Homogenous & Stable - Membrane Protein Expression Center

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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 merc@msg.ucsf.edu 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, TX100, 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.
Membrane Preparation
Cells
Lyse (high-pressure homogenizer, bead beater)
Low speed & high speed spins to access target incorporation (verses IBs)
Washes
Figure 1. Low speed spin test for membrane incorporation (Zach Newby)
BS AS
BS
Unincorporated
AS
Partial Incorporation
BS
AS
Complete Incorporation
Washed Membranes with properly targeted protein
Figure 2. Membrane Signature Gel (Coomassie & Ab, high to low concentration)
Ab
Coomassie
Accurate assessment of proteolysis and expression levels
Determine [membrane] for optimal solubilization screens
BEWARE
Ab may not bind if [protein] to high
Especially for Yeast membranes
With Tomomi Tsomeya
Detergent Solubilization
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)
Round 1 (3-5 detergents)
270mM C8G (OG),
20 mM C12M (DDM)
20mM C14PC (FC14)
And
20mM C12PC (FC12)
20mM monoC14PC (MMPC)
Figure 3. BS & AS pairs, R to L
OG, 1hr*
OG, O/N*
DDM, 1hr
DDM, 15 hr
FC14, 1hr
FC14, O/N
*Problematic
Round 2
Cymals
Sterols
POE
Zwittergents
LDAO
TX-100
Human target
Round 3, Mixtures of
Detergents
Alkyl lipids
Cholesterol, CHS
Under consideration
C13M, C14esterS, C14M, C16M
Detergent Solubilization cont.
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.
Figure 5. BS & AS pairs, R to L, of a prokaryotic transporter homolog.
Right panel = 15 hr, Left panel = 1 hr(with Tomomi Tsomeya)
FC14
SDS
DDM
MMPC
TX100
SDS
OG
DDM
LDAO
CHAPS
TX100
MMPC
Figure 6. BS & AS pairs, R to L, of a human target.
20mM DDM
50mM DDM
100mM DDM
20mM DDM, 3.2 mM CHS
7mM DDM, 5 mM CHAPS, 3.2 mM CHS
20mM FC14
20mM FC14, 20 mM DDM
20mM FC14, 3.2 mM CHS
Purification & Characterization
Parameters
Detergent/lipid
pH
Ionic strength
Reducing agent
Osmolytes
Additives
Usually start with
10% glycerol
5mM BME/2mM DTT
Ni/Affinity
Desalt
Tag Cleavage
Purification
Detergent Exchange
Cleanup
Size Exclusion
Desalt/pH change
Cation and Anion Exchange
Common mobile phases
40mM OG, 18mM NG,
8mM DM, 0.5-2mM DDM,
4mM FC12, 0.5mM FC14
Purification
Homogeneity
Ni: gravity, 1hr dwell, steps
10DG “desalting” column
Thrombin, C3 protease, TEV
Optional Co, Ni cleanup
10/300mm Superdex 200
0.75/60cm TSK G3000SW
≥ 100mM ionic strength
+/- glycerol (5-20% v/v)
10DG (3ml/4ml load/elute)
Nap 5,10 (0.5ml/1ml, 1ml/1.5ml)
Purification
pH solubility
Detergent exchange
Well behaved
HiTrap 1ml, 5ml
SP HP, pH 5-6
Q HP, pH 8-9
Salt gradient, steps
Key: assay ALL samples by SE
9-15 SE runs/ pass
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.
Serial dilutions
Figure 7. S/6/OG profiles of identical injections of a human transporter but eluted using different NaCl gradients.
Protein Concentration
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 cells
Amicon Ultracel YM; 30, 100kDa
Tangential spin
Amicon 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)
Dialysis
Chromatography (IE, Ni, SE)
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
Expression
PHS
Structure
High Priority MPEC Protein Scorecard (as of 9-08)
1.8
9
5.0
12
2.0 Å
3.8, 3.5, 5.0
8
7
S. cere, HEK, P. past, E. coli, Homologs /E.coli
6
5
4
3
2
1
0
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.
Purification summary of euk12TM/DDM expressed in Yeast
BS
AS
FT
40mM
Ni beads
300mM
desalt
Cleave, Co
Solubilization: 100% 20mM DDM, 1hr
Purification: 2mM DDM
Ni++: 1mg/L; 1hr 0mM Imidazole bind, 40mM wash, 300mM bump
Desalt into SE buffer: 20mM Tris 8.0, 150mM NaCl
Cleave: 4unit/OD thrombin, O/N, 95-100%
Benzamidine: thrombin removal; Co++: uncleaved removal
SE/8.0
PHS dilute & conc., 0.3mg/L yield; 5-10mg PHS preps
Homogenous in 0.5mM and 1mM DDM with 10% glycerol
30% lower yields due to enhanced highermers
consider assaying highermers (3 equal populations)
2mM DDM required without 10% glycerol
Gives larger yield due to minimal highermers
IE
all binds/bumps on S/6/0.5M as single peak; Q/9 good
S/6 best; stay above 20mM NaCl
Concentration: 50kDa maximum MWCO; 50kDa dialysis does not remove DDM; S/6/0.5M/4mM DDM
Pure
Homogenous & Stable
0.5 mg/ml
T1day
T47day
Well-behaved
First S/6
7.5mg/ml
1 week
human5TM/OG expressed in HEK293s
Pure
Post Ab/OG
Solubilization: 100% 50mM OG, 1hr, whole cell
Reduced
Post SE/OG
Non-reduced
Prep 2
Purification (40mM OG)
Ab affinity/50mM OG: 10CV wash
Prep 1
200uM peptide bump; 2mg/L yield
Non-cleavable tag
Serial dilutions
TSK SE: 7-9mg/8L PHS
Binds and bumps on S/6 and Q/9
Well behaved
Optimum stability and function at pH 6
S/6
DTT and glycerol not required
Concentration: 50kDa max MWt filter
Homogenous & Stable at pH 6.0, 0.3mg/ml
SDX overlay
Time 0 and 49 days
And at 11.4 mg/ml, 1 week
proZTM/OG expressed in E.coli
Solubilization: 100% 200mM OG, O/N (started with 270mM OG)
Sol
Purification
BS AS FT
40mM OG, 2 mM βME/DTT, 10% glycerol
Ni
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
bump desalt cleaved
BS AS
50kDa maximum YM MWt cut-off
Q/7.3/0.3M/40mM OG---8mM excess OG/16.7mg/ml
Well behaved
PDC Properties: 113 moles OG/tetramer, globular (IV=0.05), 4.3 nm Rh, 0.17 dn/dc
Q/7.3
Homogenous & Stable
Pure
SDX (pH 7.3, 40mM OG)
SE/7.3
S/5
Pooled SEC fractions prior to Q/7.3
16.7 mg/ml post Q/7.3, freeze thawed
6mg/ml post dialysis
Minimized [Detergent]
SDX, 7.3, 40mM OG
8mM excess OG
micelles
RI
280nm
280nm
PDC
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