Membrane Protein Expression Center © 2005 Genomic Screen for Tractable Targets in S. cerevisiae Franklin A. Hays Roadmap Meeting 26 March 2009 UCSF

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


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


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


128 MP structures from 35 Pfam’s

S. cerevisiae S. cerevisiae Pfam Pfam HomologyHomologyS. cerevisiae S. cerevisiae Pfam Pfam HomologyHomology


Empirically Based Target PipelineEmpirically Based Target Pipeline


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 )


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)


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




So what are some targets that worked?So what are some targets that worked?


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


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.


Expansion of Streamlined Empirical Expansion of Streamlined Empirical ApproachApproachExpansion of Streamlined Empirical Expansion of Streamlined Empirical ApproachApproach


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


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


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.


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?



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


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


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


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


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


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.


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


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


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


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

Documents

Membrane Protein Expression Center © 2005 Genomic Screen for Tractable Targets in S. cerevisiae Franklin A. Hays Roadmap Meeting 26 March 2009 UCSF