Shira Albeck

The Israel Structural Proteomics Center (ISPC) The Weizmann Institute of Science

www.weizmann.ac.il/ISPC

Proteins, Beyond Structure

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

Expression of Proteins for Crystallization

Domains Constructs Mutations Tags Expression system Bacterial strains Directed evolution Co-expression

Expression of Proteins for Crystallization Tricks employed at the ISPC….

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

Chemical manipulation of proteins - ubiquitylation of Proteins

Expression & purification of large complexes (slides removed from presentation)

Profinity eXactTM Protein Purification System

Outline

How can we obtain specific H2B-Ub for biological studies?

Biological Activity

of Chemically Site Specific

Monoubiquitylated Histone H2B

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

SR

Chemical conjugation to H2B-SR

Ub-SR

Ub-pept.

Intein-mediated Protein Ligation (IPL) Utilized to produce H2B-SR & Ub-SR

Chitin

CBD Intein

MeSNa

SR

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

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

Final Purification by Reverse Phase

a. b.

a

b

Ub-SR HPLC and ESI-MS

a: Ub-COOH – Calc mass 8565 (hydrolysis)

b: Ub-SR – Calc mass 8689

H6-H2b-Ubiquitin

Ubiquitinated - H2B HPLC and ESI-MS

C&EN Oct, 2010

BioRad

Profinity ExactTM Protein

Expression & Purification System (a test study)

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

Tag-free protein in a SINGLE purification process

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

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

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

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

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