One-step affinity purification and processing of fusion proteins Philip Bryan.

Slides:



Advertisements
Similar presentations
Protein Purification and Analysis Numbers of genes: Humans~40,000 genes Yeast~6000 genes Bacteria~3000 genes Solubility of proteins important for purification:
Advertisements

Chapter 5 Proteins: Primary Structure Chapter 5 Proteins: Primary Structure Biochemistry I Dr. Loren Williams Biochemistry I Dr. Loren Williams Revised.
Purification of bioengineered proteins CPSC 265 Week 12.
Affinity Chromatography: Homemade Microcystin-Sepharose Column Cindy Lee May 1, 2006.
2004 PP&CW Optimization of protein expression and solubility Alternative and novel prokaryotic expression systems Eukaryotic expression systems Methods.
Affinity Chromatography Yongting Wang Jan07. What is AC? Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect.
Supervisor: Dr. David Wishart
Protein purification Chromatography – Mikhail Tsvett (1901) pioneered the technique while attempting to separate yellow and red pigments from green leaves.
Enzymes are Proteins with Defined 3D Structures Ribonuclease A 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)  -chymotrypsin Active site:
Introduction to biotechnology Haixu Tang School of Informatics.
High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Charlotte Brink, Carina Engstrand, Eva Heldin.
AC part 1 Aug Affinity Chromatography. AC part 1 Aug What is it used for? Monoclonal and polyclonal antibodies Fusion proteins Enzymes DNA-binding.
Expression of clones genes a.o. Primrose & Twyman, 7th edition, pp Primrose, Twyman & Old, 6th edition, pp
Microbial Biotechnology Philadelphia University
Basic Ideas on Structure Biological macromolecules = biopolymers repeating subunits – 4 general types: 1.Proteins – linear chains of aa (~ 20 types) masses.
Determining if the fused product of Botox A and GFP can be used to observe the binding patterns of Botulinum toxin A. Felicia Yothers Department of Biological.
1 Lab Report: GARP 2 & Stains-All studies Fernanda Balem Department of Pharmacology 10/17/05.
A Study of Starch Metabolizing Proteins Pam Brewer-Michael 1, Tracie Hennen-Bierwagen 2, Myers /James Laboratory 2 1 Marshalltown Community School District,
Workflow of the Manual Purification of N/NC5-enriched proteins
Question: How do we know where a particular protein is located in the cell?
Week 6 Review. DNA UV Spectra DNA and RNA Bases.
BTE 204: Fundamentals of Genetic Engineering
CROMATOGRAFIA. Anion-exchange Protein at pH higher than pI (by at least 1 unit) is negatively charged and binds to anion-exchange column. Elution.
Protein Overexpression in E. coli and
Protein Overexpression in E
Protein Purification bYSY.
Protein Overexpression in E. coli and
Soybean Lipoxygenase: Which amino acids matter?
Pratistha Ranjitkar, Amanda M. Brock, Dustin J. Maly 
Target protein Additional file 3. SDS-PAGE showing the degree of purification of D1-26PtxtPL1-27 expressed in E. coli. PtxtPL1-27.
Enzymes are Proteins with Defined 3D Structures
VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13 by Koichi Kokame, Masanori Matsumoto, Yoshihiro.
Volume 32, Issue 1, Pages (October 2008)
Coordination of Rho and Rac GTPase Function via p190B RhoGAP
by Chungho Kim, Tong-Lay Lau, Tobias S. Ulmer, and Mark H. Ginsberg
Kre1p, the Plasma Membrane Receptor for the Yeast K1 Viral Toxin
Volume 7, Issue 8, Pages (August 2000)
Volume 11, Issue 17, Pages (September 2001)
Glen S. Cho, Jack W. Szostak  Chemistry & Biology 
Protein purification and quality control.
TEV protease elution from cellulose is specific and provides highly purified target protein. TEV protease elution from cellulose is specific and provides.
Volume 16, Issue 6, Pages (June 2009)
Volume 94, Issue 1, Pages (July 1998)
Dimers Probe the Assembly Status of Multimeric Membrane Proteins 
Volume 10, Issue 12, Pages (December 2003)
Volume 113, Issue 4, Pages (May 2003)
(a) (b) FhGALE M U I S W1 W2 E1 E2 E3 M - +
M.Brandon Parrott, Michael A. Barry  Molecular Therapy 
Volume 48, Issue 2, Pages (October 2005)
Volume 91, Issue 4, Pages (November 1997)
Rachel L Winston, Joel M Gottesfeld  Chemistry & Biology 
Volume 7, Issue 8, Pages (August 2000)
Volume 1, Issue 2, Pages (January 1998)
Volume 10, Issue 5, Pages (May 2002)
Scott Gradia, Samir Acharya, Richard Fishel  Cell 
p53 Protein Exhibits 3′-to-5′ Exonuclease Activity
Endonuclease-Mediated mRNA Decay Involves the Selective Targeting of PMR1 to Polyribosome-Bound Substrate mRNA  Feng Yang, Daniel R Schoenberg  Molecular.
The DnaK Chaperone System Facilitates 30S Ribosomal Subunit Assembly
Volume 3, Issue 2, Pages (August 2002)
Volume 31, Issue 6, Pages (September 2008)
Volume 76, Issue 4, Pages (April 1999)
Volume 13, Issue 4, Pages (April 2006)
Expression MosIR binding by dsRBPs TARBP and PACT.
Protein purification and quality control.
George Simos, Anke Sauer, Franco Fasiolo, Eduard C Hurt  Molecular Cell 
Bacillus subtilis Glutamine Synthetase Controls Gene Expression through a Protein- Protein Interaction with Transcription Factor TnrA  Lewis V Wray, Jill.
Neutrophil Elastase Cleaves PML-RARα and Is Important for the Development of Acute Promyelocytic Leukemia in Mice  Andrew A. Lane, Timothy J. Ley  Cell 
Volume 9, Issue 3, Pages (November 2014)
An Activator Target in the RNA Polymerase II Holoenzyme
PBMC- and T cell-dependent cytotoxic activity of DCH and variant proteins. PBMC- and T cell-dependent cytotoxic activity of DCH and variant proteins. A.
Presentation transcript:

One-step affinity purification and processing of fusion proteins Philip Bryan

One-step purification: affinity purification of fusion proteins, removal of the fusion domain isolation of the target protein

Target protein is fused onto an engineered pro-region of subtilisin

Precise fusion of target protein onto pro-region Nde I ProR58 Target protein Eco R1 Hind III Sal I Expression Vector pG58

Synthesis of fusion protein Fusion protein Cell extract

Binding of fusion protein to Sbt column Flow-through from column Loading on column

Elution of processed target protein Elution after overnight incubation Processed target protein

Purification of fusion protein Fusion protein Acid elution - No incubation

Pro-domain proR58 stripped from column with 0.1M H 3 PO 4 pH 2.1

Purified proteins Streptococcal protein G B domain 56 aa Streptococcal protein G a domain 45 aa Protein G B mutant G31156 aa Staphylococcal Protein A B domain 56 aa Protein A B mutant A21956 aa E. coli hypothetical Yab117 aa Bovine  subunit of transducin350 aa M. thermautotrophicus CDC6379 aa A. victoria Green Fluorescent Protein238 aa

Conformational switching A219 G311

15 N HSQC spectra G311 25˚C 2˚C

 -subunit bovine transducin (350aa) load Fractions pooled

 -subunit bovine transducin (350aa)

Processing activity: K d of fusion protein and SBT is < 1nM Binding of fusion protein to SBT is fast Processing is a slow, single turn-over reaction Precise N-terminus of target protein produced. K d of processed proR58 and SBT is < 0.1nM Non-specific cleavage is undetectable

Binding conditions: pH M NaCl 0-2M urea (folding on the column) 0-1M Gu-HCl

Column is tolerant of: EDTA PMSF Protease inhibitor cocktails Reducing agents

Biochemistry Patrick Alexander Kathryn Fisher Joel Hoskins Biao Ruan Sergei Ruvinov Susan Strausberg Lan Wang NIH GM42560 X-ray Orna Almog Travis Gallagher Gary Gilliland NMR John Orban Nese Sari