Expression and Purification of Membrane Proteins from Leishmania major for Structural Genomics. Nadia Fedoriw, Kathy M. Clark, Sara M. Connelly, Katrina.

Slides:



Advertisements
Similar presentations
Section H Cloning Vectors
Advertisements

Ameer Effat M. Elfarash Dept. of Genetics Fac. of Agriculture, Assiut Univ. From Gene to Protein (an overview)
Ameer Effat M. Elfarash Dept. of Genetics Fac. of Agriculture, Assiut Univ. Gene Expression.
Genomics: READING genome sequences ASSEMBLY of the sequence ANNOTATION of the sequence carry out dideoxy sequencing connect seqs. to make whole chromosomes.
The Yeast Two-Hybrid System Anne C. Luebke. What is the yeast two-hybrid system used for? n Identifies novel protein-protein interactions n Can identify.
The cloning and expression of SNAP-25a and b in zebrafish Maia Lavarias*, Dr. Wendy Boehmler Department of Biology, York College of Pennsylvania, York,
PURIFICATION OF YEAST MEMBRANE PROTEINS FOR STRUCTURAL GENOMICS Center for High Throughput Structural Biology Mark E. Dumont *†, Nadia Fedoriw *, Kathy.
Chapter 4: recombinant DNA
Purification of bioengineered proteins CPSC 265 Week 12.
2004 PP&CW Optimization of protein expression and solubility Alternative and novel prokaryotic expression systems Eukaryotic expression systems Methods.
Protein Purification and Expression MCB 130L, Lecture 2.
Protein Purification and Expression
Visual Basic and Perl Applications for Genome Project Management. Svetlana N.Yurgel* 1, Brenda K. Schroeder 1, Hao Jin 2 and Michael L. Kahn 1,3. Institute.
Affinity Chromatography Yongting Wang Jan07. What is AC? Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect.
Center for Human Genetics and Molecular Pediatric Disease
Cloning:Recombinant DNA
4 September, 2006 Chapters Methods: Proteins, Model Systems I.
Protein Production and Crystallization Workshop Structural Genomics of Pathogenic Protozoa (SGPP) Crystal Growth Lab Lori Anderson
Plasmid purification lab
Protein Purification. Why purify Proteins? Characterize Function Activity Structure Study protein regulation and protein interactions Use in assays Produce.
Construction, Transformation, and Prokaryote Expression of a Fused GFP and Mutant Human IL-13 Gene Sequence Lindsay Venditti, Department of Biological.
Towards Systematic Identification of cdiGMP Binding Proteins
Manufacture of Human Interleukin 13 Protein Using a Prokaryotic Expression System Ryan Rupp, York College of Pennsylvania, Department of Biological Sciences.
歐亞書局 PRINCIPLES OF BIOCHEMISTRY Chapter 9 DNA-Based Information Technologies.
Biochemistry February Lecture Analytical & Preparative Protein Chemistry II.
Table 5-1 Protein Purification Essential for characterizing individual proteins (determining their enzymatic activities, 3D structures, etc.) Two main.
Expression of clones genes a.o. Primrose & Twyman, 7th edition, pp Primrose, Twyman & Old, 6th edition, pp
Expression and Purification of Integral Membrane Proteins from Yeast for the Center for High-Throughput Structural Biology Kathy Clark *, Nadia Fedoriw.
1 Genetics Faculty of Agriculture Instructor: Dr. Jihad Abdallah Topic 13:Recombinant DNA Technology.
Chapter 20 Experimental Systems Dr. Capers.  In vivo ○ Involve whole animal  In vitro ○ Defined populations of immune cells are studied under controlled.
Finish up array applications Move on to proteomics Protein microarrays.
Microbial Biotechnology Philadelphia University
PREDICTING THE EXPRESSION AND SOLUBILITY OF MEMBRANE PROTEINS Center for High Throughput Structural Biology Mark E. Dumont *†, Michael A. White *, Kathy.
Plasmids Indispensable tools that allow molecular biologists to obtain essentially unlimited amounts of a DNA sequence Small circular DNA molecules that.
Proteomics The science of proteomics Applications of proteomics Proteomic methods a. protein purification b. protein sequencing c. mass spectrometry.
A facile method that allows high-throughput co- expression from plasmids with identical replication origins and antibiotic resistance markers (University.
Engineering yeast to produce proteins for X-ray Crystallography: Heterologous Expression of L. MAJOR proteins in the yeast S. cerevisiae.
1 Human metabotropic glutamate receptor 6: Expression and purification Kalyan Tirupula Graduate Student JKS Lab, UPitt.
STRUCTURAL BIOLOGY Martina Mijušković ETH Zürich, Switzerland.
Heterologous Protein Expression in Yeast CoHo7e - Green, Core and HA Malcolm Stratford & Hazel Steels MOLOGIC.
High-throughput Screening of Soluble Recombinant Proteins Protein Science, 2002, vol 11, YAN-PING SHIH,1 WEN-MEI KUNG,1 JUI-CHUAN CHEN, CHIA-HUI.
Facility I: Production and Characterization of Proteins
Membrane Protein Production: 8/06/04 One target shipped: Lmaj000191: Mitochondrial ADP/ATP translocator in 2% octyl glucoside (predominant lower MW band).
Purification and Enzymatic Activity of Cfd1 and Nbp35 Mierzhati Mushajiang, Eric Camire, and Deborah Perlstein Department of Chemistry, Boston University,
GFP-based membrane protein overexpression and purification in E. coli and S. cerevisiae Joy Kim Center for Biomembrane Research Department of Biochemistry.
Chapter 16 Microbial Genomics “If we should succeed in helping ourselves through applied genetics before vengefully or accidentally exterminating ourselves,
Molecular Cloning.
Plasmid Isolation Prepared by Latifa Aljebali Office: Building 5, 3 rd floor, 5T250.
Application of Phage Antibody Technology to Structural Genomics: Antibody Selection and Complex Formation Mark A. Sullivan Center for Human Genetics and.
Protein Purification for Crystallization Dr Muhammad Imran Forman Christian College (A Chartered University) Dr Muhammad Imran Forman Christian College.
Cloning, Over-expression and Purification of NanoLuc Luciferase
Molecular Cloning. Definitions   Cloning :   Obtaining a piece of DNA from its original source (Genome) and introducing it in a DNA vector   Sub-cloning:
What is phage display? An in vitro selection technique using a peptide or protein genetically fused to the coat protein of a bacteriophage.
Sticky Bacteria… Bacteri-adhesive… Cling-E. coli… A working title Perry Tsai June 25, 2007.
Lecture 3 – Selection of Recombinants & clone analysis The white colonies will all be recombinants, but only one of these many colonies will contain the.
MOLECULAR CLONING BY LIZ GLENN. HISTORY 1970 discovery of restriction endonucleases in bacteria DNA ligase used to join sections of DNA, termed recombinant.
Prokaryotic Expression Systems
Lecture 8 A toolbox for mechanistic biologists (continued)
Production of Recombinant Proteins
Fac. of Agriculture, Assiut Univ.
Prokaryotic Expression Systems
Fusion Proteins Fusion protein Cleavage of fusion proteins
Fac. of Agriculture, Assiut Univ.
Engineering yeast to produce proteins for X-ray Crystallography: Heterologous Expression of L. MAJOR proteins in the yeast S. cerevisiae.
Chapter 14 Bioinformatics—the study of a genome
Recombinant DNA Technology
Recombinant DNA Technology
Tagging of the endogenous Py03652 gene with the gfp gene in Plasmodium yoelii. Tagging of the endogenous Py03652 gene with the gfp gene in Plasmodium yoelii.
Presentation transcript:

Expression and Purification of Membrane Proteins from Leishmania major for Structural Genomics. Nadia Fedoriw, Kathy M. Clark, Sara M. Connelly, Katrina Robinson, Gayle Schneider, Wim G. Hol 1, and Mark E. Dumont Center for Human Genetics and Molecular Pediatric Disease and Department of Biochemistry and Biophysics. University of Rochester Medical Center. Rochester, NY Departments of Biochemistry and Biomolecular Structure, University of Washington, Seattle, Washington

9th grade Biology text

High resolution structures of transmembrane proteins (as of 1/05) (Includes multi-species) Bacterial helical membrane proteins: 26 Bacterial porin/β-barrel outer membrane proteins: 25 “Eukaryotic” helical membrane proteins: 3 Soluble structures in database (3/05) 29,956 (Source:

Membrane Proteins: Initial Strategies 1. Trypanosomatids only (initial) 2.  2 predicted transmembrane segments 3. Expression in Pichia Pastoris and E. coli 4. Ligation-Independent cloning into C-terminal cleavable double-tagged vector 5. Purified protein to be sent for crystallization in a small number of crystallography-proven detergents (~5) 6. Co-crystallization with single chain antibodies and two-hybrid binding partners

SGPP Membrane protein highlights: 3/2004-3/ Expression at  1 mg /liter of >40% of selected predicted transmembrane ORFs from L. major in an E. coli expression system. 2. Purification of 1-4 batches of 10 different L. Major predicted transmembrane proteins for crystallization 3. Detection of possible crystal hits for two ORFs at HWI 4. Confirmation of crystal hits for one ORF in optimizations set up in Rochester: 5. Partial or complete retention of PelB signal sequence in current vectors. 6. Construction and testing of a new vector allowing quantitative pelB cleavage. 7. Construction of vectors and cloning of P. falciparum ORFs for expression in Tetrahymena.

Cloning Strategy for Membrane Protein Expression Use ligation independent cloning to insert a single PCR- product into two E. coli vectors and two Pichia vectors Pichia pre-pro-α-factor signal seq. Pichia no added signal seq. E. coli pelB signal sequence E. coli no added signal seq. Single PCR product

Expression of L. major ORFs in SDS lysates of E. coli BL21(DE3) Codon plus Target selection: 3 separate groups selected for: 1) known enzymes 2) small size 3) diversity (random selection)

Evolution of a Strategy for Membrane Protein Exprssion/Purification Current StrategySteps addedSteps eliminated Targeting of full-length and signal sequence-truncated ORFs Cloning into multiple vectorsCloning into vector w/ 3C cleavable signal Transforming into Pichia Use of multiple E coli strains Transforming into expression host Transforming into Tetrahymena Testing different temperaturesSmall scale expression at 25 o C Membrane fractionation Screening of initial detergent Fos choline-16 extraction of whole-cell extract IMAC affinity - detergent exchange - 3C protease elution 6-18 liter cultures Gel Filtration - Centricon concentration ~1 liter optimization

Lmaj000817T 586 5_C0586 Potassium chloride KCl 0.1 M CAPS 0.1 M pH 10 PEG % (w/v)

E4 0.1M Tris, pH M MgSO4 PEG 4000, 15% E7 0.1M Tris, pH M KCl PEG 4000, 15%D4 0.1M Tris, pH M MgSO4 PEG 4000, 15% Crystallizations from 1% dodecylmaltoside (4 weeks) Lmaj000817T

(20% PEG M MgCl 2 0.1M Tris pH 8.5) Crystallization from 1% dodecylmaltoside (4 weeks) Crystallization from 0.8% dodecylmaltoside (12 days) 0.1 M Hepes pH 8.3, 0.1 M MgCl2, 21% PEG 4000 Lmaj000817T

PelB signal sequence: 2210 Daltons Partial removal of PelB signal sequence from Lmaj007473T MALDI-TOF

MALDI-TOF of Lmaj000817TAAA (13562 expected w/ signal) Expected for cleaved

PelB-containing LIC vectors (Insert Region) LIC Site 3C Protease Site ORF RGS-6His Calmodulin Binding Peptide STOP LIC Site ATG-Cleavable signal LIC Site 3C Protease Site ORF 6His STOP LIC Site ATG-pelB signal PSGP21: PelB + Cleavable C-terminal tags pSGP35: 3C cleavable PelB + N-terminal 6His

MALDI-TOF of purified ORFs expressed in pSGP35 (cleavable PelB) L5701 Lmaj004776T

Crystallization strategies 1. Optimization for PelB-containing Lmaj000817T- frozen crystals to be shipped or carried (Katrina Robinson) to Seattle Decreasing detergent concentration Cryopreservation Detergent mixes Additives Temperature 2. Production of antibodies to Lmaj000817T (ongoing, w/ Mark Sullivan) 3. Purification of Lmaj000817T lacking PelB from pSGP Revisiting purification of other good expressors that purify as homogeneous protein containing PelB. 5. Targeting ORFs that express well in pSGP35 lacking PelB 6. Construction of a new vector: PelB-High expressing ORF-His 6- 3C site.

Tetrahymena as a host for expression of membrane proteins from Plasmodium falciparum Advantages: 1. High membrane content coating abundant cilia. 2. High genomic AT content 3. Evolutionary relatedness of Tetrahymena to P. falciparum 4. Recently developed as a genetic system (Gaertig, Gorovsky et al.) Collaborators:Tetragenetics Inc: Donna Cassidy-Hanley, Cornell University Ted Clark, Cornell University Jacek Gaertig, University of Georgia Martin Gorovsky, University of Rochester

Vectors for Tetrahymena expression LIC Site-ATG ORF LIC Site “Soluble” 3C Protease Site ORF RGS-6His Calmodulin Binding Peptide STOP LIC Site ATG-Cleavable signal RGS-6His Calmodulin Binding Peptide STOP Metallothionein promoter “Soluble” 3C Protease Site Membranes LIC Site “Soluble” 3C Protease Site ORF 6His STOP LIC Site ATG Metallothionein promoter Soluble ORFs

Targets for membrane and secreted protein expression in Tetrahymena: LocusFunction/Target rationaleAAs MAL7P1.27pfcrt (choroquine resistance) 424 PFE1150wmdr1 (drug resist)1419 PFE1265wGPCR? 467 Pf13_0248pf47 antigen 439 PFB0405s230 antigen3135 AAF pfs25 antigen 217 AAT pfs28 antigen 218 PF11_0486MAEBL eryth binding antigen2055 PFA0125cEbl1 erythrocyte binding antigen1567 PFL1315wpfkch1 K+channel1979 PFI0955wput. Hexose transporter 476 PFA0310cCa+ATPase (atemisinin target)1228

Kathy Clark, Nadia Fedoriw, Katrina Robinson, Gayle Schneider, Sara Connelly Thanks to: Eric Phizicky, Elizabeth Grayhack, Mark Sullivan Ina Urbatsch (Texas Tech Medical Center) Michael Malkowski (HWI) Jolanta Kruczinska, Joseph Wedekind, (Crystallography- Rochester) Edward Petri (laboratory of Ravi Basavappa) (Crystallography- Rochester) Tetragenetics Rochester Membrane Protein Unit