Purification of membrane proteins

Dr.Alain Jacquet (Chulalongkorn Dr.Alain Jacquet (Chulalongkorn

University)
Account for about 40% of the proteins in the cell
Receptors, ion channels, transmembrane transporters, signal transducers, ion
pumps, free energy transducers, etc.
Approx. 60,000 protein structures in the PDB (Protein Data Bank, 2011),about
0.5% integral membrane proteins.
Why? Difficulties:
To purify, more problematic than water-soluble proteins:
Expressed in low amount
Highly hydrophobic, Sticky! (membrane-associated) , tendency to form
Membrane proteins
Highly hydrophobic, Sticky! (membrane-associated) , tendency to form
aggregates
Often more susceptible to degradation by proteases
following solubilization
To express recombinant forms
To stabilize and to crystallize
Proportion of membrane-spanning proteins in the proteome of E. coli and S.
cerevisiae associated with different cellular functions. Number of membrane
proteins classified according to the number of transmembrane helices.
2006
Proteins with a
cytoplasmic C terminus
(Cin) are plotted upwards
and those with an
extracytoplasmic C
terminus (Cout) are plotted
downwards.
Two main types of membrane proteins
Peripheral or extrinsic membrane proteins , which interact with the membrane
surface non-covalently by means of electrostatic and hydrogen bonds or with
covalent bonds through lipids or GPI anchors.
Integral or intrinsic membrane proteins , much more strongly associated with
the membrane. Interact with hydrophobic moieties of the phospholipid bilayer.
Contain one or more apolar domains that span the lipid bilayer (-helix but also
-sheet). Type I Integral membrane proteins: C-ter embedded in the cytosol or
Type II: N-ter in the cytosol.
First purification step: isolate the membrane fraction
Disrupt the harvested cells (SEE AFTER)
Remove unbroken cells with a low speed centrifugation ( 10000 g).
To remove cell debris that are not membrane, as well as some cell
organelles such as inclusion bodies (If cells are bacteria) or nuclei
Collect the membrane fraction by centrifuge the supernatant from the
last step with a high speed ultra-centrifugation (100000 g) . This
step removes all the soluble proteins.
Mammalian cell organelle
isolation by differential
centrifugation
Cell harvesting by scraping. Specific membrane markers are required to follow
the fractionation procedure, to confirm the stability of the preparation (ATPase,
cadherin,)
All Steps on Ice to prevent proteolysis!
Add Protease inhibitors in the buffer
Do not use trypsin of course for cell
detachment (membrane protein digestions) !!!
Cell pellet resuspended in a suitable buffer for cell disruption (e.g., PBS).
Addition of Dnase to reduce viscosity, useful to add a protease inhibitor cocktail
to reduce possible protein degradation.
Cell disruption methods
Gentle cell lysis to keep all organelle intact!
Cells homogenized by passage through a needle or using tight-fitting glass-
glass Potter or a Dounce homogenizer (Up to 15-20 passages of the pestle
may be required to achieve sufficient cell breakage).
..
Dounce
homogenizer
Potter
homogenizer
Low speed centrifugation (3-10000g) to prepare a post-nuclear supernatant
(PNS). Under gentle conditions of homogenization, 50-60% of a fluid phase
marker is recovered in the PNS. The rest, which consists partially of unbroken
cells, is lost to the nuclear pellet (NP).
Ultracentrifugation to isolate membranes (in the pellet or in some
zones using sucrose gradient)
Protein density: 1.3 g/cm
3
Membrane density: 1.0-1.1 g/cm
3
Peripheral Membrane Protein Extraction
Peripheral proteins (non lipid- or GPI-anchored) dissociated using relatively mild
techniques to break electrostatic or hydrogen bonds between the peripheral proteins and
the membrane, without total membrane disruption.
High salts useful to decrease electrostatic interactions between proteins and charged
lipids
Chaotropic ions disrupt hydrophobic bonds present in the membrane surface and
promote the transfer of hydrophobic groups from non-polar environment to the aqueous
phase.
High pH to disrupting sealed membrane structures without denaturing the lipid bilayer or
extracting integral membrane proteins. 1030 min extraction followed by centrifugation
(3060 min, 100000 g) to separate the released peripheral membrane proteins
(supernatant) from the remaining lipid bilayer
Na
2
CO
3
extracted membrane proteins
2-D gel electrophoresis. Silver staining
Summary for membrane isolation
Integral Membrane Protein Extraction and Purification
Necessity to disrupt the lipid bilayer, which may be achieved with organic
solvents, but is more commonly accomplished using detergents.
Detergents
Some detergents contain both
polar and nonpolar faces;
Detergents: amphipathic substances with a polar (hydrophilic) head group and
a nonpolar, (hydrophobic) tail.
measurable aqueous solubility as both aggregates and as monomers
Classified according to the polar part: nonionic, anionic, cationic, or zwitterionic
Traditional detergent monomers are generally cone shaped; hydrophilic
head groups occupying more molecular space than the linear alkyl chains
Lipids generally cylindrical; area occupied by
the two alkyl chains is similar to the area
occupied by the polar head group. Lipids have
low solubility as monomers and tend to
aggregate into planar bilayers that are water
insoluble.
Above a certain concentration (critical micellar concentration-CMC) in an
aqueous environment: detergent molecules associate to form multimolecular
complexes, micelles, with hydrophobic interiors and hydrophilic surfaces.
At a concentration = 1-3 x CMC, detergent can solubilize
integral membrane proteins
CMC inversely related to the size of the alkyl chain ,
sensitive to both temperature and [salt]
Generally, Non ionic detergents disrupt protein-lipid interactions but not protein-protein
interactions, contrary to ionic and zwitterionic detergents
detergent monomers partitioning into the bilayer. Cooperative detergent-detergent
interactions destabilize the bilayer yielding mixed lipid-detergent fragments . interactions destabilize the bilayer yielding mixed lipid-detergent fragments .
Eventually, further detergent
addition leads to bilayer dissolution and
protein solubilization
Which detergent will let membrane proteins soluble,
monodisperse, and folded (non denaturing) ?
Not only one detergent that works for all membrane proteins.
Therefore, for each membrane protein isolation: detergents screening and
particularly IF little information in the literature on the purification of
similar proteins
Of course, the choice of detergent(s) will also affect the efficiency of
downstreamprotein purification procedures but also cristallization.
Must consider the possibility of detergent exchange
Detergent structures
Centrifuge at 100 000 x g at 4C for 45 min.
Solubilization Criteria: Retention of a membrane protein in the supernatant following
centrifugation for 60 min at 100,000 x g after extraction.
Tendency of a detergent to denature membrane proteins: dependence on the
size and charge of the polar headgroup, as well as the length of the alkyl tail
(parameters also affecting the CMC and the size of the micelle)
Membrane protein extraction kit, detergent screening kit avaible.
At least from: GE Healthcare, Qiagen, Pierce
For optimal solubilization:
Membrane incubation with various
[detergent], incubation time, buffer
concentration, salt solutions, and
temperature conditions
The structural and functional properties of POI checked during the screening!
But analysis of the pellet too!
Removal of Detergents
high detergent concentrations often required during the initial extraction of
integral membrane proteins affect the stability and subsequent analysis of
the isolated membrane proteins but also the purification process
excess detergent removed or exchanged for an alternative detergent
Choice of technique depends on the unique properties of the detergent used
and the concentration range of the protein fraction.
Bio-beads SM-2 Polystyrene-divinyl-benzene
Efficiency of dialysis CMC and micelle Mw-dependent
Detergents with linear alkyl hydrophobic groups (e.g. Triton X-100) have a high
micelle Mw non dialyzable whereas detergents with a low micelle Mw and and
high CMC (e.g. bile acids and their derivatives) easily removed by dialysis.
Detergent solutions can be diluted below their CMC so that micelles
disintegrate into monomers which can be dialyzed (use of large excess of
detergent-free buffer)
Detergents with low CMCs typically removed by adsorption to hydrophobic
Beads and detergent bound beads can then be removed by filtration or
centrifugation. Gel filtration can be used to separate detergent micelles from
protein-detergent complexes and free protein based on size differences.
.
Importance of functional assays to detect POI but to be sure about the protein
integrity in the presence of detergents during purification
No single protocol for obtaining membrane protein purification
Membrane proteins usually purified as soluble protein-lipid-detergent
complexes in an aqueous environment
Possibility to use essentially the same separation techniques as used for water-
soluble proteins BUT:
-) with detergent present in all solutions (protein-detergent complexes are
dynamic and lose detergent molecules in the absence of free detergent).
Membrane protein purification by chromatographic techniques
General considerations
dynamic and lose detergent molecules in the absence of free detergent).
-)Detergent concentrations should be above the CMC but lower than what was
used during solubilization (typically in the 0.1% range).
-)All the matrices non compatible with the presence of detergents!
HIC non compatible
AF: some ligands sensitive to detergents (proteins, Abs,)
IEX: avoid using anionic detergents with anion exchange columns, and
cationic detergents with cation exchange columns
GF: ideal final purification step for membrane proteins to remove
aggregates and other impurities according to the size while
simultaneously performing buffer exchange.
Triton X-100: substantial UV absorbance at 280 nm [protein] often
overestimated by UV (use other monitoring). Alternative, CHAPS or CHAPSO
for solubilization.
Detergents with COO- groups (as N-lauryl sarcosinate, bile salts,)
precipitate with divalent cations avoiding this class of detergents in the
sample when Ca2+ or Mg2+ (No precipitation with CHAPS and CHAPSO)
Carboxylic acid-containing detergents (bile salts and N-lauryl sarcosinate)
Influence on the pH and the charge
Carboxylic acid-containing detergents (bile salts and N-lauryl sarcosinate)
may not be suitable in protein separation where pH values vary (isoelectric
focusing, pH gradient elution from ion-exchange resin, )
Such detergent expected to protonate and become insoluble in aqueous
media at weakly acid pH value.
Non-ionic and zwitterionic detergents do not move in electrical fields, don't
bind to ion exchange resins, and do not contribute to the net charge of
macromolecules to which they are bound use of non-ionic or zwitterionic
detergents for charge-related separation techniques such as ion-exchange
chromatography and preparative electrophoresis
A lot of techniques to estimate the quality of purified membrane
proteins
Nanodisc: patch of 130160 lipids organized as a bilayer and surrounded by
stabilizing proteins (called membrane scaffolding proteins (MSPs)) forming
amphipathic helical protein belt.
MSP derived from human high-density lipoprotein apoA-1, modified to remove
undesired domains and to duplicate other domains to increase the proteins
length and, thereby, the perimeter of the ND.
Nanodisc to solubilize membrane proteins
MP inside nanodisc
Membrane protein isolation in a nanodisc
MP transiently solubilized with a detergent in the presence of phospholipids and
MSP. After detergent removal, MP simultaneously assembles with
phospholipids into a discoidal bilayer with the size controlled by MSP length
Soluble membrane protein expression using Nanodisc
Proteins expressed in the presence (blue) or absence (red) of Nanodiscs.
For the analyzed data set, the overall solubility increased from 17.3 2.2% (in the
absence of Nanodisc) to 78.8 3.4% (in the presence of NLPs), notably G protein-
coupled receptors (GPCRs)
TMS = transmembrane segments.