Lessons from the Quest for Quality Protein (and Crystals) Pure Homogeneous & Stable Monomer Dimer 8mM excess OG micelles PDC 280nm SDX, 7.3, 40mM OG RI Minimized [Detergent] Well behaved IE Low [OG]/no phase sep SEC microinjections 280nm Dilute Concentrated Over time High [OG]/phase sep

Perspective The Challenge Detergents/lipids complicated & barely understood PDC not homogenous Protein, Detergent belt, Micelle and Crystal packing dependant on many parameters Primary and secondary detergent/lipid (type & conc.) Ionic strength (type and conc.) Osmolytes, additives, precipitating agents Temperature, protein Burov et al 2008 C8TMA-Cl C16TMA-Cl Sansom et al KvAP/DM Simulation 250 ns snapshot OG phase diagram (Zulauf 1991) PEG

Pebay-Peyroula et al 1995 Crystal packing dependant on detergent C10DMA0 OG Sauer et al, Acta Crystal D, 2002 C8E12/C8-2-HES OmpF

Working Foundation Do whatever it takes to obtain/maintain PHS with minimized detergent May take 2-4 steps which can remove “all” endogenous lipids Not concerned with initial lipid removal +/- lipid will not inhibit xtal growth, but xtal quality It’s requirement will be determined thought the purification process Secondary detergents/lipids can be added back during crystal trial Empirical No one/few magical condition amendable to all targets Every protein needs to be considered independently Map out solubility/crystallization space using different crystallization methods VD, batch, dialysis, LPC, counter diffusion If quality protein but poor or no crystals Systematically modify the PDC 1 st : detergent belt 2 nd : protein Experimental style Chromatography Quality Output (careful, complete and methodical; slow) Go-fast, streamlined med-high throughput very important

Purification & Characterization Detergent Solubilization Concentration Membrane Preparation 250mM OG -- 40mM OG solvent 20mM DDM mM C14PC/C12PC/MMPC-- Ni, Affinity Desalt Tag Cleavage Cleanup Size Exclusion Desalt/pH change Cation and Anion Exchange Tetra Detector Array/Analysis Concentration: Abs & RI Shape (IV), size (Rh): Viscometer Mass: RALS Size Exclusion Key Parameters Detergent/lipid pH Ionic strength Reducing agent Osmolytes Additives Max MWt cut-off filter IE, Ni, Affinity Dialysis Crystallization & Crystallography (CSMP) Core Purification Approach Properly targeted Over expressed ≥ 500ml culture All fractions 9-15runs /exp/3day (Minimal 3 Detergent Screen) Concentrate 100kDa start 0.5-2mM DDM solvent Start 10% glycerol 5mM BME/2mM DTT if Cys present Purification pH/salt solubility/homogeneity Detergent exchange Well behaved Wash, Lyse Optional buffer & high salt wash Membrane Signature Gel +TLC and/or +Dilution Factor

With Corey Anderson, André Bachmann, Sotiri Banakos, Akanksha Bapna, Sarika Chaudhary, Melissa Del Rosario, Vladimir Denic, Robert Edwards, Pascal Egea, Franz Gruswitz, Frank Hays, Joe Ho, David Julius, Monty Krieger, Witek Kwiatkowski, John Lee, Min Li, Bipasha Mukherjee, Vinod Nair, Zach Newby, Roger Nicoll, Sabrina Noel, Joseph O’Connell, Yaneth Robles, Edwin Rodriquez, Zygy Roe-Zurz, Renee Robbins, David Savage, Shimon Schuldiner, Tomomi Tsomeya, Linda Vuong, Jonathan Weismann, and Ronald Yeh. People with italicized names are no longer working with us. High Priority MPEC Protein Progress Structure Diffraction Crystal PHS SE, IE Tag Cleave Affinity Solubilize Expression S. cere, HEK, P. past, E. coli, Homologs /E.coli MPEC Targets (>128, 32 PHS) Å S. cere HEK P. past E. coli Homologs /E.coli Workflow

Membrane Preparation Purification Approach Properly targeted Over expressed ≥ 500ml culture Wash, Lyse Optional buffer & high salt wash Membrane Signature Gel ≥ 500ml culture (scale up issues) Membrane signature gel (high to low conc.) Wash membranes as much as needed Low and high salt washes Rachel Bond

Detergent Solubilization 250mM OG -- 40mM OG solvent 20mM DDM mM C14PC/C12PC/MMPC-- Purification Approach Start 10% glycerol 5mM BME/2mM DTT if Cys present Only small subset of detergents initially required 7 for full gel OG, DDM, FC12, MMPC, CHAPSO, C12E8, LDAO Keep in mind the Large available arsenal for solubilization & purification (Thank you Anatrace, Qinghai Zhang, Sam Gellman) Common purification solvents 40mM OG, 18mM NG, 8mM DM, 0.5-2mM DDM, 2-4mM FC12, 0.5mM FC14 exploring MMPC, MMPG, mixtures

Purification & Characterization Size Exclusion Desalt/pH change Cation and Anion Exchange Key Parameters Detergent/lipid pH Ionic strength Reducing agent Osmolytes Additives Purification Approach All fractions 9-15runs /exp/3day Concentrate 100kDa start Start 10% glycerol 5mM BME/2mM DTT if Cys present Purification pH/salt solubility/homogeneity Detergent exchange Well behaved Concentration (find max kDa) 100 to 50 to 30 kDa spin gel, spec Remove heterologous residues Always test different pHs Minimally desalt into other pHs Ni, Affinity Desalt Tag Cleavage Cleanup Ni bump desalted to pH 6, 8, and 9 Post TEV cleavage at pH 6, 8, and 9 First pass of human/DDM (Rachel Bond) pH 5 pH 7 pH 9

Always ask if SEC peaks run true Stable GlpF/OG; 2.2 Å Post Ni, no desalt Fraction 4 Day 1 Day 2 SEC/OG SEC/DDM Fraction 3+5 Human/DDM Rebecca Robbins 1 st purification pass F4

Concentration Tetra Detector Array/Analysis Concentration: Abs & RI Shape (IV), size (Rh): Viscometer Mass: RALS Size Exclusion Max MWt cut-off filter IE, Ni, Affinity Dialysis Crystallization & Crystallography Purification Approach Always follow SEC profile during protein concentration Does purified, homogenous protein remain stable upon concentration? Good Bad

Concentration Tetra Detector Array/Analysis Concentration: Abs & RI Shape (IV), size (Rh): Viscometer Mass: RALS Size Exclusion Max MWt cut-off filter IE, Ni, Affinity Dialysis Crystallization & Crystallography Purification Approach “Universal Calibration Method” Vh=IV*M vs. retention time Still some exemptions SE matrix not inert Chromatography can be successfully to concentrate while minimizing [detergent] Poster/ manuscript/website: 4 proteins, 3 detergents, 4 different methods RI detectors ROCK! Every workstation should have one! Quantitate excess [detergent] while following PDC homogeneity PDC systems must be used when studying micelle behavior on MWCO filters +TLC and/or +Dilution Factor

Sold on the concept of multi detection for SEC PDC mass, size, shape and % binding partners using SEC Micelle mass, size, shape But Tetra detection not ready for mainstream MP work Acquisition fine Slow and involved No fraction collection (when analyzing) Analysis OK, but complicated Requires calibration standard Very sensitive (sees everything) Accuracy problematic (assumptions must be verified) Calibration Standard: dn/dc, dA/dc, Mass Unknown MP: dA/dc, dn/dc Totally willing to keep moving forward with tetra detection

Ovalbumin MWt (44,300 Da actual) Mw (weighted avg) whole peak analysis peak slice analysis For PDC analysis, obtain single UV peak before proceeding with Multi detection Peak slice analysis can only be used when Mass constant throughout peak humanMP/OG 191,012 PDC/110,792 protein (3.2 monomers/PDC whole peak analysis 179,652 PDC/ protein (3.3 monomers/PDC) peak slice analysis Mw/Mn of single peaks = = monodisperse MWt distribution Mw/Mn = = monodisperse

Acknowledgments MPEC Subproject 6: Protein Purification Bill Harries & John Lee (infrastructure, management) Pat Greene (consulting, grants) Olivia Viloria (finance) Suzan Betheil (admin) and Robert Stroud (Commander & Chief) Andrew Sandstrom (University of Chicago) Collaborators Rebecca Robbins, Mimi Ho, Rachel Bond