Part 1. Production of Pure, Homogeneous and Stable Membrane Protein
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Transcript of Part 1. Production of Pure, Homogeneous and Stable Membrane Protein
Part 1. Production of Pure, Homogeneous and Stable Membrane Protein
Larry Miercke, Rebecca Robbins, Mimi Ho, Andrew Sandstrom*, Rachel Bond, Bill Harries and Robert Stroud
Department of Biochemistry and Biophysics, UCSF, San Francisco, CA; *Present address is Biochemistry & Molecular Biology, University of Chicago, Chicago, IL
This work was supported by NIH Roadmap grant P50 GM073210
Please e-mail Larry at [email protected] for questions, comments ect.
Introduction
Our approach to growing quality protein crystals is the production of Pure, Homogeneous and Stable Protein. For it is only when identical crystal building blocks are used that the probability of growing large well ordered crystals is optimized.
Protein production consists of 4 basic steps: membrane preparation, solubilization, purification & chromatographic characterization, and concentration.
Starting with washed membranes, the target is solubilized, then purified to a stable and homogenous state, and finally concentrated while maintaining homogeneity and ultimately minimizing the detergent concentration before entering crystallization trials.
Membrane Preparation: Targets enter the purification workflow once membranes from ≥ 500ml cultures with over-expressed and properly targeted constructs are produced. Most membranes are lysed and washed using a single centrifugation step (harvested cells resuspended directly into high salt buffer prior to lysis, and unwashed pelleted membranes resuspended in glycerol containing buffer). If target proteolysis and protein contaminants are problematic, multiple centrifugation steps are performed (wash cells, lyse in low salt with separate high salt membrane wash; additional steps may include repeated buffer washes and a final sucrose density gradient). A membrane signature gel (series from high to low protein concentration) is run to give an accurate assessment of proteolysis and expression levels, and to determine the membrane concentration required for optimal solubilization screens.
Solubilization: Initial solubilization screens consist of three or up to five different detergents (270mM OG, 20mM DDM and 20mM FC12/FC14/MMPC). All properly targeted constructs tested have been found to be solubilized to >80% by at least one of these detergents. Even though OG is generally poor for extraction, it is always included since it is a favorite for crystallization and function. Additional solubilization screens such as Cymals, sterols, POEs, zwittergents, LDAO, TX-100, and diC6PC will be performed next if required for purification. If solubility, homogeneity and
stability post affinity purification continue to be problematic, a third approach uses mixtures of detergents, alkyl lipids, and cholesterol. It is not until these all fail to produce quality crystals that selective target extraction studies are implemented.
Purification and characterization: Using a combination of affinity, size exclusion and ion exchange chromatography, six key purification parameters (detergent, pH, ionic strength, reducing agent, osmolytes, and additives) are iteratively examined to find a condition which maintains a PHS concentrated population of protein-detergent-complexes or protein-detergent-lipid-complexes. Affinity chromatography is used for purification and detergent exchange, and size exclusion chromatography is used for purification and for assaying homogeneity. Ion exchange chromatography at low and high pH is used for purification, detergent exchange, assaying solubility and homogeneity dependence on pH and ionic strength, and to help determine if the protein is well behaved (a target which chromatographs on multiple formats indicates a well behaved and identically-folded population). Since oligomerization is the major purification problem, analyzing all generated protein fractions throughout the purification by SEC, including the effects of additional additives and sample storage at 4º and -80ºC, is essential to this purification approach.
Concentration: A combination of ultra filtration MWt cut-off filters (centrifugal and high-pressure), chromatography (ion exchange and affinity) and dialysis are all utilized to concentrate protein while minimizing the final detergent concentration. Thin layer chromatography ( is used to estimate the final detergent (and lipid) concentration, while SEC coupled with a tetra detector (absorbance, refraction, viscometer, and light scattering) is used to quantitate the excess micelle concentration while following protein homogeneity and measuring hydrodynamic properties.
The focus of this purification approach is quality output, and requires 3-4 days and 7-14 SEC runs per experiment. It is also used following medium throughput expression and purification screens such as 2 detergents for solubilization and SEC (Savage et al 2008), 1 detergent and SEC condition (Min et al 2009), or 1 detergent and 6 different SEC runs (Stroud Lab, unpublished). Over 67 membrane proteins have been purified to PHS using this approach, and 12 crystal structures solved.
Figure 2. Membrane Signature Gel (Coomassie & Ab, high to low concentration)
CellsLyse (high-pressure homogenizer, bead beater)Low speed & high speed spins to access target incorporation (verses IBs) Washes
Membrane Preparation
Washed Membranes with properly targeted protein
BEWAREAb may not bind if [protein] to high
Especially for Yeast membranes
CoomassieAb
Accurate assessment of proteolysis and expression levelsDetermine [membrane] for optimal solubilization screens
Figure 1. Low speed spin test for membrane incorporation (Zach Newby)
Unincorporated
BS AS
Partial Incorporation
BS AS BS AS
Complete Incorporation
With Tomomi Tsomeya
Well suspended membranes
300μl Vt, centrifuge tube, stir bar (transfer sup to new tube for mixing)
pH 8, 50mM Tris (dictated by first purification step), 300mM NaCl
1 hour, 6 hour, O/N
SDS-PAGE Before and After Spin (BS and AS)
Detergent Solubilization
Figure 3. BS & AS pairs, R to LOG, 1hr*OG, O/N*DDM, 1hr
DDM, 15 hrFC14, 1hrFC14, O/N
*Problematic
Human target
Figure 4. BS & AS pairs, R to L,
of Human target.
Top panel = 1hr, bottom panel = O/N
270 mM OG
20 mM DDM
20 mM FC14
20 mM DDM, 3.2 mM CHS
7 mM DDM, 5 mM CHAPS, 3.2 mM CHS
40 mM C12 Sucrose
Detergent Solubilization cont.
SDSOG DDMLDAOCHAPSTX100 MMPC
FC14SDSDDMMMPCTX100
20mM DDM50mM DDM100mM DDM20mM DDM, 3.2 mM CHS
Figure 5. BS & AS pairs, R to L, of a prokaryotic transporter homolog. Right panel = 15 hr, Left panel = 1 hr(with Tomomi Tsomeya)
Detergent Solubilization cont.
Figure 6. BS & AS pairs, R to L, of a human target. 7mM DDM, 5 mM CHAPS, 3.2 mM CHS20mM FC1420mM FC14, 20 mM DDM20mM FC14, 3.2 mM CHS
Ni/Affinity
Desalt
Tag Cleavage
Cleanup
Size Exclusion
Desalt/pH change
Cation and Anion Exchange
Purification Homogeneity
PurificationpH solubilityDetergent exchangeWell behaved
Purification Detergent Exchange
Parameters Detergent/lipidpHIonic strengthReducing agentOsmolytesAdditives
10/300mm Superdex 2000.75/60cm TSK G3000SW≥ 100mM ionic strength+/- glycerol (5-20% v/v)
HiTrap 1ml, 5ml SP HP, pH 5-6Q HP, pH 8-9Salt gradient, steps
Purification & Characterization
Ni: gravity, 1hr dwell, steps10DG “desalting” columnThrombin, C3 protease, TEVOptional Co, Ni cleanup
Common mobile phases40mM OG, 18mM NG, 8mM DM, 0.5-2mM DDM, 4mM FC12, 0.5mM FC14
Key: assay ALL samples by SE9-15 SE runs/ pass
Usually start with10% glycerol
5mM BME/2mM DTT10DG (3ml/4ml load/elute)Nap 5,10 (0.5ml/1ml, 1ml/1.5ml)
Comment on IE: Beware that multi IE peaks may not be real.Since pH and ionic strength are important parameters involved with homogeneity and stability, running S and Q gradients are performed, initially using SEC purified sample. However, as shown in Figure 6 below, different gradients may be needed to verify that multiple peaks are real, or if they are due to a combined effects of [salt] on micelle and detergent belt size/shape thus giving different detergent-protein ratios and shielding charges used in binding.
Figure 7. S/6/OG profiles of identical injections of a human transporter but eluted using different NaCl gradients.
Serial dilutions
A combination of ultrafiltration, dialysis and chromatography (Ni, IE, SE) is used.Our most popular MWCO (MWt cut-off) membranes are colored in red.
MWCO filters (filter type, method, MWCO)Polyacrylonitrile/polyvinyl chloride (Millipore Amicon XM 50, stir)
Favorite; but no longer available (we still have some boxes)Regenerated cellulose
Stirred cellsAmicon Ultracel YM; 30, 100kDa
Tangential spinAmicon Ultracel YM; 30, 50, 100kDa Sartorius (Vivaspin Hydrosart) 30kDa Orbital Biosciences 60, 150kDa
Polyethersulfone filters (currently retesting, and testing) Stirred cell
Amicon Biomax (PM) 30, 50, 100kDa Pall Omega (modified PES) 30, 50, 100kDa
Tangential spin Amicon Biomax (PM) 30, 50, 100kDa Sartorius (Vivaspin PES) 30, 50, 100kDa PALL Omega 30, 50, 100kDa
Pressure-fugation Sartorius (Vivaspin, Vivacell; spin +/- pressure; pressure + rock)
DialysisChromatography (IE, Ni, SE)
Protein Concentration
Nine Step Expression to Structure Workflow
Experimental Step Value
Structure 9
Diffraction 8
Crystals 7
Pure Homogenous Stable 6
SE, S, Q 5
Tag Cleavage 4
Affinity 3
Solubilization 2
Expression 1
Major Milestones ExpressionPHSStructure
S. cere, HEK, P. past, E. coli, Homologs /E.coli
5.0 12
1.8 2.0 Å
3.8, 3.5, 5.0
High Priority MPEC Protein Scorecard (as of 9-08)
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.
Solubilization: 100% 20mM DDM, 1hr Purification: 2mM DDM
Ni++: 1mg/L; 1hr 0mM Imidazole bind, 40mM wash, 300mM bumpDesalt into SE buffer: 20mM Tris 8.0, 150mM NaClCleave: 4unit/OD thrombin, O/N, 95-100%Benzamidine: thrombin removal; Co++: uncleaved removalSE/8.0
PHS dilute & conc., 0.3mg/L yield; 5-10mg PHS prepsHomogenous in 0.5mM and 1mM DDM with 10% glycerol
30% lower yields due to enhanced highermersconsider assaying highermers (3 equal populations)
2mM DDM required without 10% glycerol Gives larger yield due to minimal highermersIE
all binds/bumps on S/6/0.5M as single peak; Q/9 goodS/6 best; stay above 20mM NaCl
Concentration: 50kDa maximum MWCO; 50kDa dialysis does not remove DDM; S/6/0.5M/4mM DDM
PureWell-behaved
First S/6
BSAS
FT
40mM
Ni beads
300mM
desalt
Cleave, Co
0.5 mg/ml
T1day
T47day
Homogenous & Stable
7.5mg/ml
1 week
Purification summary of euk12TM/DDM expressed in Yeast
human5TM/OG expressed in HEK293s
Solubilization: 100% 50mM OG, 1hr, whole cell
Purification (40mM OG)
Ab affinity/50mM OG: 10CV wash
200uM peptide bump; 2mg/L yield
Non-cleavable tag
TSK SE: 7-9mg/8L PHS
Binds and bumps on S/6 and Q/9
Optimum stability and function at pH 6
DTT and glycerol not required
Concentration: 50kDa max MWt filter
SDX overlayTime 0 and 49 days
Homogenous & Stable at pH 6.0, 0.3mg/ml
Pure
Reduced Non-reduced
Post Ab/OG Post SE/OG
Serial dilutions
Prep 1
Prep 2
Well behaved
S/6
And at 11.4 mg/ml, 1 week
Solubilization: 100% 200mM OG, O/N (started with 270mM OG)
Purification
40mM OG, 2 mM βME/DTT, 10% glycerol
Ni++: 3mg/L yield; 2hr 35mM Imidazole bind, 40mM wash, 300mM bump
Desalt into Ni buffer: 20mM Tris 8.0, 500mM NaCl
Cleave: TEV, >5x excess, O/N
Co++: TEV removal
SE/7.3: PHS dilute mg/ml; 2-2.5mg/L yield; >20mg PHS preps
IE: all binds and bumps on S/5,6 and Q/7.3, 8, 9 as single peak
single pH 7.3 SE peaks using dilute mg/ml from pH 5-8 (highermers at pH 9)
Concentration
50kDa maximum YM MWt cut-off
Q/7.3/0.3M/40mM OG---8mM excess OG/16.7mg/ml
PDC Properties: 113 moles OG/tetramer, globular (IV=0.05), 4.3 nm Rh, 0.17 dn/dc
proZTM/OG expressed in E.coli
Homogenous & StablePure
SE/7.3 S/5
bump desalt cleaved
BS AS FT
SolNi
Well behaved
Q/7.3
BS AS
Pooled SEC fractions prior to Q/7.316.7 mg/ml post Q/7.3, freeze thawed6mg/ml post dialysis
SDX (pH 7.3, 40mM OG)
280nm
8mM excess OG micelles
PDC
RI
280nm
SDX, 7.3, 40mM OG
Minimized [Detergent]