1. Presented by Subproject 6 of the MPEC Roadmap Grant P50 GM073210 Membrane Protein Production for Crystallization Goals, Approach, Progress and Examples

2. Production Goals Concentrated Protein which is Pure, Homogeneous and Stable at > 2mg/prep Identical crystal building blocks >95% PH and S for 2 weeks dilute and 1 week concentrated > 200ul 10mg/ml Minimized Detergent Concentration Detergent tends to concentrate with protein during concentration High detergent concentrations produce phase separation during crystallization Detergent concentration critical for quality crystals Initially avoid detergent phase separation during crystallization Allowing trials below, at and above phase boundary Utilize SEC Tetra Detector Array/Analysis (TDA) Monitor and optimize detergent concentration while assaying PDC homogeneity during purification and final concentration for crystallization Measure physical properties of the Protein Detergent/Lipid Complex (PDC) Mass (oligomeric state), size (Rh), shape (IV), Detergent:protein ratio Motto: Quality Output

3. Purification & Characterization Detergent Solubilization Concentration Membrane Preparation 250mM OG --- 40mM OG solvent 20mM DDM ----------------------- 20mM C14PC/C12PC/MMPC -- Ni, Affinity Desalt Tag Cleavage 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 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 residues present Purification pH/salt solubility/homogeneity Detergent exchange Well behaved Wash, Lyse Optional buffer and high salt wash Membrane Signature Gel