Proteins, Beyond Structure - P4EUSlow flow rate to prevent leakage of the uncleaved H2B/Ub from the...
Transcript of Proteins, Beyond Structure - P4EUSlow flow rate to prevent leakage of the uncleaved H2B/Ub from the...
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Shira Albeck
The Israel Structural Proteomics Center (ISPC) The Weizmann Institute of Science
www.weizmann.ac.il/ISPC
Proteins, Beyond Structure
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Structure
Functional Studies
Different entry points
Different exit points
Target Protein
Bioinformatics analysis
Cloning
Purification
Expression E.coli, Yeast, Insect & 293 cells
Crystallization
ISPC Strategy - From gene to 3D structure
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Expression of Proteins for Crystallization
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Domains Constructs Mutations Tags Expression system Bacterial strains Directed evolution Co-expression
Expression of Proteins for Crystallization Tricks employed at the ISPC….
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Animal Studies endotoxins, formulations, BBB
Biochemical and Biophysical Studies
Study of large proteins and complexes by EM & Structural MS
SSNMR
NMR
In cell NMR
Emerging Structural Studies
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Chemical manipulation of proteins - ubiquitylation of Proteins
Expression & purification of large complexes (slides removed from presentation)
Profinity eXactTM Protein Purification System
Outline
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How can we obtain specific H2B-Ub for biological studies?
Biological Activity
of Chemically Site Specific
Monoubiquitylated Histone H2B
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Auxiliary-Mediated Site-Specific Peptide Ubiquitylation
McGinty et al, Nature 453, 2008, 812-816
H2B-SR
H2B(1-116)-α-thioester
Ub-SR
Ubiquitin(1-75)-α-thioester
peptide 117-125
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SR
Chemical conjugation to H2B-SR
Ub-SR
Ub-pept.
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Intein-mediated Protein Ligation (IPL) Utilized to produce H2B-SR & Ub-SR
Chitin
CBD Intein
MeSNa
SR
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Non effective binding
to chitin beads (FT)
Non-specific cleavage
(before addition of MeSNa)
CBD-Ub elutes upon addition of MeSNa
(without cleavage)
Ub-CBD
CBD
Ub-SR
FT
Beads
Elution
Problems encountered producing Ub-SR & H2B-SR
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Add 1M Gd-HCl for the binding and cleavage to avoid precipitation of H2B-SR.
Slow flow rate to prevent leakage of the uncleaved H2B/Ub from the column.
Use pH 6.5 to prevent non-specific hydrolysis.
100mM MESNA improves cleavage.
Avoid Tris containing buffer – Use HEPES instead (to prevent Ub-S-Tris)
Final product purified by Reverse Phase to remove CBD and uncleaved Ub-CBD
Optimization to obtain working protocol
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Final Purification by Reverse Phase
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a. b.
a
b
Ub-SR HPLC and ESI-MS
a: Ub-COOH – Calc mass 8565 (hydrolysis)
b: Ub-SR – Calc mass 8689
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H6-H2b-Ubiquitin
Ubiquitinated - H2B HPLC and ESI-MS
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C&EN Oct, 2010
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BioRad
Profinity ExactTM Protein
Expression & Purification System (a test study)
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EEDKLFKAL
subtilisin
Subtilisin prodomain
Agarose Beads
Mutant subtilisin (S189) is immobilized on Superflow agarose resin.
The subtilisin mutant exhibits high binding affinity to an 8 kDa subtilisin prodomain, fused to the N-terminal of the recombinant protein.
The fused protein is separated by the 8 kDa tag by 9 amino acids at its C-terminal which are recognized and cleaved by the subtilisin protease.
Cleavage is triggered by 100mM potassium fluoride.
Ruan B. et al Biochemistry, 2004, 43 (46), pp 14539–14546
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Tag-free protein in a SINGLE purification process
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Cloning is done by the Restriction Free (RF) or Transfer-PCR (TPCR) methods
pPAL7/pPAL8
Tag Profinity-tag MCS
pET28-Profinity
pETTrx-Profinity
6xHis
Trx-tag
AmpR
KanR
KanR 6xHis
Cloned at the ISPC
Profinity ExactTM -based
expression vectors
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Protein expression using the Profinity eXact system
commercial pPAL7/pPAL8
M Elute
Wash
1 2 3
Lys F.T E Elute
M Lys F.T Wash
2 1 E
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pET28-Profinity pETTRx-Profinity pPAL8
No expression with pPAL8 Non complete binding Elution of non cleaved fusion
Protein expression using new vectors vs.
commercial pPAL7/pPAL8
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Additional linker (Thr-Ser) following the Profinity-tag might be needed to improve cleavage.
Buffer for cell lysis should NOT contain NaCl or KCl or Tris-Cl which trigger the cleavage process. We use 100 mM Sodium phosphate buffer pH-7.2 for binding/washing.
Cleavage can also be performed by 10 mM sodium azide.
Column regeneration- washing with 0.1 M H3PO4 following re-equilibration with binding buffer containing 100 mM sodium phosphate buffer.
Comments for the Profinity eXact system
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Joel Sussman Yigal Burstein Gideon Schreiber Israel Silman Shira Albeck Orly Dym Yoav Peleg Tamar Unger Reut Bernheim Ada Dantes Dikla Hiya Yossi Jacobovitch Shelly Rogotner Meital Yona-Rubin