAG TAC CTG GAG AAA GCT TTA AAC AAG CGC AGA CGA CCG AAA GGT AGA
GGC AAG AGG AGA CGA GAG AAG CAG AGG CCG ACC GAT AGT CAT CTG
1. CXCL12 –SHL Rev1 (57 bp)
5’-CGTCTCCTCTTGCCTCTACCTTTCGGTCGTCTGCGCTTGTTTAAAGCTTTCTCCAGG-3’
2. Rev2 SHL+R (66 bp)
5’- ATAATATGGATCCGCGCAGATGACTATCGGTCGGCCTCTGCTTCTCTCGTCTCCTCTTGCCTCTAC-3’
1-2. MCS CXCL12 Fwd (40 bp)
5’-AATTACCGGTGAC AAGCCCGTCAGCCTGAGCTACAGATGC-3’
SHL-CXCL12 CGC AGA CGA CCG AAA GGT AGA GGC AAG AGG AGA CGA GAG AAG CAG AGG CCG ACC GAT AGT CAT CTG AAG CCC
GTC AGC CTG AGC TAC AGA TGC CCA TGC CGA TTC TTC GAA AGC CAT GTT GCC AGA GCC AAC GTC AAG CAT CTC AAA
ATT CTC AAC ACT CCA AAC TGT GCC CTT CAG ATT GTA GCC CGG CTG AAG AAC AAC AAC AGA CAA GTG TGC ATT GAC
CCG AAG CTA AAG TGG ATT CAG GAG TAC CTG GAG AAA GCT TTA AAC AAG
1. SHL-CXCL12 Fwd (50 bp)
5’-CAGAGGCCGACCGATTGTCATCTGAAGCCCGTCAGCCTGAGCTACAGATG-3’
2. Fwd A - SHLdom (55 bp)
5’- GAAAGGTAGAGGCAAGAGGAGACGAGAGAAGCAGAGGCCGACCGATAGTCATCTG-3’
3. Fwd B – SHLdom (58 bp)
5’- TATATTACCGGTGACCGCAGACGACCGAAAGGTAGAGGCAAGAGGAGACGAGAGAGGC-3’
1-3. MCS CXCL12 Rev (50 bp)
5’-GCCTGCTGGATCC CTTGTTTAAAGCTTTCTCCAGGTACTCCTGAATCCAC-3’
-21-
S3. Methods to ligate CXCL12α and Fc
tag
1. CXCL12α + Fc then add plasmid vector in one reaction
after 1hr of incubation
1.
Varied ratio of CXCL12 to Fc
2. CXCL12α + Fc with two restriction sites
3. CXCL12α + Fc with one restriction site
4. Cut band from ligation of only CXCL12α and Fc at
correct size and amplify by PCR
-22-
S4. Restriction of CXCL12α variants
5
1
2
3
1.
2.
3.
4.
5.
6.
7.
CXCL12-SHL (AgeI & BamHI)
CXCL12noHindIII (BamHI)
Fc tag (BamHI)
Fc tag (BamHI &HindIII)
pXLG (BamHI & AgeI)
pXLG (AgeI & HindIII)
CXCL12-Fc (AgeI & HindIII)
1.
2.
3.
4.
CXCL12-SHL (BamHI &BgIII)
CXCL12-Fc (BamHI &BgIII)
SHL-CXCL12 (BamHI &BgIII)
CXCL12-Fc (BamHI &BgIII)
4
8
9
6
7
10 11
-23-
S5. SDS-page for western blots
1 1* 2 2* 3 4 4* 5 5* 6 6* 7 8
1 1* 2 2* 3 4 4* 5 5* 6 6* 7 8
50
25
10
3. Western blot anti-his
SDS-page staining of gel after WB transfer
1.
2.
3.
4.
Before his purification
After his purification
Dialysis in 1X PBS
After EK cleavage
Western blot with anti-CXCL12
1. Size exclusion 12-16
2. Size exclusion 29-31
3. Size exclusion 32-35
-24-