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 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): 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 2: Objective 1: identify eukaryotic integral membrane proteins amenable to purification and crystallization efforts Objective 2: Do the above with minimal effort/expense

Membrane Protein Expression Center © MP structures from 35 Pfam’s S. cerevisiae Pfam Homology

Membrane Protein Expression Center © 2005 Empirically Based Target Pipeline

Membrane Protein Expression Center © 2005 Target Selection in S. cerevisiae 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 ( (2 x 86) + 3 )

Membrane Protein Expression Center © 2005

Empirically Based Target Pipeline - Priotiziation Use only one detergent for solubilization : DDM Use 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

So what are some targets that worked?

Membrane Protein Expression Center © 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. expression of Human IMP’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 Approach

Membrane Protein Expression Center © 2005 Top Funnel – Extensive Prioritization Phase Bottom Inverse Funnel – Intensive Production Phase  Protein f unction 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 f unction 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 CONCLUSIONSCONCLUSIONS 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 AcknowledgementsAcknowledgements 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?

Membrane Protein Expression Center © 2005

Priority List 3X

Membrane Protein Expression Center © 2005 Gene Amplification Genes 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 Construction 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 Validation (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 Test 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,

Membrane Protein Expression Center © 2005 Cell Lysis and Membrane Preparation 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 500  L buffer.

Membrane Protein Expression Center © 2005 Small Scale DDM Solubilization 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) 1:2000 Anti-His 1:5000 Anti-FLAG

Membrane Protein Expression Center © 2005 What is gene redesign? Optimization of a gene sequence for increased expression in a heterologous host. Optimization of a gene sequence for increased expression in a heterologous host. What is “optimization”? Modify codon usage Modify codon usage Eliminate unfavorable codon pairs or high GC/AT content Eliminate unfavorable codon pairs or high GC/AT content Avoid unfavorable mRNA secondary structural elements. Avoid unfavorable mRNA secondary structural elements. Eliminate repetitive sequences Eliminate repetitive sequences Cloning and expression elements (e.g. restriction sites, etc.) Cloning and expression elements (e.g. restriction sites, etc.) Alter characteristics of leading ~45-90 bases Alter characteristics of leading ~45-90 bases

Membrane Protein Expression Center © 2005 Aquaporin from Plasmodium falciparum Ni Purified Protein Ni Purified Protein after 3 days at 4ºC. 32.5’ 2.0 Å resolution Zach Newby, Joe O'Connell, Yaneth Robles 2Fo-Fc Native gene  No expression Optimized gene  ~33 μg/L (!)  Structure

Membrane Protein Expression Center © st Injection Reinjection (6 days later) hAQP4: PHS and Structure – OPTIMIZE? Yield Post Nickel: Native - 8 OD per Liter; Optimized – ~1.5 OD per liter 1 st Injection Monomer Dimer Conc Pure Homogenous & Stable Joe Ho and Bill Harries