Proteins, Beyond Structure

Transcription

1 Proteins, Beyond Structure Shira Albeck The Israel Structural Proteomics Center (ISPC) The Weizmann Institute of Science

2 ISPC Strategy - From gene to 3D structure Target Protein Different entry points Bioinformatics analysis Cloning Different exit points Expression E.coli, Yeast, Insect & 293 cells Purification Crystallization Functional Studies Structure

3 Expression of Proteins for Crystallization

4 Expression of Proteins for Crystallization Tricks employed at the ISPC. Domains Constructs Mutations Tags Expression system Bacterial strains Directed evolution Co-expression

5 Emerging Structural Studies Study of large proteins and complexes by EM & Structural MS SSNMR NMR In cell NMR Animal Studies endotoxins, formulations, BBB Biochemical and Biophysical Studies

6 Outline Chemical manipulation of proteins - ubiquitylation of Proteins Expression & purification of large complexes (slides removed from presentation) Profinity exact TM Protein Purification System

7 Biological Activity of Chemically Site Specific Monoubiquitylated Histone H2B How can we obtain specific H2B-Ub for biological studies?

8 Auxiliary-Mediated Site-Specific Peptide Ubiquitylation McGinty et al, Nature 453, 2008, Ub-SR Ubiquitin(1-75)-α-thioester peptide H2B-SR H2B(1-116)-α-thioester

9 Ub-pept. SR Ub-SR Chemical conjugation to H2B-SR

10 Intein-mediated Protein Ligation (IPL) Utilized to produce H2B-SR & Ub-SR Intein CBD SR Chitin MeSNa

11 Problems encountered producing Ub-SR & H2B-SR Non effective binding to chitin beads (FT) Ub-CBD CBD Non-specific cleavage Ub-SR (before addition of MeSNa) CBD-Ub elutes upon addition of MeSNa Elution Beads FT (without cleavage)

12 Optimization to obtain working protocol 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

13 Final Purification by Reverse Phase

14 a. b. Ub-SR HPLC and ESI-MS b a a: Ub-COOH Calc mass 8565 (hydrolysis) b: Ub-SR Calc mass 8689

15 H 6 -H2b-Ubiquitin Ubiquitinated - H2B HPLC and ESI-MS

16 C&EN Oct, 2010

17 Profinity Exact TM Protein Expression & Purification System (a test study) BioRad

18 Agarose Beads subtilisin EEDKLFKAL Subtilisin prodomain 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

19 Tag-free protein in a SINGLE purification process

20 Profinity Exact TM -based expression vectors Tag Profinity-tag MCS ppal7/ppal8 Amp R pet28-profinity 6xHis Kan R pettrx-profinity Trx-tag 6xHis Kan R Cloned at the ISPC Cloning is done by the Restriction Free (RF) or Transfer-PCR (TPCR) methods

21 Protein expression using the Profinity exact system commercial ppal7/ppal8 M Elut E e Lys F.T Wash Elut E e Wash 2 1 F.T Lys M

22 Protein expression using new vectors vs. commercial ppal7/ppal8 pet28-profinity pettrx-profinity ppal8 No expression with ppal8 Non complete binding Elution of non cleaved fusion

23 Comments for the Profinity exact system 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 H 3 PO 4 following reequilibration with binding buffer containing 100 mm sodium phosphate buffer.

24 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