Emily Buck Whitaker Conference April 30, 2015 Engineering the growth factor, CXCL12 α, for heart...
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Transcript of Emily Buck Whitaker Conference April 30, 2015 Engineering the growth factor, CXCL12 α, for heart...
Emily Buck
Whitaker Conference
April 30, 2015
Engineering the growth factor, CXCL12α, for heart tissue regeneration
Cardiovascular diseases account for the highest number of deaths worldwide1.
MAJOR CONSEQUENCES:
Loss in cardiac function
Possibility of complications
Heart tissue is damaged when blood supply is totally cut off from heart tissue
HEART ATTACK (MYOCARDIAL INFARCTION)
Blockage of coronary arteries causes a reduction in blood supply to heart tissue
CORONARY (ISCHEMIC) HEART DISEASE
1. World Health Organization, “Global status report on noncommunicable diseases,” p. 9, 2011 -2-
Ischemic tissue transiently expresses growth factors, such as VEGF and CXCL12α (SDF-1α), to initiate mechanisms for revascularization in the damaged area
2. C. Cencioni et al, Cardiovascular Research, 94: 400-407, 20123. M. Zhang et al, The FASEB Journal, 21: 3197-3207, 2007
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
-4-
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
How can we improve the regenerative capacity of CXCL12α?
-5-
My lab in Lausanne
Dr. Jeffrey Hubbell
Priscilla Briquez
Laboratory for Regenerative Medicine and Pharmacobiology (LMRP)
-6-
1. Design and Clone 2. Produce and Purify
3. Characterize ECM-binding affinity and bioactivity
5. D. Czajkowsky et al, EMBO Mol Med, 4: 1015-1028, 2012.
How will we engineer CXCL12α?
DESIGN:Establish sequence with the
PlGF-2 domain fused toN- OR C-terminus of CXCL12α
AND Fc tag to produce wild-type5
CLONE:PCR, agarose gel electrophoresis
Restriction, ligation and transformation into plasmid
Sequence verificationAmplify plasmid for production
PRODUCE:Transient gene expression of
CXCL12α variants in mammalian cells
SDS-Page, Western blotting
PURIFY:Separate CXCL12α from
contaminants through
His-tag affinity chromatography
Size exclusion chromatography
CHARACTERIZE:
Binding affinity for ECM components through…
ELISARelease from fibrin hydrogels
Bioactivity testing through…
MSC migration assay
Phosphorylation assay
-7-
Ligate
in plasmid by restrictionenzymes
I. DESIGN AND CLONEKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKwtCXCL12α
68 AA
SHL-CXCL12α90 AA
CXCL12α-SHL90 AA
RRRPKGRGKRRREKQRPTDSHLKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKRRRPKGRGKRRREKQRPTDSHL
PlGF-2123-14422 AA
RRRPKGRGKRRREKQRPTDCHL
CXCL12α-SHL (288 bp)
SHL-CXCL12α(288 bp) CXCL12-SHL
ORSHL-CXCL12
PLASMID:pXLG*Chis
AgeI
BamHI
200300
200300
Transforminto DH5α competent
bacteria
Verify DNA sequence
-8-
Ligate
in plasmid by restrictionenzymes
I. DESIGN AND CLONEKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKwtCXCL12α
68 AA
RRRPKGRGKRRREKQRPTDSHLKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK
PlGF-2123-14422 AA
RRRPKGRGKRRREKQRPTDCHL
CXCL12-SHL OR
SHL-CXCL12
PLASMID:pXLG*Chis
AgeI
BamHI
Transforminto DH5α competent
bacteria
200300
200300
Verify DNA sequence
CXCL12α-SHL (288 bp)
SHL-CXCL12α(288 bp)
SHL-CXCL12α90 AA
CXCL12α-SHL90 AA
-9-
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKRRRPKGRGKRRREKQRPTDSHL
Ligate
in plasmid by restrictionenzymes
I. DESIGN AND CLONEKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKwtCXCL12α
68 AA
Fc domain6
227 AA
Transform
into DH5α competent
bacteria
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK + FLAG cleavage site
CX
CL12-FcPLASMID:
pXLG*Chis
AgeI
HindIII
CXCL12α (204 bp)
200
300
Fc Tag (786 bp)
700
800 Ligate
two fragmentsby restrictionenzymes
CXCL12α-Fc (1056 bp)
Amplify
band of correct size
by PCR
CXCL12α-Fc (984 bp)
Verify DNA sequence
6. J.W. Murphy et al, J Biol Chem, 282: 10018-10027, 2007 -10-
50 kDa 15 kDa 10 kDa
II. PRODUCE AND PURIFYHEK ± VPA CHO ± DMSO
wt CXCL12α9.2 kDa
1. Supernatant +2. Supernatant –3. PBS wash +4. PBS wash –
5. NaCl wash +
6. NaCl wash –7. Pellet +8. Pellet -
HEK
CHO
CXCL12α-Fc36.6 kDa
CXCL12α-SHL12.1 kDa
SHL-CXCL12α12.1 kDa
SDS-page for Day 7 supernatant, PBS wash, NaCl wash, and pellet
Need to add Fc for PlGF-2 variants-11-
II. PRODUCE AND PURIFYFc tag for PlGF-2 variants
CXCL12-SHL
OR
SHL-CXCL12
PLASMID:pXLG*Chis Fc
Tag
BamHI
BgI II
Ligate Fc tag in PlGF-2 variant plasmids with restriction
enzymes
Transform into DH5α competent
bacteria
Verify DNA sequence
AgeI
-12-
1. CXCL12α-SHL-Fc (39 kDa)2. SHL-CXCL12α-Fc (39 kDa)
50 kDa 15 kDa 10 kDa
1 1* 2 2*
HEK + VPA
II. PRODUCE AND PURIFY His-affinity for CXCL12α-Fc
His purification fractions(left to right) A6, A6*, A7, A11, A13, B15, B15*, B13, B11, B5, B3, B1, B1*, and C3
Supernatant with CXCL12α-Fc
Contaminants
1. 2.
1. 2.
Elution buffer
Protein in elution buffer
As-received Day 7
supernatant
-13-
4 8 16 4 8 16* 4 8 16 0
50
25
10
Enterokinase (%) per weight CXCL12α-Fc
1. 0.0001% 2. 0.00064% (1 unit)
3. 0.01%
II. PRODUCE AND PURIFY Cleave Fc tag from CXCL12α-Fc 0.0001% 0.00064% 0.01%
CXCL12α Fc TagFLAG
+ Enterokinase (31 kDa)
CXCL12α + Fc TagFLAG
37 kDa
9 kDa 28 kDa
Incubate RT
Incubation time at room temperature
1. 4h2. 8h3. 16h
VARIABLE 1: VARIABLE 2:
EK to CXCL12α-Fc
-14-
3. Western blot anti-his
1 1* 2 2* 3 4 4* 5 5* 6 6* 7 8
10
25
50
12 12* 13* 14*14 29 30 30* 31 32 33 34 35 36
Size exclusion chromatographyTo separate cleaved Fc tag from CXCL12α
II. PRODUCE AND PURIFY Separate Fc tag from CXCL12α
Fc tag + EK CXCL12α
Western blotTo confirm presence of CXCL12α after cleavage
1. Before his purification2. After his purification3. Dialysis in 1X PBS4. After EK cleavage
5. Size exclusion 12-16
6. Size exclusion 29-317. Size exclusion 32-35
-15-
NEXT STEPS…II. PRODUCE AND PURIFY 1. CXCL12α-SHL-Fc
Optimize cleavage of Fc tag from CXCL12α-SHLSeparate CXCL12α-SHL from tag and enzymesPerform western blot to confirm presence of
CXCL12α
2. CXCL12αMass spectrometry and amino acid sequencing
3. SHL-CXCL12α-FcOptimize production
III. CHARACTERIZE
Binding to ECM componentsStudy bioactivity of CXCL12αCompare binding and bioactivity of CXCL12α-
SHL with that of CXCL12γ -16-
-17-
In-vivo model
1. Induce infarction by coronary artery ligation and reperfuse
2. Inject saline, wtCXCL12α, wtCXCL12γ, OR CXCL12α-PlGF2 variant into infarcted area after surgery
3. Study regeneration of myocardium through
H&E stainingImmunohistochemistryFlow cytometry
Acknowledgements
Dr. Jeffrey Hubbell Priscilla Briquez Jean-Phillipe Gaudry
Protein Expression Core Facility
Fellow members of LMRP
-18-
ERC Cytrix Grant
S1. Previous studies by LMRP
GF binding to ECM proteins, measured by ELISA4
4. M. Martino et al, Science, 343: 885-888, 2014
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
-20-
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
CXCL12-SHL
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’
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
SHL-CXCL12
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’
S2. Primers
-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 variants5
8
741 2 3
1. CXCL12-SHL (AgeI & BamHI)2. CXCL12noHindIII (BamHI)3. Fc tag (BamHI)4. Fc tag (BamHI &HindIII)5. pXLG (BamHI & AgeI)6. pXLG (AgeI & HindIII)7. CXCL12-Fc (AgeI & HindIII)
8. CXCL12-SHL (BamHI &BgIII)9. CXCL12-Fc (BamHI &BgIII)10. SHL-CXCL12 (BamHI &BgIII)11. CXCL12-Fc (BamHI &BgIII)
6
9 10 11
-23-
S5. SDS-page for western blots
3. Western blot anti-his
1 1* 2 2* 3 4 4* 5 5* 6 6* 7 8
10
25
50
SDS-page staining of gel after WB transfer Western blot with anti-CXCL12
1 1* 2 2* 3 4 4* 5 5* 6 6* 7 8
1. Before his purification2. After his purification3. Dialysis in 1X PBS4. After EK cleavage
5. Size exclusion 12-16
6. Size exclusion 29-317. Size exclusion 32-35
-24-