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
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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.
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Lessons from the Quest for Quality Protein(and Crystals)
Pure
Homogeneous & Stable
Monomer
Dimer
8mM excess OG micelles
PDC280nm
SDX, 7.3, 40mM OG
RI
Minimized [Detergent]
Well behaved
IE
Low [OG]/no phase sep
SEC microinjections
280nm
DiluteConcentrated
Over time
High [OG]/phase sep
PerspectiveThe Challenge
Detergents/lipids complicated & barely understoodPDC not homogenousProtein, 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 agentsTemperature, protein
Burov et al 2008
C8TMA-Cl
C16TMA-Cl
Sansom et al. 2005
KvAP/DM Simulation250 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 FoundationDo 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 qualityIt’s requirement will be determined thought the purification processSecondary detergents/lipids can be added back during crystal trial
EmpiricalNo one/few magical condition amendable to all targetsEvery 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 crystalsSystematically modify the PDC
1st : detergent belt2nd: protein
Experimental styleChromatographyQuality Output (careful, complete and methodical; slow)
Go-fast, streamlined med-high throughput very important
Wash, LyseOptional buffer & high salt washMembrane Signature Gel
+TLCand/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)
2.1 1.8 2.0 Å
3.8 3.5 10
S. cere HEK P. past E. coli Homologs /E.coli
Wo
rkfl
ow
Membrane Preparation
Purification ApproachProperly targeted Over expressed ≥ 500ml culture
Wash, LyseOptional buffer & high salt washMembrane Signature Gel
≥ 500ml culture (scale up issues)
Membrane signature gel (high to low conc.)
Wash membranes as much as neededLow and high salt washes
Rachel Bond
Detergent Solubilization
250mM OG -- 40mM OG solvent20mM DDM -----------------------20mM C14PC/C12PC/MMPC--
Purification ApproachStart
10% glycerol 5mM BME/2mM DTT if Cys present
Only small subset of detergents initially required7 for full gel
OG, DDM, FC12, MMPC, CHAPSO, C12E8, LDAO
Keep in mind theLarge available arsenal for solubilization & purification