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 lease e-mail Larry at [email protected]for questions, comments ect.
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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.
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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 [email protected] for questions, comments ect.
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
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.
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.