Membrane Protein Expression Center © 2005 Genomic Screen for Tractable Targets in S. cerevisiae...
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Transcript of Membrane Protein Expression Center © 2005 Genomic Screen for Tractable Targets in S. cerevisiae...
Membrane Protein Expression Center © 2005
Genomic Screen for Tractable Targets in Genomic Screen for Tractable Targets in S. S. cerevisiaecerevisiaeGenomic Screen for Tractable Targets in Genomic Screen for Tractable Targets in S. S. cerevisiaecerevisiae
Franklin A. HaysRoadmap Meeting26 March 2009UCSF
Membrane Protein Expression Center © 2005
Li M., Hays F. A., Roe-Zurz Z., Vuong L., Kelly L., Robbins R., Ho C., Pieper U., O’ Connell J. D., Miercke L. J., Giacomini K. M., Sali A. and Stroud R. M. (2009) “Selecting optimum eukaryotic integral membrane proteins for structure determination by rapid expression and solubilization screening”. J. Mol. Biol., 385 (3):820-830
Hays F. A., Roe-Zurz Z., Li M., Kelly L., Gruswitz F., Sali A., Stroud R. M. (2009) “Ratiocinative screen of eukaryotic integral membrane protein expression and solubilization for structure determination”. J. Struct. Funct. Genomics, 10 (1):9-16
Membrane Protein Expression Center © 2005
Objective 1: Objective 1: identify eukaryotic integral membrane proteins amenable to purification and crystallization efforts
Objective 2:Objective 2: Do the above with minimal effort/expense
Membrane Protein Expression Center © 2005
128 MP structures from 35 Pfam’s
S. cerevisiae S. cerevisiae Pfam Pfam HomologyHomologyS. cerevisiae S. cerevisiae Pfam Pfam HomologyHomology
Membrane Protein Expression Center © 2005
Empirically Based Target PipelineEmpirically Based Target Pipeline
Membrane Protein Expression Center © 2005
Target Selection in Target Selection in S. S. cerevisiaecerevisiaeTarget Selection in Target Selection in S. S. cerevisiaecerevisiae
Four sets of 96 targets were represented by color. Targets are connected if their profiles are significantly related.
1. 3 TMH’s or more
2. Less than 100kDa MW
3. No introns
4. Parent of each node• 622 of 6600 S. cer. Protein sequences predicted to be 3TM or more.
• 130 sequences with no Pfam
• 165 unique Pfams
• 79 Pfam singletons
• 86 Pfams w > two members
384 targets selected (130 + 79 + (2 x 86) + 3 )
Membrane Protein Expression Center © 2005
83nu6579 bp
AmpR
HIS3
Flag Tag
3C site
LIC cassette
Thrombin Site
10xHis Tag
Gal-1
p(LAC)
2 MICRON
PMB1
F1 ORI
Cyc
Sma I (4175)
Membrane Protein Expression Center © 2005
Empirically Based Target Pipeline - PriotiziationEmpirically Based Target Pipeline - Priotiziation
Use only one detergent for solubilization : DDMUse only one buffer condition for SEC void checks: 20 mM TRIS-HCl pH 7.4RT, 200 mM NaCl, 1 mM DDM, and 10% v/v glycerol
Membrane Protein Expression Center © 2005
Membrane Protein Expression Center © 2005
Membrane Protein Expression Center © 2005
So what are some targets that worked?So what are some targets that worked?
Membrane Protein Expression Center © 2005
18 scaled-up 6 crystal trials 4 crystallized
Hurdles for targets during intensive phase:
• obtaining complete tag cleavage• buffer stability• non-specific binding to IMAC• peak profile during SEC• protein stability
Membrane Protein Expression Center © 2005
S. cer. S. cer. expression of Human expression of Human IMP’sIMP’sS. cer. S. cer. expression of Human expression of Human IMP’sIMP’s
Human integral membrane proteins can be overexpressed in S. cer., a system with many positive attributes for high-throughput screening and subsequent functional studies.
Even a scarce 8% return on human targets, vs. the 24% for yeast, would yield > 200 targets for the intensive production phase.
Membrane Protein Expression Center © 2005
Expansion of Streamlined Empirical Expansion of Streamlined Empirical ApproachApproachExpansion of Streamlined Empirical Expansion of Streamlined Empirical ApproachApproach
Membrane Protein Expression Center © 2005
Top Funnel – Extensive Prioritization Phase
Bottom Inverse Funnel – Intensive Production Phase
Protein function NOT important Use one detergent (e.g. DDM) S. cer. for episomal expression (GAL1) Membrane prep. and solubility screens Medium to large scale test expression Desalt after IMAC No cleavage of expression tags Speed and consistency is important MAIN OBJECTIVE: list of targets ordered by expression level, detergent solubility and SEC profile
Protein function VERY important Cleave expression tags Large scale expression (e.g. fermentors) Develop membrane prep. protocol Develop protein purification protocol Consider switch to Pichia pastoris Screen various buffer conditions Develop concentration scheme Obtain pure/homogenous/stable protein Broad crystallization screens/methods MAIN OBJECTIVE: Obtaining a structure
Membrane Protein Expression Center © 2005
CONCLUSIONCONCLUSIONSSCONCLUSIONCONCLUSIONSS
1.Extensive genomic screen sufficient to identify tractable targets for crystallization efforts.
2.Target focused effort follows extensive screen prior to crystallization
3.DDM solubilization sufficient to capture ~25% of yeast targets
4.Yeast is viable system for the overexpression of eukaryotic IMP’s
Membrane Protein Expression Center © 2005
AcknowledgemeAcknowledgementsntsAcknowledgemeAcknowledgementsnts
Stroud Lab:– Bob Stroud– Min Li– Zygy Roe-Zurz– Linda Vuong– Joseph O’Connell– Mimi Ho– Zach Newby– Dave Savage– Renée Robbins
Advanced Light Source– James Holton– George Meigs
Sali Lab:– Andrej Sali– Libusha Kelly– Ursula Pieper
Giacomini Lab:– Kathy Giacomini– Ying Chen
F.A.H is supported by postdoctoral fellowships from NIGMS (F32 GM078754) and the Sandler Family Foundation.
This work was supported by the Center for Innovation in Membrane Protein Production (GM73210) and the Center for Structures of Membrane Proteins (GM074929) to R.M.S.
Membrane Protein Expression Center © 2005
QUESTION:QUESTION: Can generalized procedures be implemented to identify and produce eukaryotic integral membrane proteins for structural studies?
QUESTION:QUESTION: Can generalized procedures be implemented to identify and produce eukaryotic integral membrane proteins for structural studies?
Membrane Protein Expression Center © 2005
Membrane Protein Expression Center © 2005
Priority ListPriority List
83nu6579 bp
AmpR
HIS3
Flag Tag
3C site
LIC cassette
Thrombin Site
10xHis Tag
Gal-1
p(LAC)
2 MICRON
PMB1
F1 ORI
Cyc
Sma I (4175)
3X
Membrane Protein Expression Center © 2005
Gene AmplificationGene AmplificationGene AmplificationGene AmplificationGenes amplified from S288C genomic DNA
Modbase uniprot IDs
Conversion to yeast Gene ID
Batch download coding sequences from Saccharomyces Genome Database
Peruse and emend list
Upload into Vector NTI
Construct forward and reverse 39-mer removing ATG and TCA codons respectively
Y96-II Y96-IV
Membrane Protein Expression Center © 2005
Expression Plasmid Expression Plasmid ConstructionConstructionExpression Plasmid Expression Plasmid ConstructionConstruction
Gel purification (96-well format) of genes
LIC Reaction– T4 polymerase mediated 3’-5’
exonuclease reaction in the absence of deoxynucleotide triphosphates
– ‘chewedback’ gene and 83nu annealed to construct expression plasmid
Transformation into competent DH5 Escherichia coli house stock
Pick two colonies
Membrane Protein Expression Center © 2005
Insert ValidationInsert Validation(on each of two colonies)(on each of two colonies)Insert ValidationInsert Validation(on each of two colonies)(on each of two colonies)
Y96-I Colony PCR with Gal and Cyc primers
For plasmids with inserts 96-well mini-prep
Membrane Protein Expression Center © 2005
Expression with Solubilization Expression with Solubilization TestTestExpression with Solubilization Expression with Solubilization TestTest
Transformation into W303 strain
Skipping the small scale: 500ml growths for each target
Cell lysis
Membrane preparation
Detergent screen (with DDM)
White, M.A., et al. (2007) J. Mol. Biol. 365, 621-636
Membrane Protein Expression Center © 2005
Cell Lysis and Membrane Cell Lysis and Membrane PreparationPreparationCell Lysis and Membrane Cell Lysis and Membrane PreparationPreparation
Disrupt cells with 0.5mm beads in blenders (~1:1) on ice
Low speed centrifugation at 6000xg for 15 min.
Harvest membranes at 138,000xg for 60 min.
Membranes resuspended in 500L buffer.
Membrane Protein Expression Center © 2005
Small Scale DDM Small Scale DDM SolubilizationSolubilizationSmall Scale DDM Small Scale DDM SolubilizationSolubilization
300 L scale. 40 mM DDM and 20 L membranes.
1 hour solubilization at 4°C
15 min. 42,000 rpm spin in TLA-55
SDS-PAGE and western blot (membranes ~30X on gel)
1:2000Anti-His
1:5000Anti-FLAG
Membrane Protein Expression Center © 2005
What is gene What is gene redesign?redesign?
What is gene What is gene redesign?redesign? Optimization of a gene sequence for increased Optimization of a gene sequence for increased
expression in a heterologous host. expression in a heterologous host.
What is What is “optimization”?“optimization”?
What is What is “optimization”?“optimization”?
Modify codon usageModify codon usage
Eliminate unfavorable codon pairs or high GC/AT Eliminate unfavorable codon pairs or high GC/AT contentcontent
Avoid unfavorable mRNA secondary structural Avoid unfavorable mRNA secondary structural elements.elements.
Eliminate repetitive sequencesEliminate repetitive sequences
Cloning and expression elements (e.g. restriction Cloning and expression elements (e.g. restriction sites, etc.)sites, etc.)
Alter characteristics of leading ~45-90 basesAlter characteristics of leading ~45-90 bases
Membrane Protein Expression Center © 2005
Aquaporin from Aquaporin from Plasmodium Plasmodium falciparumfalciparum
50372520
Ni Purified Protein
Ni Purified Protein after 3 days at 4ºC.
32.5’
2.0 Å resolution
Zach Newby, Joe O'Connell, Yaneth Zach Newby, Joe O'Connell, Yaneth RoblesRobles
2Fo-Fc
Native gene Native gene No expression No expression
Optimized gene Optimized gene ~33 ~33 μμg/L (!) g/L (!) Structure Structure
Membrane Protein Expression Center © 2005
1st Injection
Reinjection (6 days later)
hAQP4: PHS and Structure – OPTIMIZE?hAQP4: PHS and Structure – OPTIMIZE?hAQP4: PHS and Structure – OPTIMIZE?hAQP4: PHS and Structure – OPTIMIZE?
Yield Post Nickel: Native - 8 OD per Liter; Optimized – ~1.5 OD per liter
1st Injection
Monomer
Dimer
150
250
10075
37
50
25
20
15
10
Conc
PureHomogenous & Stable
Joe Ho and Bill HarriesJoe Ho and Bill Harries