lessons from the quest for quality protein (and crystals) pure homogeneous & stable monomer...
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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
Purification & CharacterizationDetergent Solubilization
Concentration
Membrane Preparation
250mM OG -- 40mM OG solvent20mM DDM -----------------------20mM C14PC/C12PC/MMPC--
Ni, AffinityDesalt
Tag CleavageCleanup
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/lipidpHIonic strengthReducing agentOsmolytesAdditives
Max MWt cut-off filter
IE, Ni, Affinity
Dialysis
Crystallization & Crystallography(CSMP)
Core Purification ApproachProperly targeted Over expressed ≥ 500ml culture
All fractions 9-15runs /exp/3day
(Minimal 3 Detergent Screen)
Concentrate100kDa start
0.5-2mM DDM solvent
Start10% glycerol
5mM BME/2mM DTT if Cys present
PurificationpH/salt solubility/homogeneityDetergent exchangeWell behaved
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
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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
(Thank you Anatrace, Qinghai Zhang, Sam Gellman)
Common purification solvents40mM OG, 18mM NG, 8mM DM,0.5-2mM DDM, 2-4mM FC12, 0.5mM FC14exploring MMPC, MMPG, mixtures
Purification & Characterization
Size Exclusion
Desalt/pH change
Cation and Anion Exchange
Key Parameters Detergent/lipidpHIonic strengthReducing agentOsmolytesAdditives
Purification Approach
All fractions 9-15runs /exp/3day
Concentrate100kDa start
Start10% glycerol
5mM BME/2mM DTT if Cys present
PurificationpH/salt solubility/homogeneityDetergent exchangeWell behaved
Concentration (find max kDa)100 to 50 to 30 kDa spingel, spec
Remove heterologous residuesAlways test different pHs
Minimally desalt into other pHs
Ni, AffinityDesalt
Tag CleavageCleanup
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
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
-5 5 15 25 35 45 55 65 75 85 95
Time (min)
OD 280nm (2.5mm path)
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/DDMFraction 3+5
Human/DDM Rebecca Robbins1st 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 concentrationDoes 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
+TLCand/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)44183 whole peak analysis34392 peak slice analysis
For PDC analysis, obtain single UV peak before proceeding with Multi detectionPeak slice analysis can only be used when Mass constant throughout peak
humanMP/OG
191,012 PDC/110,792 protein (3.2 monomers/PDCwhole peak analysis
179,652 PDC/113540 protein (3.3 monomers/PDC) peak slice analysis
Mw/Mn of single peaks = 1.009 = monodisperse
MWt distribution Mw/Mn = 1.001 = 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