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Lessons from the Quest for Quality Protein (and Crystals)

Dimer. Monomer. Lessons from the Quest for Quality Protein (and Crystals). Pure. Homogeneous & Stable. Dilute Concentrated Over time. SEC microinjections. SDX, 7.3, 40mM OG. 8mM excess OG micelles. 280nm. Minimized [Detergent]. 280nm. PDC. Well behaved. RI. IE.

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Lessons from the Quest for Quality Protein (and Crystals)

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

  2. 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 250 ns snapshot KvAP/DM Simulation C8TMA-Cl C16TMA-Cl PEG Sansom et al. 2005 OG phase diagram (Zulauf 1991) Burov et al 2008

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

  4. 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 1st : detergent belt 2nd: protein Experimental style Chromatography Quality Output (careful, complete and methodical; slow) Go-fast, streamlined med-high throughput very important

  5. Properly targeted Over expressed ≥ 500ml culture Core Purification Approach Start 10% glycerol 5mM BME/2mM DTT if Cys present Membrane Preparation Detergent Solubilization Purification & Characterization Wash, Lyse Optional buffer & high salt wash Membrane Signature Gel 250mM OG -- 40mM OG solvent 20mM DDM ----------------------- 20mM C14PC/C12PC/MMPC-- 0.5-2mM DDM solvent Key Parameters Detergent/lipid pH Ionic strength Reducing agent Osmolytes Additives (Minimal 3 Detergent Screen) Ni, Affinity Desalt Tag Cleavage Cleanup Concentration Max MWt cut-off filter IE, Ni, Affinity Dialysis All fractions9-15runs /exp/3day Size Exclusion Tetra Detector Array/Analysis Concentration: Abs & RI Shape (IV), size (Rh): Viscometer Mass: RALS Concentrate 100kDa start Size Exclusion +TLC and/or +Dilution Factor Desalt/pH change Crystallization & Crystallography (CSMP) Purification pH/salt solubility/homogeneity Detergent exchange Well behaved Cation and Anion Exchange

  6. High Priority MPEC Protein Progress 2.1 1.8 2.0 Å Structure 3.8 3.5 10 Diffraction S. cere HEKP. past E. coli Homologs /E.coli S. cere, HEK,P. past, E. coli, Homologs /E.coli Crystal PHS SE, IE Workflow Tag Cleave Affinity Solubilize Expression MPEC Targets (>128, 32 PHS) 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.

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

  8. Purification Approach Start 10% glycerol 5mM BME/2mM DTT if Cys present Detergent Solubilization Common purification solvents 40mM OG, 18mM NG, 8mM DM, 0.5-2mM DDM, 2-4mM FC12, 0.5mM FC14 exploring MMPC, MMPG, mixtures 250mM OG -- 40mM OG solvent 20mM DDM ----------------------- 20mM C14PC/C12PC/MMPC-- 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)

  9. 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 Purification Approach Start 10% glycerol 5mM BME/2mM DTT if Cys present Purification & Characterization Ni bump desalted to pH 6, 8, and 9 Post TEV cleavage at pH 6, 8, and 9 Key Parameters Detergent/lipid pH Ionic strength Reducing agent Osmolytes Additives Ni, Affinity Desalt Tag Cleavage Cleanup First pass of human/DDM (Rachel Bond) All fractions9-15runs /exp/3day Size Exclusion Concentrate 100kDa start pH 5 pH 7 pH 9 Desalt/pH change Purification pH/salt solubility/homogeneity Detergent exchange Well behaved Cation and Anion Exchange

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

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

  12. Purification Approach 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 Concentration “Universal Calibration Method” Vh=IV*M vs. retention time Still some exemptions SE matrix not inert Max MWt cut-off filter IE, Ni, Affinity Dialysis Tetra Detector Array/Analysis Concentration: Abs & RI Shape (IV), size (Rh): Viscometer Mass: RALS Size Exclusion +TLC and/or +Dilution Factor Crystallization & Crystallography

  13. 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

  14. For PDC analysis, obtain single UV peak before proceeding with Multi detection Peak slice analysis can only be used when Mass constant throughout peak Mw (weighted avg) 44183 whole peak analysis 34392 peak slice analysis Ovalbumin MWt (44,300 Da actual) MWt distribution Mw/Mn = 1.001 = monodisperse humanMP/OG 191,012 PDC/110,792 protein (3.2 monomers/PDC whole peak analysis 179,652 PDC/113540 protein (3.3 monomers/PDC) peak slice analysis Mw/Mn of single peaks = 1.009 = monodisperse

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

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