Tricks and improvements in Structural Genomics Chantal Abergel Structural and Genomic Information Laboratory UMR 2589-CNRS, IBSM, 31 chemin J. Aiguier,

Slides:

Advertisements

Similar presentations

Antibodies Analytical Techniques Utilizing Antibodies: flow cytometry

Advertisements

Recombinant Expression of PDI in E. coli

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

Ameer Effat M. Elfarash Dept. of Genetics Fac. of Agriculture, Assiut Univ. Gene Expression.

Abstract Enterococci are a part of the normal flora of the human intestine and can reach concentrations of up to 10 8 CFU/gram in feces. As an opportunistic.

Growing Protein Crystals

Western Analysis Laboratory procedure that allows you to:

Protein Purification Molecular weight Charge Solubility Affinity.

Review: Amino Acid Side Chains Aliphatic- Ala, Val, Leu, Ile, Gly Polar- Ser, Thr, Cys, Met, [Tyr, Trp] Acidic (and conjugate amide)- Asp, Asn, Glu, Gln.

Protein Purification and Analysis Day 4. Amino Acids, Peptides, and Proteins.

© Johan Zeelen M.P.I. of Biophysics The crystallization of (membrane) proteins, using a flexible Sparse Matrix Screen. Soluble proteins and membrane proteins.

Bio 98 - Lecture 4 Amino acids, proteins & purification.

Southern Analysis: Hybridization, Washing, and Detection.

Structural Interaction of Thioredoxin (Trx) and Thioredoxin- interacting Protein (Txnip) Haydeliz Martínez-Ruiz University of Puerto Rico at Mayagüez Dr.

Chapter 10: Analysis of proteins. Purification schemes: 1. soluble recombinant proteins 2. insoluble recombinant proteins that are produced as inclusion.

Affinity Chromatography Yongting Wang Jan07. What is AC? Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect.

Updates on Prion Protein Purification Donovan Duggan Bow Suriyamongkol Supervisor: Dr. David Wishart Prion Group Meeting 2 February, 2007.

Plasmid purification lab

Plasmid DNA Isolation Exercise 8.

Extraction of Human DNA

Ion Exchange Laboratory

Towards Systematic Identification of cdiGMP Binding Proteins

High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Charlotte Brink, Carina Engstrand, Eva Heldin.

23 May June May 2002 From genes to drugs via crystallography 19 May 1996 Experimental and computational approaches to structure based.

PSI Data Management and Reporting: Expectations, Standards and Utility J. Michael Sauder Director, Bioinformatics NYSGXRC Project Leader.

18.7 Isolation, Purification, and Fractionation of Proteins (1)

Biochemistry February Lecture Analytical & Preparative Protein Chemistry II.

AC part 1 Aug Affinity Chromatography. AC part 1 Aug What is it used for? Monoclonal and polyclonal antibodies Fusion proteins Enzymes DNA-binding.

Table 5-1 Protein Purification Essential for characterizing individual proteins (determining their enzymatic activities, 3D structures, etc.) Two main.

Chapter 3.2 and 3.3: Peptides, Proteins, and Working with Proteins CHEM 7784 Biochemistry Professor Bensley.

Antimicrobials from Lactobacillus plantarum isolated from turmeric (Curcuma longa linn.) and their applications as biopreservative and in edible film Melada.

BIOCHEMICAL METHODS USED IN PROTEN PURIFICATION AND CHARACTERIZATION

Ion Exchange Laboratory

EXPRESSION / PURIFICATION ER ER / TIF2 COMPLEX FORMATION GR CONSTRUCT TIF2 CONSTRUCT ER CONSTRUCT EXPRESSION / PURIFICATION TIF2 CO-EXPRESSION GR / TIF2.

Proteomics Module Day 1 Tech talk. Experiment: Yeast protein expression changes caused by H 2 O 2 exposure. ► 2 Control groups (A and B): nothing added.

Figure S1 pEG + pJG pEG + pJG DUG2 pEGDUG3 + pJG pEGDUG3 + pJGDUG2 pEG202 + pJGDUG WD40 domain pEGDUG3 + pJGDUG2 WD40 domain +ve control -Ve control CM+Leu.

Workflow Analysis for the Northeast Structural Genomics Consortium at the CABM/Rutgers University/RWJMS Protein Production Facility October 22, 2002 Celia.

Finish up array applications Move on to proteomics Protein microarrays.

Antibody Detection. Part II Detection of Antigens Western Blotting “Dot-Blot Assay” Positive Control Primary antibody (IgY) Lysed E. Coli Cells Lysed.

Structure to Elucidate Mechanism

Protein Primary Sequence Protein analysis road map: Bioassay design Isolation/purification Analysis Sequencing.

Proteomics The science of proteomics Applications of proteomics Proteomic methods a. protein purification b. protein sequencing c. mass spectrometry.

Heterologous Protein Expression in Yeast CoHo7e - Green, Core and HA Malcolm Stratford & Hazel Steels MOLOGIC.

DESIGN AND CHARACTERIZATION OF SYMMETRIC HALF BARRELS

Presented by Subproject 6 of the MPEC Roadmap Grant P50 GM Membrane Protein Production for Crystallization Goals, Approach, Progress and Examples.

Facility I: Production and Characterization of Proteins

Separation techniques ?. Molecules can be separated: Chemically: by charge, by action with specific reagents Physically: by solubility, by molecular weight,

1 Protein-Protein Interactions High-throughput strategy –Prediction from sequence In silico analysis –Protein A from species A: domain 1 and 2 –Protein.

Genomic Screening Princess Kara Parker-Smith Marlyn Gonzalez and Dr. Peter Lipke Hunter College, CUNY Harlem Children Society.

Rochester Data in Sesame Logging in to Genie to handle 96-well plates

Saida Almashharawi Basic Biochemistry CLS 233 Ch4: Protein Purification 1.

Bringing structural biology services through collaboration.

Protein Overexpression in E. coli and

Protein Overexpression in E

Protein Overexpression in E. coli and

WESTERN BLOT Reagents: 2x SDS buffer Running buffer Transfer buffer

Characterization of the acid phosphatase from Staphylococcus aureus strain 154 and its development as a reporter system.

Page 1 Protein expression system in Profacgen(1) by Profacgen.

Figure S1. Production of recombinant NS1 protein

Amino Acids, Peptides, and Proteins

Fac. of Agriculture, Assiut Univ.

Protein Seperation Methods

Ion Exchange Laboratory

Plasmid DNA Isolation.

Different applications of protein electrophorasis

Bioinformatics, Vol.17 Suppl.1 (ISMB 2001) Weekly Lab. Seminar

Characterization of a Novel Isoform of α-Nascent Polypeptide-associated Complex as IgE-defined Autoantigen Roschanak Mossabeb, Susanne Seiberler, Irene.

Flora Ambre Honoré, Vincent Méjean, Olivier Genest Cell Reports

Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins Toshimitsu Kawate, Eric Gouaux Structure

Plasmid DNA Isolation.

Presentation transcript:

Tricks and improvements in Structural Genomics Chantal Abergel Structural and Genomic Information Laboratory UMR 2589-CNRS, IBSM, 31 chemin J. Aiguier, Marseille, FRANCE

Microbial Genomics Bioinformatics Structural Genomics Mimivirus - T. whipplei - M. timonensis - A. baumannii Functional Genomics Experiments R. felis - R. africae - R. belli - R. massiliae, R. conorii - Exploration of microbial genomes Fundamental: surprises Directed: biomedical

Structural genomics projects: TargetDB Escherichia coli Orfan genes Conserved genes of unknown function search for new antibiotic targets PROFUN : search for new antifungal targets Saccharomyces cerevisiae: Essential genes conserved in fungi absent in vertebrate genomes Candida albicans: Conserved in fungal pathogens absent in vertebrate genomes

MasterDB

~ 300 Targets

Incomplete Factorial Design: Experimental matrix (SAmBa) Automated protocol, 12 conditions (out of 96 possible) 4 strains 3 temperatures 3 growth media

Dot Blot for recombinant protein detection  Total extracts and soluble extracts (supernatant after centrifugation) deposited on nitrocellulose membranes  Use of anti (his) 5 antibody coupled HRP (Qiagen)  ECL detection Dot blot statistical analysis Soluble expression condition Up to 12 proteins tested a day 90% Rosetta 17°C 2YT

Microdialysis Affinity purification Scale up (1L culture) pI Non ionic ionic pH Overnight dialysis OD total OD soluble

Desalting Concentration Best buffer: OD total /OD soluble ~1 λ nm Absorbance

Electrophoresis (native, IEF, SDS) N-terminal Sequencing /Mass Spectrometry Circular Dichroïsm Dynamic light scattering Stability over time Biochemical & Biophysical Characterization Purification optimization Crystallization Stability of produced proteins ? Folding ? Protein solution monodispersity

Combinatorial approach Functional & Structural predictions Functional & Structural predictions Bioinformatics Bioinformatics sequence homologies (BMCD) binding motif, hypothetical ligand, co-factors… Electrophoresis (native, IEF…) Electrophoresis (native, IEF…) Set of variables to be tested

SAmBA: For crystallization prospection

Tecan diluting distributing robot 8 needles Greiner crystallization plates 5x96 wells

Promising crystals...

In house testing...

Poor diffraction

Soaking/desiccation

YbgL Step 1

Step 2 YbgL

Final YbgL

YggV Step 1

Final YggV

CA0996 Step 1

Final CA0996

What do they have in common ? (1) Mol. weight (kDa) Oligomerization Space group Cell dimension (Å) Molecule per AU YbgL26monomerC2C2 a = 70.3 b = 36.7 c = 79.7 β = YggV21monomerP a = 78 c = CA099616dodecamerP222 a = 93 b = c =

What do they have in common ? (2) Crystallization conditions CryoprotectantSoaking time Dataset Resolution ESRF YbgL Sodium citrate pH 9.15 Ethylene glycol 10% 2h FIP :2.29 Å R merge : 5.8% (28.7) YggV (NH 4 ) 2 SO 4 pH 9 glycerol 10%30 mins ID14: 2.0 Å R merge : 6.4 % (29.3) CA0996 PEG8000 LiSO 4 pH 7 glycerol 10 %15 minsFIP: 3.0

Experimental Team Sabine Chenivesse Sophie Cozzani Céline Deregnaucourt Sandra Jeudy Marjorie Varagnol Bioinformatics Team Hiroyuki Ogata Stéphane Audic Olivier Poirot Pr. Jean-Michel Claverie