GFP-based membrane protein overexpression and purification in E. coli and S. cerevisiae Joy Kim Center for Biomembrane Research Department of Biochemistry.

Slides:

Advertisements

Similar presentations

Structural Biology of Membrane Proteins Problems of structure determination & Membrane-specific solutions KcsA structure Mechanistic insights KvAP and.

Advertisements

Cell-free Bacterial Yeast Insect Mammalian Protein Expression Systems.

PURIFICATION OF YEAST MEMBRANE PROTEINS FOR STRUCTURAL GENOMICS Center for High Throughput Structural Biology Mark E. Dumont *†, Nadia Fedoriw *, Kathy.

Prediction of protein localization and membrane protein topology Gunnar von Heijne Department of Biochemistry and Biophysics Stockholm Bioinformatics Center.

2004 PP&CW Optimization of protein expression and solubility Alternative and novel prokaryotic expression systems Eukaryotic expression systems Methods.

Expression and purification of membrane proteins: Initial screening of Thermotoga maritima α-helical membrane proteins for NMR structural studies This.

Center for Human Genetics and Molecular Pediatric Disease

1 Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network Kai Simons group, 2009, J. Cell Biology Deniz.

Large-scale expression and automated purification of G-protein-coupled receptors for structure determination Jim F. White 1, Loc B. Trinh 2, Joseph Shiloach.

Chapter 6 Manipulating Cells in Culture. Advantages of working with cultured cells over intact organisms More homogeneous than cells in tissues Can control.

1 General consideration about Gene Expression and expression studies Expression studies Expression Host -> Expression System Promoter system -> expression.

MCB 7200: Molecular Biology

Molecular Cell Biology Purifying Proteins Cooper.

High-throughput, flourescent-based optimisation of eukaryotic membrane protein overexpression and purification. Simon Newstead, Joy Kim, Gunnar von Heijne,

The Gateway® Cloning System Gene Expression Contents Description of destination vectors Examples of expression clones in destination vectors Effects of.

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

Recombinant protein expression. Other alternatives

Expression and Purification of Integral Membrane Proteins from Yeast for the Center for High-Throughput Structural Biology Kathy Clark *, Nadia Fedoriw.

Definition of Biotechnology in Canadian Legislation Biotechnology is the application of science and engineering in the direct or indirect use of living.

Bacillus subtilis expression/secretion systems

Jan-Willem de Gier Center for Biomembrane Research, Stockholm, Sweden Green Light for Membrane Proteins.

Finish up array applications Move on to proteomics Protein microarrays.

PREDICTING THE EXPRESSION AND SOLUBILITY OF MEMBRANE PROTEINS Center for High Throughput Structural Biology Mark E. Dumont *†, Michael A. White *, Kathy.

IGEM: Measurement Techniques for Pathway Output Noah Helman Lim Lab May 2007.

MCB 720: Molecular Biology Biotechnology terminology Common hosts in biotechnology research Transcription & Translation Prokaryotic gene organization &

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

Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Can’t rely on glycosylation, disulphide.

Provider of Global Contract Research Services Accelerating Preclinical Research, Drug Discovery & Therapeutics Services > Generation of Stable Cell Lines.

Protein Overexpression in E. coli and

Protein Overexpression in E

Protein Overexpression in E. coli and

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

Protein Expression Systems

Page 1 Yeast Expression System — Creative BioMart.

Production of Recombinant Proteins

Protein engineering and recombinant protein expression

Page 1 Yeast Expression Systems in Creative BioMart — Creative BioMart.

Membrane protein expression Proteins are the building blocks of all living organisms and play crucial roles in biological processes. To investigate how.

Membrane protein expression Proteins are the building blocks of all living organisms and play crucial roles in biological processes. To investigate how.

Custom membrane protein antibody As a pioneer and the undisrupted global leader in antibody discovery and engineering, Creative Biolabs proudly introduces.

Antibody proteins As a pioneer and the undisrupted global leader in antibody discovery and engineering, Creative Biolabs proudly introduces our professional.

Yeast Tgl3p expressed in HeLa cells localizes to lipid droplets.

Kirby R. Siemering, Ralph Golbik, Richard Sever, Jim Haseloff

Biotechnology and Genetic Engineering PBIO 450/550

Department of Chemical Engineering

1- Progress on folding reporters

Protein Complex Discovery

TEV protease elution from cellulose is specific and provides highly purified target protein. TEV protease elution from cellulose is specific and provides.

The ΔrlmA mutant strain has impaired CWI pathway activation.

Testing the iGM insertion module.

Secretion assays are shown for pCASP plasmid, strain variations, and silks. Secretion assays are shown for pCASP plasmid, strain variations, and silks.

Volume 16, Issue 1, Pages (June 2016)

Experimental setup.A, topology models of the overexpressed membrane protein GFP fusions. Experimental setup.A, topology models of the overexpressed membrane.

Membrane protein overexpression affects the pH of the culture and protein secretion.A, the pH of the culture of GFP fusion-overexpressing cells and the.

Characterization of aggregates isolated from E

Analysis of whole cell lysates by Western blotting and 2D gel electrophoresis.A, cells overexpressing GFP fusion proteins and control cells were cultured.

Protein Complex Discovery

Ras1 is a specific target of Ram1.

1- Progress on folding reporters

Volume 12, Issue 5, Pages (May 2007)

TIPI mediates rapid degradation of proteins in yeast.

Western blotting analysis of purified cytoplasmic membranes.

Partial recovery of TBSV RNA replication in yeast overexpressing selected subverted proviral host proteins. Partial recovery of TBSV RNA replication in.

Optimization of technical aspects for the genomic tagging approach.

Yeast Msp1 and human ATAD1 are required to limit the level of mitochondrially mislocalized TA proteins Pex15 and PEX26, respectively Representative images.

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

Characterization of various mammalian TF-Cub-Baits in yeast

Volume 3, Issue 2, Pages (August 2002)

WW domain of Rsp5p blocks TBSV RNA replication in yeast.

Presentation transcript:

GFP-based membrane protein overexpression and purification in E. coli and S. cerevisiae Joy Kim Center for Biomembrane Research Department of Biochemistry and Biophysics Stockholm University Stockholm, Sweden

Soluble protein vs. membrane protein structures

Solubilization with suitable detergent Purification Crystallization Structure determination Homologs of a target protein Overexpression screening Overexpression optimization Detergent screening GFP-based

E. coli

In-gel fluorescence vs. Western Blotting IGF WB

Overexpression of eukaryotic membrane proteins in E. coli Differences in lipid compositions Post translational modifications Folding environments Alternative overexpression hosts Yeast (P. pastoris, S. cerevisiae) Insect and mammalian cells Cell-free system Easy genetic manipulations Well known genetics ER quality control system

TEV site GFPHisX8GFPHisX8 GAL1 GFPHisX8 TEF Initial tests In two different yeast strains, one with Pep4 deletion In two different promoters, one inducible and the other constitutive

Fluorescence of whole-cell lysates from 10 ml vs. 1L cultures

Comparison of the upscale expression in 2.5 L shaker flask vs. 15 L fermenter

Time course for the overexpression of MP-GFP fusions

Fluorescence of whole-cell lysates vs. membrane fractions

Fluorescence In gel band intensity Fluorescence in solution vs. in-gel fluorescence

Combination of Gal1, an inducible promoter/Pep4, a vacuolar protease deletion strain gave the overall best overexpression for majority of test membrane proteins. Overexpression screening of yeast MP-GFP fusions

Optimizing expression with chemical chaperones

N-terminal vs. C-terminal GFP tagging 1: Rer1-GFP 2: GFP-Rer1

Assessing the quality of MP-GFP fusions by subcellular localization ConfocalWide field

Assessing the quality of MP-GFP fusions by FSEC Newstead et al., 2007, PNAS 104: Aggregation MP-GFP fusion Free GFP

Purification of a nucleotide sugar transporter Newstead et al., 2007, PNAS 104: Detergent solubilized membranesPurified MP-GFPPurified MP

GFP-based overexpression and purification of eukaryotic membrane proteins in S. cerevisiae Drew et al., Nat. Protocol, in press

Overexpression screening Solubilization with suitable detergent Purification Crystallization Structure determination Overexpression optimization Detergent screening Homologs of a target protein GFP-based

Acknowledgements Jan Willem de Gier Gunnar von Heijne David Drew Simon Newstead So Iwata Center for Biomembrane Research Stockholm University Stockholm, Sweden Membrane Crystallography group Imperial College London, U. K.