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Membrane Protein Purification

The challenge of Membrane Protein

Purification

According to Calbiochem: Biological Detergents Guide for solubilization of membrane proteins and selecting tools for detergent removal

Workflow for membrane protein production

Solubilization of Cell Membranes with Detergents

Main features of detergents - Classification of Detergents

Recommended detergents for solubilization of membrane

proteins

Guidelines for Selecting a Detergent

Purification Strategy

Removal of Unbound Detergents - Detergent Exchange

Practical aspects of overexpressing membrane

transporters for structural studies

Detergents in crystallography

Introduction

Membrane proteins already cover more than 50% of the current

drug targets and about 30% of a typical cell’s proteins

Solubilization of membrane proteins can be accomplished by

using amphiphilic detergents.

But preservation of their biological and functional activities can be

a challenging process as many membrane proteins are susceptible

to denaturation during the isolation process.

One of the most important challenges in structural studies

Workflow overview for membrane protein isolation and purification for structural and functional studies

GE Healthcare: Purifying Challenging Proteins.Principles and Methods

Natural source

Solubilization

Final Conditions Buffer Exchange

Desalting Concentration

Detergent exchange

Check Purity, Activity and

Oligomeric state

Purification

Cell disruption and

membrane isolation

Cloning and expression

Detergent screening

Expression screening

Structural and/or

functional studies

Cell harvest

Classification of Membrane Proteins

Integral Membrane Proteins

Amphiphilics: hydrophobics parts in the lipid bilayer and hydrophilics parts to the aqueous environment

Solubilized with detergents or organic solvets.

Group I: Anchored by covalent attachment to fatty acid or lipids

Group II: Anchored by 1 or 2 transmembrane

Group III: Single membrane spanning segment

Group IV: Traverse the bilayer several times. Mayor part within the hydrophobic phase

Findlay,J.B.C.. 1990 Purification of Membrane Proteins in Protein Purification Applications…IRL

Press

Peripheral Membrane Proteins Mainly hydrophylics. Solubilized with buffers that interrupt electrostatic interactions.

Main features of detergents

• Detergents are amphipathic in nature and contain a polar group at one end and long hydrophobic carbon chain at the other end.

• The polar group forms hydrogen bonds with water molecules

The hydrocarbon chains aggregate via hydrophobic interactions.

• At low concentrations, detergent molecules exist as monomers. When the detergent monomer concentration is increased above a critical concentration, detergent molecules self associate to form aggregates known as micelles.

• The critical micelle concentration (CMC) is an important parameter for selecting an appropriate detergent.

• The effective CMC of a detergent can also be affected by other components of the biological system, such as lipids, proteins, pH, ionic strength, and temperature of the medium.

• Aggregation Number: is the number of monomeric detergent molecules contained in a single micelle


At low concentrations, detergents bind to the membrane by partitioning into

the lipid bilayer.

As the concentration of detergent increases, the membrane bilayer is

disrupted and lysed.

Micelles formed by the aggregation of detergent molecules are analogous to

the bilayer of the biological membranes, and proteins can incorporate into

these micelles by hydrophobic interactions.

Hydrophobic regions of membrane protein, normally embedded in the

membrane lipid bilayer, are surrounded by a layer of detergent molecules and

the hydrophilic portions are exposed to the aqueous medium.

This property allows hydrophobic membrane

proteins to stay in solution

Criteria for solubility after detergent extraction

A membrane protein is considered solubilized if it is present in the

supernatant after one hour centrifugation of a lysate or a homogenate at

100,000 x g.

Hence, the appropriate detergent should yield the maximum amount of

biologically active protein in the supernatant.

Another criteria could be behavior in gel filtration (insoluble protein will

elute in the void volume, while protein detergent complex will separate

according to their MW)

Classification of Detergents Non-ionic Detergents

They are considered non-denaturant since they solubilize membrane proteins in a gentler manner,

allowing the solubilized proteins to retain native subunit structure, enzymatic activity and/or non-

enzymatic function.

Non-ionic detergents contain uncharged, hydrophilic head groups that consist of either

polyoxyethylene moieties as in BRIJ® and TRITON® or glycosidic groups as in octyl glucoside and

dodecyl maltoside.

Unlike ionic detergents, salts have minimal effect on the micellar size of the non-ionic detergents,

but increases substantially with rising temperature.

Detergents containing aromatic rings absorb in the ultraviolet region and may interfere with

spectrophotometric monitoring of proteins at 280 nm.

Alkyl glycosides have become more popular as nonionic detergents in the isolation of membrane

proteins.

Can be used with ion exchange columns

Classification of Detergents Non-ionic Detergents

Polyoxyethylenes Alkyl glycosides

Glucamides

Classification of Detergents Zwitterionic Detergents

Offer combined properties of ionic and non-

ionic detergents.

Do not possess a net charge, they lack

conductivity and electrophoretic mobility.

Do not bind to ion-exchange resins.

Like ionic detergents, they are efficient at

breaking protein-protein interactions.

Classification of Detergents Ionic Detergents

Contain head group with a net charge. Cannot be use in EIX

Either anionic (- charged) like Sodium dodecyl sulfate (SDS) or cationic (+ charged)

like Cetyl methyl ammonium bromide (CTAB)

Useful for dissociating protein-protein interactions.

The CMC of an ionic detergent is reduced by increasing the ionic strength of the

medium, but is relatively unaffected by changes in temperature.

Classification of Detergents: Ionic Detergents

Bile acids

Sodium alkyl sarcosine

Sodium dodecyl sulfate

Some recommended detergents for solubilization of membrane

proteins

GE Healthcare: Purifying Challenging Proteins.

Principles and Methods

Some recommended detergents for solubilization of membrane

proteins

A Practical Guide to Membrane Protein

Purification by G. Von Jagow and H.

Schagger Academic Press

Workflow for membrane protein production for structural

and functional studies




proteins







Guidelines For Selecting a Detergent

The appropriate detergent should yield the maximum amount of biologically active protein in the supernatant of a lysate or a homogenate after 100,000 x g

Try first a detergent that has been used previously for the isolation and characterization of a protein with similar biochemical or enzymological properties.

Some “trial and error” may be required for determining optimal conditions for isolation of a membrane protein in its biologically active form.

Not only the type but also the quantity of the detergent used will affect the protein activity. For some proteins biological activity is preserved over a very narrow range of detergent concentration. Below this range the protein is not solubilized and above a particular concentration, the protein is inactivated.

Maintaining a high detergent to protein ratio to make sure the protein is well dispersed (the detergent concentration will be much higher than the CMC).

Guidelines For Selecting a Detergent Consider downstream applications

Consider the method of detergent removal:

If dialysis is to be employed, a detergent with a high CMC is clearly preferred.

If ion exchange chromatography is utilized, a non-ionic detergent or a ZWITTERGENT® is the detergent of choice.

Ionic detergents should be avoided if the proteins are to be separated by charge columns (IEX, Hydroxyapatite, etc)

For gel filtration of proteins, use detergents with smaller aggregation numbers.

Consider detergent purity. Some detergents such as TRITON®X-100 are generally known to contain peroxides as contaminants.

TRITON® X-100 contains aromatic rings that absorb at 260-280 nm, this detergent should be avoided if the protocols require UV monitoring of protein concentration.

Guidelines For Selecting a Detergent

If the protein is not completely detached from other proteins during solubilization, and from most of the phospholipids, the protein will behave in an unpredictable way in a chromatographic separation (sometimes will behave heterogeneously).

A detergent that is good for solubilizing intact membranes and removing excess lipid may not be the best for continuing to purify the “naked” protein (may be too harsh).

For the initial steps, you may want to choose a relatively cheap, and relatively pure detergent that can be readily exchanged for another.

Even if a protein is not active in a particular detergent, if you can demonstrate that you can reverse this inactivity by adding back lipid or another detergent, you may still be able to use it.

High or reasonable yields must be combined with preservation of activity and stability in detergent solution after solubilization

Workflow overview for membrane protein isolation and purification for structural and functional studies

GE Healthcare: Purifying Challenging Proteins.Principles and Methods

Natural source

Solubilization

Final Conditions Buffer Exchange

Desalting Concentration

Detergent exchange

Check Purity, Activity and

Oligomeric state

Purification

Cell disruption and

membrane isolation

Cloning and expression

Detergent screening

Expression screening

Structural and/or

functional studies

Cell harvest

Purification Strategy - I

Over-expression is a major bottleneck in the overall workflow for membrane protein production. It is often useful to try different hosts or host strains in parallel for a particular target protein to increase the likelihood of success

In addition, homologous membrane proteins from several sources can be cloned in parallel to be able to select those that express well

“High” expression levels for functional membrane proteins are usually more than an order of magnitude lower than for over-expression of water-soluble proteins in E. coli. Membrane proteins need to be inserted into membranes, and the availability of membrane structures in most cells is limited

A modest growth and expression rate is beneficial to avoid the formation of inclusion bodies when using E. coli as a host. This can be achieved by the use of a weak promoter, a low concentration of inducer and/or lowering the growth temperature after induction

Overexpression systems used for prokaryotic and eukaryotic membrane protein production

GE Healthcare: Purifying Challenging Proteins. Principles and Methods

Expression system

Advantages Disadvantages

E.Coli The most widely used

over-expression system for

(prokaryotic) membrane protein

production.

Often not suitable for over-expression of eukaryotic membrane proteins

No glycosylation and limited posttranslational modifications

Yeast Can perform some posttranslational modifications

Several different yeast systems have been used for membrane protein production

Does not produce high cell densities

(S. cerevisiae)

Hyperglycosylation can occur

(S. cerevisiae)

Different lipids (compared with mammalian cells)

Insect Cells Less complex growth conditions compared with mammalian cells

Relatively high expression levels

Glycosylation

More costly and complex than E. coli or yeast; different lipids (compared with mammalian cells)

Overexpression systems used for prokaryotic and eukaryotic membrane protein production


Expression system

Advantages Disadvantages

Mammalian cells

CHO, BHK and other cell lines are often used for functional studies of receptors

Authentic (mammalian) protein is produced

High cost and complex work

Rhodobacter spp.

High expression levels through coordinated synthesis of foreign membrane proteins with synthesis of new internal membranes

Different lipids (compared with mammalian cells)

Cell free Allows expression of toxic proteins and proteins that are easily degraded in vivo

Allows incorporation of labeled and non-natural amino acids.

High cost

Membrane protein insertion in membrane or detergent micelle has not been fully developed

Purification Strategy - II What is known about the properties of the protein. Literature

Small scale extraction and solubilization experiments OPTIMIZATION: detergents, and then salt concentration, pH, protease inhibitors, additives, activity, etc

Choice of a suitable protein properties to follow target purification

Isolation time as short as possible + 4°C + protease inhibitors

Dissolve the protein at the minimum detergent/protein radio

Avoid intensive delipidation: could affect activity without affecting structure

Consider a first step of enrichment without the use of detergents: Isolating organelles and membranes at the beginning of the purification

Purification Strategy – III

Chromatographic Separation: narrow spectrum

Affinity: Affinity tagging greatly facilitates expression screening and purification.

Longer histidine tags (with 8 or 10 histidine residues) are often used for membrane

proteins to increase the binding strength, and placed on the C-terminal to reduce

risk of affecting the membrane insertion process based on the N-terminal signal

peptide.

Ultrafiltration and Gel Filtration: consider aggregation number of the detergent

Ion Exchange or Hydroxylapatite: do not use ionic detergents. Can cause

aggregation. Excellent for homogeneity characterization of purified proteins.

Hydrophobic exchange: limited use

Blue Native preparative electrophoresis

Purification Strategy - IV

For some proteins a batch procedure is prefer to column chromatography

Detergent concentration in all buffers should be above the CMC.

A relatively low NaCl concentration (e.g., PBS is 150 mM NaCl) is recommended because membrane proteins tend to be less soluble at higher ionic strengths.

Protein aggregation is a common issue with membrane proteins. Aggregation often appears to be irreversible and it may occur slowly over time but also rapidly and unexpectedly with modest changes in ionic strength, protein : detergent ratio, pH, and other factors.

SDS-PAGE analysis: Boiling of the sample with SDS can cause aggregation of membrane proteins. Incubate at 60°C for 30 min or at 37°C for 60 min for preparing the sample. There are some exceptions

Screening of pH and ion strength conditions for optimal homogeneity and stability of a detergent-protein complex


Gel filtration is the method of choice for rapid detection of

aggregation since SDS (in PAGE-SDS) solubilizes most aggregates.

Charge homogeneity characterization of histidine-tagged cytochrome bo3 ubiquinol oxidase using anion exchange chromatography with

Mono Q of two fractions obtained from Ni purification GE Healthcare: Purifying Challenging Proteins. Principles and Methods

Analytical Ion Exchange: Excellent for homogeneity characterization of purified proteins

Removal of Unbound Detergents Detergent Exchange

Dialysis or Ultrafiltration When detergent solutions are diluted below the CMC, the micelles are dispersed

into monomers of low molecular weight that can be easily removed by dialysis. (If a large dilution is not practical, micelles can be dispersed by other techniques such as the addition of bile acid salts).

Very good option for removal or exchange detergents with high CMC.

Less suited for non-ionic detergents. Long time.

Size Exclusion Chromatography

Takes advantage of the difference in size between protein-detergent, detergent-lipid, and homogeneous detergent micelles. The elution buffer should contain a detergent below its CMC.

Usually for detergents with low aggregation number.

Limited volume sample.

Removal of Unbound Detergents Detergent Exchange

Detergent Hydrophobic Adsorption This method exploits the ability of detergents to bind to hydrophobic commercial

resins. The resin with the bound detergent can be removed by centrifugation or filtration.

This technique is effective for removal of many detergents. If the adsorption of the protein to the resin is of concern, the resin can be included in a dialysis buffer and the protein dialyzed.

Be sure not to lost the protein

Selected Protein Adsorption Target protein binding to Ion Exchange Chromatography or Affinity

Chromatography. Adsorbed protein is washed or replaced with another detergent and eluted.

This technique is the best option to removal or replaced of detergents with low CMC. Bonus: protein concentration and purification

Workflow for membrane protein production for structural

and functional studies




proteins







Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies

Da-Neng Wang et al. Biochimica et Biophysica Acta 1610 (2003) 23– 36

Expressing several homologues of the target protein simultaneously.

Use various expression vectors Ideally, the vector should have a tightly regulation to prevent leaky expression, which can

lead to in vivo proteolysis, or even cell death when expressing toxic membrane proteins Moderately strong promoter, to avoid inclusion body formation A wide range of usable inducer concentrations allows better control of expression

Try Several host strains / Colony selection Expression levels can vary as much as 2- to 5- fold between strains

Careful optimization of cell culture conditions Can drastically improve the expression level and homogeneity of the target protein. Culture medium cell density at induction and harvesting inducer concentration induction temperature Avoid inclusion bodies formation.



Protein purification, stability and functionality optimization

Size exclusion chromatography is an efficient method in screening detergent, pH an other

conditions required for maintaining the stability and monodispersity of the protein.

Protein construct

Flexible termini and loops in a protein often obstruct the formation of high-quality crystals.

Using new sample preparation techniques for mass spectrometry of membrane proteins

have enabled to identify the rigid protein core, which can be subsequently over-expressed.

Protein homogeneity

Protein impurity, detergents (free, unbound detergent in solution) and co-purified lipid.

Complete delipidation of membrane proteins often causes denaturation and aggregation. It

is therefore necessary to identify the lipids that are critical for the protein’s stability and

subsequently to control their composition in the purified sample



Stability and monodispersity: detergent, lipid, pH, and

temperature

Use GF to see aggregation or change in oligomeric state

Glycerol at concentrations of 20% is highly effective in

increasing stability

Monitore activity and integrity to ensure that the

structure determined is of physiological relevance:

A) reconstitution of the purified protein into proteo-

liposomes, followed by transport assays or

B) substrate binding to the solubilized protein in

detergent solution

Monodispersity after incubation 2hrs 25°C with different detergents

An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins

Eshaghi et al Protein Science (2005) Vol 14

HTP strategy for cloning and expression screening of membrane proteins in their

detergent solubilized form

Three expression vectors using Gateway technology: 6-His-, a FLAG-, or an MBP- at the

NH2 terminus and a 6-His tag coding sequence followed by three stop codons at the

COOH terminus that allowed dot-blot detection

Transformormation: C41, C43, and BL21 strains.

HTP detection of expressed membrane proteins in 96-well plates

Direct detergent extraction of membrane proteins in the noncentrifuged cell lysate,

followed by affinity purification of both, sup and pellet separately

Dot-blot analysis and then PAGE-SDS

Effects of temperature, induction time, and inducer concentrations

HTP screen of various detergents in extraction and purification of selected proteins.

Evaluation of expression results by

medium-scale protein production

Intactness and homogeneity of

the proteins purified on gel filtration

with either FC12 or DDM.

The amounts of purified proteins

after gel filtration were calculated to

be 3–5 mg/L culture for high

expression, 1–3 mg/L for medium

expression, and 0.2–1 mg/L for low

expression

An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins

Eshaghi et al Protein Science (2005) Vol 14

An overall scheme of the detergent screen platform using 96-well plates

D. Niegowski et al. / International Journal of Biological Macromolecules 39 (2006) 83–87

Same platform used to purification optimizations before

large-scale purifications : various imidazole concentrations,

four different types of metal affinity columns, etc.

Detergent exchange was successfully performed during

IMAC purification followed by gel filtration

Detergents in crystallography So Iwata, Methods and Results in Crystall. Of Membranes Proteins - IUL Biotechnology Series

The detergent concentration used during initial crystallization screens for membrane proteins remains

an important consideration (Methods in Enzymology, Volume 557, 95-115: Quantification of Detergent

Using Colorimetric Methods in Membrane Protein Crystallography - Chelsy Prince, Zongchao Jia)

Minimization of excess detergent and “empty” detergent micelles would be favorable to crystallization

efforts

Concentrations near a detergent’s cloud point (i.e., the phase boundary in which intermicellar attractive

forces drive micelles into a separate phase) can show correlation with crystallization

Proteins are often more soluble and stable in detergents with long alkyl chains such as Triton X-100 or

dodecylmaltoside, because they more closely mimic the cell membrane but they have lower crystall

quality

So, it is useful to prepare the sample in a long chain detergent where the protein is happy, and add a

short chain detergent for crystallization

Most common detergents used in crystallography: OG (1%), DDM (0.03%), UDM or decylmaltoside

(0.1%), C12E9 (0.03%) and LDAO (0.1%). Usually 2-3 times the CMC.

The use of stabilizing mutants has had a revolutionary impact on increasing the crystallization propensity of

some membrane protein targets

Incorporating fusion partner proteins such as T4 lyzozyme (T4L)1 has been particularly important in

structural studies of G protein-coupled receptors (GPCR)

The improved understanding of cellular pathways controlling translation and protein folding, and how they

influence functional recombinant protein yields, means it is now possible to select (or even design) better

expression strains

New methods for extracting and solubilizing membrane proteins from the cell membrane using styrene

maleic anhydride (SMA) co-polymers have enabled traditional detergents to be circumvented. The

benefits of this approach include improved thermostability of the solubilized protein and retention of

protein–lipid interactions that are normally disrupted during detergent-extraction

Finally, most GPCR crystal structures have been obtained using a fusion protein strategy where the flexible

third intracellular loop isreplaced by T4L, with modified T4L variants having been developed to optimize

crystal quality or promote alternative packing interactions

Recent experimental breakthroughs in structural studies of G protein-coupled receptors (GPCR)

S.J. Routledge et al., Methods (2015), http://dx.doi.org/10.1016/j.ymeth.2015.09.027

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