Affinity Chromatography (AC) - Hebrew University of...

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Affinity Chromatography

(AC)

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Affinity Chromatography (AC)

• Principles of AC

• Main stages in Chromatography

• How to prepare Affinity gel - Ligand Immobilization - Spacer arms – Coupling methods – Coupling tips

• Types of AC

• Elution Conditions

• Affinity Tags and Fusion Proteins: Chelating, Strep-tag, GST, MBP, etc

• Cleavage sites – Proteases

• Parameters for development optimization (Troubleshooting): binding, washing, elution

• Examples

• Binding equilibrium, competitive elution, kinetics

• Future Considerations

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What is affinity chromatography?

Affinity chromatography is a technique of liquid chromatography which

separates molecules through biospecific interactions.

The molecule to be purified is specifically and reversibly adsorbed to a

specific ligand

The ligand is immobilized to an insoluble support (“matrix”): resin, “chip”,

Elisa plate, membrane, western, etc

Introduction of a “spacer arm” between the ligand and the matrix to

improve binding

Elution of the bound target molecule: a) non specific or b) specific elution

method

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What is it used for?

Monoclonal and polyclonal antibodies

Fusion proteins

Enzymes

DNA-binding proteins . . . . . .

ANY protein where we have a binding partner!!

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Sample: 600 ml mouse monoclonal IgG 2a Gel: rProtein A Sepharose Fast Flow Elution: 20 mM Sodium Citrate, pH 4.0 Result: 83 mg Mab, recovery 95%

AU

2.0

1.5

1.0

0.5

0.0

0 200 400 600 Volume (ml)

Starting material

Eluate

94

43

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SDS-PAGE reduced

Purifying monoclonal antibodies

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Designing and preparing an affinity gel

Choosing the matrix

Designing the ligand - Spacer arms

Coupling methods

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Ligand Immobilization

Matrix

+ Activating

agent +

Ligand

Activated matrix

Immobilised ligand

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Designing the ligand

Essential ligand properties: interacts selectively and

reversibly with the target

Carries groups which can couple it to the matrix without

losing its binding activity

Available in a pure form

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Steric considerations & spacer arms

Small ligand (<1,000)

Risk of steric interference with

binding between matrix and

target molecule

Often need spacer arm

but watch out for

adsorption to the spacer! Spacer arm

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Design of spacer arms

Alkyl chain Real risk of unspecific interactions between spacer and target molecule

Hydrophilic chain Risk of unspecific interactions greatly reduced No coupling reaction will use 100% of the available binding sites. Using a gel with pre-attached spacer arms will leave some uncoupled spacers, increasing the possibility of non-specific interactions. The unused spacer arms should not be too much of a problem if they are hydrophilic. If available, this problem may be eliminated by using a ligand with a pre-attached spacer arm.

OH

O

O

H

H

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Choosing a coupling group

• Ligands are coupled to the matrix via reactive groups

Amino -NH2

Hydroxyl -OH

Aldehyde -CHO

Thiol -SH

Carboxyl -COOH

• The group coupled must not be too near the site for binding the target

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Useful Coupling Methods

Coupling chemistry Kinds of group coupled Ligand

N-hydroxysuccinimide -NH2 Protein ,Peptide. Sugar.Polynucleotide.Cofactor

CNBr -NH2 Protein , Sugar.Polynucleotide.Cofactor

Carbodiimide -NH2 Small ligands. Non polar ligand in organic

via hydrophilic spacer arm solvents. Derivatized material

Epoxide -SH-NH2-OH Sugars. Small ligands

via hydrophilic spacer arm

Thiol exchange -SH Protein ,Peptide. Polynucleotide

Mild Conditions Low MW thiol containig ligand

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Useful Coupling Methods

Sepharose Cyanogen bromide

Cyanate ester formed

C N

Br+O

Gel HO

Gel C N + H Br

Cyanogen bromide activation

+ Lig NH2

OGel C N Gel

OC

NH

NH

Lig

CNBr-activated Sepharose

Protein ligand N-substituted isourea

Cyanogen bromide coupling

Reaction at pH 8. Isourea may be cleaved by nucleophilic attack at high pH.

Multiple bonds formed with protein ligands. No spacer arm present.

WARNING!: Cyanogen bromide is dangerous. Consult safety data before start working

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A general protocol for ligand coupling

Prepare the ligand solution in coupling buffer

Prepare the activated matrix

Mix the ligand solution and the activated gel in coupling buffer until

coupling is complete

Check total ligand before and after coupling: ligand/ml resin

Block any remaining active groups

Wash the coupled gel alternately at high and low pH to remove adsorbed

ligand

Transfer to storage buffer

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Tips: Preparing the activated matrix - couple buffer

If the activated matrix is freeze-dried, allow it to swell before washing

Always use the recommended swelling and washing solutions!

The swelling, washing and equilibration are best performed on a sintered

glass filter. Facilitates quickly removal of excess liquid from the media

Couple buffer: make sure there are no components, e.g. amines, that can

couple in place of the ligand, e.g. Tris buffer has a primary amine

To suppress ionic adsorption, add NaCl, 0.5 M.

pH is important. Always use the recommended pH.

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Tips: Coupling reaction, washing and storage

Ligands which are poorly soluble in water, may usually be added in an organic solvent.

The ligand should be available at a high level of purity. Any contaminants present in the

ligand sample will likely be coupled to the matrix.

Mix the activated gel and the ligand solution by swirling or end-over-end mixing - Avoid

magnetic stirrers as these can crush the gel and produce fines!!

Take samples of ligand before and after reaction: to calculate ligand per ml resin

Time and temperature are in the standard protocol. Most coupling reactions are

performed at room temperature

Block any remaining active groups with a small neutral ligand (Tris buffer for amino

reactions)

Wash the coupled gel at alternating high and low pH to remove adsorbed substances.

Keep in storage buffer (Na Azide , Thimerosol 0.02% or ethanol if possible)

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7

20

2408

18647

A. Tobin et al. (1996) J. Biol. Chem. 271, 3907-3916

• 10 mg homogenous protein

G Protein Receptor Kinase

Technique

Ppt

HIC

AIEX

CIEX

AC

Pig brain homogenate

RESOURCE Q

RESOURCE S

HiTrap Heparin

Purification factor

Comment

Ammonium sulfate precipitation

Butyl Sepharose Fast Flow

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Placental extract in 1.5% Triton X-100

Membrane Protein

Technique

Blue Sepharose

DEAE Sephacel

SP Sepharose

Muc2 Sepharose

Mono S

AC

AIEX

CIEX

AC

CIEX

Purification factor

Comment

• Main purification step

• Final polishing and purity check, 20 mg obtained

T. White et al. (1995) J. Biol. Chem. 270, 24156-24165

3

4

6

242

1442

• Concentration and capture

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AC

DNA-binding Protein

Technique

DNA-1 Sepharose

DNA-2 Sepharose

Mono S

CIEX

AC

AC

CIEX

Purification factor

Comment

• General AC step for DNA-binding proteins

• Removal step, non-specific DNA binding activity removed

• Main purification step

• Final polishing, 20 mg obtained

J. Berthelsen et al. (1996) J. Biol. Chem. 271, 3822-3830

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8

9

2447

4943

HeLa cell nuclear extract

SP Sepharose High Performance

Heparin Sepharose Fast Flow

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Advantages and Disadvantages of affinity chromatography

Easy to achieve otherwise difficult

separations

Often high purity in one step

(but not always)

Fast separations (depends of the

kinetics of binding and elution)

Can be use to remove specific

contaminants

Economics (scale-up)

Buffer / elution limitations

Sometimes needs harsh or

denaturizing elution conditions

Do not resolve all the problems

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The main stages in affinity chromatography

Equilibration Of gel and sample to binding conditions

Sample application

Wash contaminants

Elution of the target

Re-equilibration Of the gel to binding conditions Or keep in storage buffer (20% Ethanol or 0.02%NaAzide in Buffer)

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Columns and equipment

Column volume: according to amount of target and gel capacity

Avoid using excess of resin: to avoid low affinity binding of

impurities

Column length: not usually critical, short and wide

Equipment: no special demands

Equilibrate column before applying sample and follow standard

protocol

Tips

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Sample preparation

Adjust pH, buffer salts and additives to promote binding

Filter or centrifuge to remove particles

Make sure that components known to interfere with binding

are absent

Consider alternative capture steps before affinity purification

(cost of resin, crude impurities or interfering substances,

concentrate sup, etc)

Tips

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Sample application

Binding buffer: usually neutral pH, and high salt concentration when is

possible (0.3-0.5M NaCl) to avoid hydrophobic interactions of non specific

proteins to the resin. Use additives only if necessary.

For batch binding, incubation of around 1.5 hours at 4°C is enough. For

extremely slow binding kinetics, incubate overnight at 4°C.

Loading directly on column

- Strong affinity and fast binding: High flow rate

- Weak affinity and/or slow binding: Low flow rate

Tips

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Elution and re equilibration

Follow standard protocol

Optimize alternative wash strategies to increase final purity

If eluting at extreme pH: collect the target protein in a small

amount of concentrated buffer at a neutralizing pH

Re-equilibrate column immediately with binding or storage

buffer

Regeneration: use buffers that do not harm the ligand

Tips

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Column Regeneration

Wash with basic and acidic buffers.

– 10 col. vol. 0.1 M Tris.HCl, 0.5 M NaCl, pH 8.5

– 10 col. vol. 0.1 M NaAc, 0.5 M NaCl, pH 4.5

– Use chaotropic agents or detergents if needed

Re-equilibrate with 10 column volumes starting buffer. Use more if needed

Ni columns can be washed with 0.5N NaOH, neutralized, destriped with

100mM neutral EDTA, wash, charge with 100mM Ni SO4, wash and store

with 20% Ethanol

Tips

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Column Storage

To maximize the useful lifetime of an affinity resin, choose a

storage buffer that does not harm the ligand

Typical buffers are 20% ethanol solution or binding buffer

containing a bacteriostatic agent (e.g. 0.02% Thimerosal or

0.02% sodium azide)

Many of the commercial resins can be used hundreds of times

Tips

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• Types of AC


• Affinity Tags and Fusion Proteins: Chelating, Strep-tag, GST, MBP, etc



• Examples



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Type of Affinities

Mono-specific ligands

• Specific for a single substance

Antigen antibody

Hormone receptor

• Usually home-made gels

• Elution scheme must be worked

out for each case:

• Often general elution

• Little help from the literature

Group-specific ligands

• Specific for a group of structurally

or functionally similar substances:

Lectins glycoproteins

Protein G IgG antibodies

Dye-stuffs enzymes

• Often ready-made gels

• Known elution schemes

• Standard tested elution protocols

• Often competitive elution

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Group-specific ligands - 1

Ligand Specificity

Protein A Fc region of IgG

Protein G Fc region of IgG

Concanavalin A Glucopyranosyl & Mannopyranosyl groups

Peanut Lectin Terminal galactose group

Cibacron Blue Broad range of enzymes, serum albumin

Procion Red NADP+ dependent enzymes

Lysine Plasminogen, ribosomal RNA

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Group-specific ligands - 2

Ligand Specificity

Arginine Serine proteases

Benzamidine Serine proteases

Calmodulin Proteins regulated by calmodulin

Heparin Coagulation factors, lipoproteins, lipases,

hormones, steroid receptors, protein synthesis

factors, Nucleic acid-binding enzymes

Metal chelate resins Proteins and peptides which contain

(Co, Ni, Zn, Cu, Fe) 6-12 Histidines

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Designing the elution scheme

General elution conditions

– Low pH, salt, GuHCl, Urea etc.

– Often harsh conditions

Specific eluents

– Competing free ligand

– Competing binding substances

– Sometimes expensive compounds (like peptides)

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Reconstituting

buffer

+

Changing buffer conditions

Usually decrease pH or increase ionic strength

decrease polarity adding up to 10 % dioxane or up to 50 % ethylene glycol

Denaturing buffer

Usually extremes of pH or chaotropic agents

General elution conditions

+

There are no guaranteed general elution methods in affinity chromatography

Low pH, e.g. glycine HCl, pH 2.8, is the closest approach

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General elution conditions • pH • 100 mM glycine•HCl, pH 2.5-3.0 • 100 mM citric acid, pH 3.0 • 50-100 mM triethylamine or triethanolamine, pH 11.5 • 150 mM ammonium hydroxide, pH 10.5

• Ionic strength and/or chaotropic effects • 3.5-4.0 M magnesium chloride, pH 7.0 in 10 mM Tris • 5 M lithium chloride in 10 mM phosphate buffer, pH 7.2 • 3.0 M Potassium chloride • 2.5 M sodium iodide, pH 7.5 • 0.2-3.0 sodium thiocyanate

• Denaturing • 2-6 M guanidine•HCl • 2-8 M urea • 1% deoxycholate • 1 % SDS

• Organic • 10% dioxane • 50% ethylene glycol, pH 8-11.5 (also chaotropic)

• Competitor • >0.1 M counter ligand or analog

Elution conditions are intended to break

the ionic, hydrophobic and hydrogen bonds

that hold the ligand and the target together

Successful eluting conditions will be

dependent upon the specific ligand-target

interaction that is occurring.

Ideally, an elution condition effectively

releases the target without causing

permanent damage, but all eluting

conditions result in some loss of functionality

Empirical evidence is needed to determine

which elution condition is the best.

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Elution at Low pH IgG antibodies on rProtein A Sepharose

Column: HiTrap rProtein A

Binding: 20 mM sodium phosphate, pH 7.0

Elution: 0.1 M glycine HCl, pH 3

Note: Collect IgG into a small volume of strong buffer, pH 7, to

preserve its antibody activity.

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Elution at High salt concentration DNA-binding proteins on Heparin Sepharose

Column: HiTrap Heparin

Binding: 20 mM Tris-HCl, pH 8, 0.5 M NaCl

Elution: Binding buffer + 1 to 2 M NaCl

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Competing binding substance in solution Elution of glycoproteins from Con A Sepharose by a-D-methylmannoside Elution of 6His-Proteins from chelating columns (Ni-NTA) with Imidazole

Specific eluents

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Competing ligand in solution Elution of enzymes from Blue Sepharose by free NADH Glutathione elution of GST fusion protein from Glutathione-Agarose columns

+

Competitive soluble ligands or binding substances can elute the bound target specifically

Are usually far more gentle than general methods

But sometimes could be very expensive (like specific peptides for antigen elution)

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Lectins

Lectin Specificity

Concanavalin A α-mannose, α-glucose

Glycine Max (soybean) N-Acetyl Galactosamine

Ulex Europaeus I α-L-fucose

Wheat germ lectin N-Acetyl Glucosamine & NeuNAc

Peanut lectin α-mannose

Ricinus Communis, RCA 120 ß-Galactose

Arachis hypogaea ß-Gal(1->3)GalNAc

Bandeira Simplicifolia, BS-I α-Gal & α Gal NAc

Maackia amurensis Sialic acid

Lymulus polyphemus NeuNAc

Lectins are proteins

which bind well-defined

sugar residues in

polysaccharides,

glycoproteins, etc

Elution with competing

free binding substance:

sugar

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Dye-stuffs

Dye-stuff Sites recognised

Cibacron Blue 3G-A NADH-binding and similar

Procion Red NADPH-binding and similar

Many dye-stuffs mimic binding sites of natural biologically active molecules

Elution with High Salt

The structural analogies are not exact.

Related structures are also recognized.

Blue Sepharose also binds serum albumin.

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• Types of AC


• Affinity Tags and Fusion Proteins

• Chelating, Strep-tag, GST, MBP, etc



• Examples



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Affinity Tags - I

Small stretches of amino acids added to the N-terminal or C-terminal end

of a protein with a high affinity for a specific biological or chemical ligand

Allow purification and detection of the expressed protein

Enables different proteins to be purified using a common method (HTPS)

Most popular: His-tag, comprised of six histidine that provide specific

binding to metal chelate resins

Polyarginine or polyaspartic acid tags can be used to alter binding of a

protein on ion-exchange resins

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Affinity Tags - II

The Strep-tag II (8 amino acids, WSHPQFEK), which binds with high

selectivity to Strep-Tactin.

The tag may be placed at either end of the protein or in a region with

appropriate surface exposure to allow binding or recognition.

To enable removal of the tag, a linker region is typically included between

the tag and the native protein sequence.

The linker contributes to increased accessibility of the affinity tag and is

often required for effective endoprotease cleavage.

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Fusion proteins Solubility-enhancement tags (SETs)

Use to overcome some of the problems of bacterial expression: protein aggregation, poor

expression levels and difficult purification.

The most popular fusion systems employ maltose-binding protein (MBP), glutathion S-

tranferase (GST), thioredoxin (TRX), SUMO and NusA. These genes are well expressed and

the proteins are highly soluble and provide specific characteristics to aid purification.

Typically, the gene of interest is inserted “in frame” at the 3’ end of the carrier protein, in

place of the termination codon.

To enable removal of the fusion protein, a linker region is typically included between the

fusion protein and the native protein sequence.

Specific recognition of the linked regions with specific proteases: TEV, Prescission, SUMO

protease, Enterokinase

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Advantages / disadvantages of fusion proteins

In several cases

facilitate protein purification

facilitate protein refolding /

increase solubility

improve protein yield (high

expression)

prevent proteolysis

But in other cases

Target is not soluble after cleavage

change in protein conformation

lower protein yields

alteration in biological activity

undesired flexibility in structural studies

Toxicity

Extra work if you need to cleave

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IMAC: Immobilized metal affinity chromatography Chelating-Chromatography for poly-His fusion proteins

The most widely used.

It’s small in size. Less immunogenically active

It does not need to be removed for downstream applications

Availability of a large number of expression vectors

Tag may be placed at either the N or C terminus

A protease cleavage site allows the tag to be removed after purification

The interaction of the tag with the Ni2+column does not depend of the tag structure,

making it possible to purify otherwise insoluble proteins using denaturating conditions

(8 M urea or 6 M GuHCl )

Avoid use of chelating: EDTA, DTT, etc. (cell chelating molecules in low expression)

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Immobilized metal ion affinity chromatography (IMAC) – for poly-His fusion proteins

The basis of the purification is the interaction of the imidazole moiety of the poly His with

the metal (Nickel in most cases).

The metal is immobilized to a support through complex formation with a chelate that is

covalently attached to the support

In some resins we can use

Co , Cu , Zi or Fe instead of Ni

and obtain different results

Ni-NTA, Co resins, etc

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IMAC Binding Conditions:

20 mM phosphate or TrisHCl buffers

+ (0.5 M NaCl) or low imidazol

concentrations (10-20mM) to avoid non-

specific binding of contaminants to the resin.

Buffers may include 8 M urea or 6 M GuHCl

when purifying inclusion bodies solubilised

proteins.

Elution Conditions:

Usually Imidazol (Histidine analoge) or

low pH (~4.5)

Alternatives: EDTA, Histidine

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Strep-tag /Strep-Tactin system According to Expression and purification of proteins using Strep-tag and/or 6xHistidine-tag. A comprehensive manual

from IBA GmbH

The Strep-tag II is a short peptide (8 mino acids, WSHPQFEK), which binds with high

selectivity to Strep-Tactin, an engineered streptavidin.

This technology allows one-step purification of almost any recombinant protein under

physiological conditions, thus preserving its bioactivity (elution with 2.5mM desthiobiotin).

The Strep-tag system can be used to purify functional Strep-tag II proteins from any

expression system including baculovirus, mammalian cells, yeast, and bacteria.

The Strep-tag/Strep-Tactin interaction is compatible with a variety of reagents (detergents,

reducing agents, etc.) making the system attractive for purifying metallo- and membrane

proteins, large proteins and protein complexes.

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Strep-tag/

Strep-Tactin system

According to Expression and purification of

proteins using Strep-tag and/or 6xHistidine-tag.

A comprehensive manual

from IBA GmbH

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Strep/6 x Histidine system (double-tag) IBA and QIAGEN

Useful for full length recombinant proteins purification at high purity under

standardized and non-denaturative conditions (Imidazol and desthiobiotin

elution)

Especially useful to eliminate “difficult to resolve” protease cleavage

fragments of target protein.

Recombinant proteins that carry 6xHistidine-tag at the N-terminus and

Strep tag II at the C-terminus (or vice versa).

Efficiently expressed in E. coli, yeast, insect, or mammalian cells.

Recommendation: use IMAC as first capture step. No buffer exchange is

required for the second purification step: Strep-Tactin resin

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A panel of commonly used affinity tags selected for purification of recombinant fusion proteins and their

associated characteristics

Preparative Purification of Recombinant Proteins: Current Status and Future Trends Mayank Saraswat et al. Hindawi Publishing Corporation - BioMed Research International Volume 2013, Article ID 312709,

http://dx.doi.org/10.1155/2013/312709

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Gluthathione S-transferase or GST-fusion proteins

• Column: Glutathione Sepharose 4B, pre-packed

• Binding: PBS (+ 1% Triton X-100)

• Elution: 5-10 mM Glutathione, 50 mM Tris.HCl, pH 8.0

r-Protein

Cleavage site

Specific ligand Matrix Affinity tag Target protein

GST: 26kDa cytoplasmic protein. Binds specifically to glutathione

Highly use in immunoprecipitation

Less use for increasing solubility

Commercial vectors containing either factor Xa, Prescision or a thrombin

recognition site to allow cleavage and removal of the GST.

Useful for dimerization

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Maltose-binding protein pMAL ™

MBP: a 43kDa secreted protein from E. coli,

binds specifically to maltose

Purification: Maltose or Amylose agarose

columns and Dextrin Seph (most recommended)

Can be secreted to the periplasm

Or expressed without the signal peptide in the

cytoplasm.

Very effective for solubility enhancement

TEV protease cleavage site

Sometimes additional 6His in N-terminal

Use sometimes to aid crystallization of target

protein

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The Mechanism of Solubility Enhancement by MBP Sreejith Raran-Kurussi and David S. Waugh PLOS ONE (2012) 7 (11): e49589 - doi:10.1371

Recently, MBP has also been used to maintain proteins that contain

disulfide-bonds in a soluble state in the E. coli cytoplasm so that

they could be acted upon by appropriate redox enzymes that were

co-expressed in the same cellular compartment

MBP serves as a passive participant in the folding process; passenger proteins either fold spontaneously or with the

assistance of chaperones.

Chaperones and/or chaperonins seem to come into play after a passenger protein has been rendered soluble by MBP.

MBP serves primarily as a ‘‘holdase’’, keeping the

incompletely folded passenger protein from forming

insoluble aggregates until either spontaneous or

chaperone-mediated folding can occur.

A third class of passenger proteins is unable to fold

and exists in an incompletely folded state and

typically precipitate after they are cleaved

11/19/2014 55

SUMO (small ubiquitin-like

modifier)

Increased expression

Increased solubility

Acidic protein ~10kDa

Both the tag and the protease have 6xHis tags

SUMO Protease leaves no unwanted residues on the N-terminus

SUMO Protease extremely efficiently (1:500)

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Thioredoxin

Thioredoxin: 12 kDa intracellular E. coli protein. Very soluble, and highly over-expressed

Periplasmic or cytoplasmatic expression

Most popular to increase disulfide bonds

6His Tag can be add to the N terminal of TRX for metal-chelator purification

Since TRX is thermostable, and some heat-stable fusion proteins can be purified by thermal

denaturation of contaminants by thermoosmotic shock (osmotic shock coupled with heat-

treatment) According to Q.-R. Guo et al. / Protein Expression and Puriffication 49 (2006) 32–38

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Fusion Tag Comparison Study LifeSensors (http://www.lifesensors.com/r_and_d/protein_expression.php3)

SUMO (Small Ubiquitin-like MOdifier) and NusA fusion tags dramatically outperform

glutathiones transferase (GST), maltose binding protein (MBP), thioredoxin (TRX), and

ubiquitin (Ub). The protein target tested in this study is GDF-8, a growth/differentiation factor.

UN: un-induced, IN: induced, S: soluble, IB: inclusion body.

UN IN S IB UN IN S IB UN IN S IB UN IN S IB UN IN S IB UN IN S IB UN IN S IB

GDF8 ALONE Ub-GDF8 SUMO-GDF8 MBP-GDF8 GST-GDF8 TRX-GDF8 NUS A-GDF8

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IMPACT™-CN System (NEB)

Intein Mediated Purification with an Affinity Chitin-

binding Tag protein purification system.

Use inducible self-cleavage activity of a protein splicing

element (intein) to separate the target protein from the

affinity tag without the use of a protease.

A target protein is fused to a tag consisting of the

intein and the chitin binding domain.

In the presence of thiols such as DTT,

b-mercaptoethanol or cysteine, the intein undergoes

specific self-cleavage which releases the target protein

from the chitin-bound intein tag

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PinPoint™ Xa from Promega Production and purification of fusion proteins that are biotinylated

in vivo

In vivo Biotinylated Affinity Tags: biotinylation reaction in E. coli through

biotin ligase holo-enzyme.

Fusion purification tag with a single biotin specifically on one Lys residue

The system use a monomeric avidin (Soft Release Avidin Resin): allows

protein elution with a non-denaturing 5mM biotin buffer.

(Avidin-biotin interactions are very and requires denaturing conditions)

Tag cleavage with Factor Xa

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Comparison of Affinity tag technologies According to J.J. Lichty et al. / Protein Expression and Puriffication 41 (2005) 98–105

mg10Tag Size(aa) Resin Eluting agent Source Capacity Cost Cost/

MBP 396 Amylose Maltose Biolabs 3mg/ml $105/10ml $12

HIS 6 Talon Imidazole Clontech 5–14mg/ml $220/25ml $18

Ni–NTA Imidazole Qiagen 5–10mg/ml $257/25ml $21

GST 218 GSH–Sepharose Glutathione Amersham 10 mg/ml $396/25ml $36

CBP 28 Calmodulin EGTA Stratagene 2mg/ml $227/10ml $114

Strep II 8 Strep-Tactin Desthiobiotin IBA 50–100 nmol/ml $1100/25ml $293

FLAG 8 Anti-FLAG M2 FLAG peptide Sigma 0.6mg/ml $1568/25ml $1045

HPC 12 Anti-Prot.C Ab EDTA Roche 2–10 nmol/ml $299/1ml $4983

61

Fusion protein cleavage methods

Chemicals cleavage is effective but often requires extreme conditions (low

pH or high temp.), and is often non-specific (cyanogen bromide,

hydroxylamine, etc).

Enzymatic digestion is the method of choice for soluble fusion protein

cleavage. Reaction is carried out under relative mild conditions.

Commonly used endo-proteases : thrombin, factor Xa, enterokinase

Best sellers: TEV, SUMO and rhinovirus 3C protease (Prescission)

Exoprotease: TAGZyme, carboxypeptidase A (C-terminal tag)

Intein cleavage by reduction agents such as DTT, or low pH

62

Purification of Fusion Proteins

Desorption

Proteolytic cleavage

Linker sequence

Gel

Ligand

r-Protein Affinity

tail + L

r-Protein Affinity

tail

Gel L

r-Protein Affinity tail

Proteolytic cleavage

r-Protein Affinity

tail +

63

Cleavage Sites • Thrombin Amersham-Biosciences, Novagen, SIGMA, Roche

Leu-Val-Pro-Arg▼Gly-Ser Protease Capture: Benzamidine-Agarose

Secondary cleavage sites. Biotinylated form available for removal with immobilized streptavidin.

• Factor Xa Amersham-Biosciences, NEB, Roche

Ile-Glu/Asp-Gly-Arg▼ Protease Capture: Benzamidine-Agarose

Will not cleave if followed by proline and arginine. Secondary cleavage sites following Gly-Arg sequences.

• Enterokinase NEB, Novagen, Roche

Asp-Asp-Asp-Asp-Lys▼ Protease Capture: Trypsin Inhibitor-Agarose

The site will not cleave if followed by a proline residue. Secondary cleavage sites at other basic residues, depending on conformation of protein substrate. Active from pH 4.5 to 9.5 and between 4°C and 45°C

• TEV protease Invitrogen – Life Technologies

Glu-Asn-Leu-Tyr-Phe-Gln▼Gly Protease Capture: Ni-NTA (6His recomb. TEV)

Seven-residue recognition site, making it a highly site-specific protease. Active over a wide range of temperatures. Protease available as a His-tag fusion protein, allowing for protease removal after recombinant protein cleavage.

• PreScission Amersham-Biosciences

Leu-Glu-Val-Leu-Phe-Gln▼Gly-Pro

Genetically engineered form of human rhinovirus 3C protease with a GST fusion tag, allowing for facile cleavage and purification of GST-tagged proteins along with protease removal after recombinant protein cleavage. Enables low-temperature cleavage of fusion proteins containing the eight residue recognition sequence.

• TAGZyme Quiagen: His-tag removal by Exoproteolytic Digestion

Protease Capture: Ni-NTA (6His recomb. enzyme)

The TAGZyme System is an efficient and specific solution for the complete removal of small N-terminal His tags and other amino acid tags by the use of exopeptidases. These recombinant enzymes contain a C-terminal His tag and can therefore be bound to Ni-NTA matrices.

• Intein Site (dithiothreitol cleavage) NEB

DTT elimination by dialysis

Uses self-cleavable affinity tags. Even after cleavage un-natural termini are present on the protein of interest.

64

Incubation of HLT-435aaCtermBP2 with TEV-protease

Shajar Rotem, Assaf Friedler

Protein µl 5 5 5 5 5 5 5 5 5 5

TEV-pr. µl 0 0.2 1 2.5 5 0 0.2 1 2.5 5

Temp ºC 4 4 4 4 4 22 22 22 22 22

HTL-435aaCtermBP2

435aaCtermBP2

TEV-protease

HTL

65

Disadvantage of Proteolytic Cleavage

Spurious, non-specific proteolytic cleavage of the fusion protein

Non-covalent forces maintain proteins connected after cleavage: separation problems

Some proteases need elevated temperatures (25-37°C) for efficient cleavage: can denature

or cause aggregation of the fusion protein

Incomplete cleavage, which reduces the yield and/or introduces heterogeneity to the

purified protein

Buffer and additive restriction (mainly detergents)

$ and time: The need for additional steps to separate the cleaved fusion protein from the

fusion tag, remove the protease, and exchange buffer or desalt

Increase purity (negative column or other chromatographic procedure)

Targets are mainly C-terminal of the solubility protein (exoproteases as carboxypeptidase A)

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Self-cleaving elastin-like polypeptide tag Purification of recombinant protein without the need for affinity

chromatography or proteolytic tag removal

Simple bioseparations using self-cleaving elastin-like polypeptide tags. Banki M. et al. - Nature Methods 2, 659 - 662 (2005) Self-cleaving ELP tags consisting of repeating pentapeptides of VPGXG (X = any amino acid) fused to a controllable self-cleaving intein

ELPs can be designed to undergo a reversible transition from soluble to insoluble upon temperature upshift

The reversibility of the precipitation is distinct from simple irreversible aggregation or misfolding, and can be used by several groups to purify ELPs and protein products fused to ELPs

Intein-fused affinity tags get inducible self-cleave under mild conditions (room temperature and slightly acidic pH)

PROBLEMS: Slow growth.

Expression level

Optimization will be required for new, uncharacterized products on a case-by-case basis

68





• Types of AC

• Elution Conditions: Affinity Tags and Fusion Proteins

• Chelating, Strep-tag, GST, MBP, etc



• Examples



69

Parameters for optimization during binding and washings- I

If protein does not bind to the resin, there are several options to choose

Check the quality of the resin (use an His-tag control protein) or check for the presence of reagents that avoid binding

Partial binding: use more resin, or bind for longer time (BUT: the longer the duration of purification, the greater the risk of protein degradation). Use unbound material for a new purification.

Try next time additives as glycerol, detergents or more NaCl (soluble aggregates??)

Tag is inaccessible: try purification under denaturating conditions (for poly-His fusion proteins)

Check by western-blot if the tag has been degraded; if this is the case, try to work all the time at 4°C and use more protease inhibitors during lysis

Construct a new vector with the tag in the opposite end of the protein.

70

Parameters for optimization during binding and washings- II

If multiple proteins bands are seen in the elution try:

Protein degradation (you can check previously with western blot) try to work all the time at 4°C and

use protease inhibitors during lysis.

Use more stringent competitive conditions during binding and washing (example: use low Imidazole concentrations during binding and washings to increase competition for the same sites on the resin)

Decrease resin volume (allows higher competition between fusion protein and contaminants for

the same sites on the resin)

If contaminants are associated with the tagged protein, try to disrupt the non-

specific interaction by adding to the wash buffer before elution additives as ß-ME, glycerol up to 50%, detergents as Triton X-100, NP40 or Tween 20 up to 2% or increase ionic strength up to 1.5M NaCl or KCl.

Increase the washing step volume.

Consider an additional purification step before or after purification.

Consider the use of a pre-column of beads without ligand to adsorb proteins that bound to

beads non-specifically

71

Parameters for optimization during elution

Optimize resolution

Adjust gradient slope/shape, or step elution with increasing competitor concentration

Include additives in buffers

Adjust flow rate

Adjust column volume

Bead size and quality of the resin

If the protein slightly elutes or does not elute from the column

Use higher competitor concentration (Examle: up to 1M Imidazol for chelating columns),

or additives

Reduce elution flow-rate

Change elution conditions, consider elution under denaturating conditions

72

Parameters for optimization during protease cleavage

and purification

Cleavage depends of target/protease ratio, volume, temperature, time, buffer, etc

If possible try to cut at low temperature

Cleavage can be done inside dialysis bags under dialysis to prepare protein to next step

If necessary add additives in buffers to avoid aggregation of the target after cleavage

High aggregation can affect cleavage

Some targets slightly bound to the IMAC resin in the negative step: increase competition

(Imidazol, etc)

Cleavage is OK, but proteins elute together because of other interactions. Try to reduce

these interactions (high salt, detergents, etc)

Alternatives to negative affinity: IEX, HIC, GF

73

Lysis of 10ml culture

Bound and washed (in the presence of 10, 20, 30 mM imidazol) to nickel resin

Elution was with 300 mM imidazol

stop of ARNO-(His tag) cc17 pDest Optimization during binding

Each well contains 10 micro liter of the desired step (from 40 microliters )+ 5 micro liter sample buffer.

Each of the marker proteins is 2 micro-grams.

20kDa

14 kDa

6.5 kDA

His6-cc

Tamar Shultz

Altschuler lab

Elution 300 mM Imidazole

Ste

p 1

Ste

p 2

Ste

p 2

Ste

p 2

Ste

p 2

Ste

p 1

Ste

p 1

Ste

p 1

0 10 20 30

Elution marker

Binding and washing- x mM imidazol

11/19/2014 74

P38 CAPTURE -Optimization

Incubate each fraction (batch binding) 60min 4°C with 150µl Ni-NTA (equilibrated with different [Imidazol]).

Spin 3min 3000rpm 4°C. Discharge unbound material.

Wash resin 6x1ml washing buffer + different [Imidazol].

Elution 3x300µl elution buffer (250mM Imidazol). Run 12%PAGE-SDS of each elution.

Ni-NTA equilibration (mM Imidazol) 10 10 10 20 20 20 30 30 30 40 40 40

[Imidazol] for binding (mM Imidazol) 10 10 10 20 20 20 30 30 30 40 40 40

[Imidazol] for washing (mM Imidazol) 10 10 10 20 20 20 30 30 30 40 40 40

[Imidazol] for elution (mM Imidazol) 250 250 250 250 250 250 250 250 250 250 250 250

Elution number 1 2 3 1 2 3 MW 1 2 3 1 2 3

Cells lysis from 250ml culture.

Spin 30min 20000g 4°C.

Divide supernatant in 4 fractions,

add to each one different [Imidazol].

11/19/2014 75 100gr cells (6L culture). Cell disruption with Mountain Goulin in 900ml lysis buffer. Batch binding to 15ml Ni-NTA 90min 4°C in

the presence of 20mM Imidazol. Wash with 20mM Imidazol buffer and elution with 5cv gradient 20-250mM Imidazol.

Pool P38

61 OD280nm

P38 CAPTURE - Affinity Chromatography

Unb Wash 4 5 6 7 MW 8 9 10 11 12

11/19/2014 76

P38 INTERMEDIATE PURIFICATION: Anion Exchange

60 OD 280nm P38 after Ni-NTA and dialysis ON vs buffer A. Load on Resource Q-30 6ml column. Wash with 10cv 50mM

NaCl buffer. Elute with gradient 5cv 50-300mM + 40cv 300-750mM + 5cv 750-1000mM NaCl

P38

35 OD280

Bef 22 24 32 33 34 35 MW 36 37 38 40 42 44 46

11/19/2014 77

P38 FINAL POLISHING: SEC

35 OD280nm P38 after Ni-NTA, Res.Q & ultrafiltration cut-off 10000. Load on Superdex 75 60x1.6cm column (2 runnings).

Elution 1ml/min

P38 30

OD280nm

MW 5 7 9 10 11 12 13

P38

78

AbrBHiTrapNiHP1mlb001:1_UV1_280nm AbrBHiTrapNiHP1mlb001:1_UV2_260nm AbrBHiTrapNiHP1mlb001:1_Conc AbrBHiTrapNiHP1mlb001:1_Fractions AbrBHiTrapNiHP1mlb001:1_Inject AbrBHiTrapNiHP1mlb001:1_Logbook

0

100

200

300

400

mAU

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Load + 35 cv 0%B + 8cv 4%B + 8cv 10%B + 5cv 10-100%B + 10cv 100%B (500mM Imidazol)

IMAC purification: Low Imidazol step washing before elution Case study: AbrB- Collaboration with A.Kaplan & D. Schatz

Sol 1 3 5 9 10 12 13 14 15 16 17 19 20

34

26

17

10

AbrB

11/19/2014 79

HLT-p53CT- Affinity start with pellet of 1.5L culture

Ni-Sepharose FF 14ml Ronen Gabizon – Assaf Friedler Group

HLTp53CTNiNTA16ml004:1_UV1_280nm HLTp53CTNiNTA16ml004:1_UV2_260nm HLTp53CTNiNTA16ml004:1_Conc HLTp53CTNiNTA16ml004:1_Fractions HLTp53CTNiNTA16ml004:1_Inject HLTp53CTNiNTA16ml004:1_Logbook

0

500

1000

1500

2000

2500

mAU

200 250 300 350 400 450 ml

F4 Waste 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Load + 10cv 0%B + 3cv 8%B + 4cv 15%B + 4cv 100%B

POOL 17-22: 3.5OD x 35ml ~ 276mg

11/19/2014 80

HLT-p53CT- Cation Exchange after TEV protease cleavage

ON 4ºC SP-Sepharose FF 5ml

HLTp53CTHiTrapSP5mlml005:1_UV1_280nm HLTp53CTHiTrapSP5mlml005:1_UV2_260nm HLTp53CTHiTrapSP5mlml005:1_Cond HLTp53CTHiTrapSP5mlml005:1_Conc HLTp53CTHiTrapSP5mlml005:1_Fractions HLTp53CTHiTrapSP5mlml005:1_Inject HLTp53CTHiTrapSP5mlml005:1_Logbook

0

50

100

150

mAU

500 550 600 650 700 ml

F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

11/19/2014 81

HLT-p53CT- GF after Cation

Exchange

Sephacryl S100 FF 500ml

HLTp53CTSephacrylS100of500ml004:1_UV1_280nm HLTp53CTSephacrylS100of500ml004:1_UV2_260nm HLTp53CTSephacrylS100of500ml004:1_Fractions HLTp53CTSephacrylS100of500ml004:1_Inject HLTp53CTSephacrylS100of500ml004:1_Logbook

0

50

100

150

mAU

60 80 100 120 140 160 180 200 220 min

F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Waste

POOL 7-14

82

Binding equilibrium

At equilibrium

K

L T

LTD

KD is the equilibrium dissociation constant, [L] is the concentration of free ligand [T] is the concentration of free target [LT] is the concentration of the ligand/target complex

83

Binding equilibrium

It can be shown* that

Bound target

Total targetL

K L0

D 0

KD is the equilibrium dissociation constant, L0 is the concentration of ligand, usually 10-4 - 10-2 M

*Graves, DJ, Wu, YT Meth. Enzymol 34 (1974) 140-163

For good binding, KD should be at least two orders of magnitude less than L0, i.e. 10-6 - 10-4 M

84

Binding equilibria

L + T LT KD 10-6 - 10-4 M

Binding

L + T LT KD 10-1 - 10-2 M

Elution

We can change KD by changing pH, temperature, salt concentration etc.

Target elutes as a sharp peak

85

Elution by changing KD - Unexpected results

KD too high during binding (low affinity)

Target is retarded as a broad peak and elutes under binding conditions as a broad, low peak

Find better binding conditions or use ligand with higher affinity

KD not low enough during elution

Target elutes in a long, low 'peak'

Try different elution conditions or use ligand with lower affinity

KD too low during binding (extremely high affinity)

Difficult to elute target. Difficult or impossible to increase KD enough to elute the target

without destroying its activity

Use ligand with lower affinity

86

Competitive elution

+

Binding

Release

C + T CT

Elution +

87

Binding equilibrium for competing ligand

At equilibrium

K

C T

CTDComp

KDComp is the equilibrium dissociation constant [C] is the concentration of free competing ligand [T] is the concentration of free target [CT] is the concentration of the competing ligand/target complex

88

Competitive elution

For good elution: Increase competitor concentration to get a sharp peak if necessary

If KDComp and KD are similar then the concentrations of competing and coupled ligand should be similar

If KDComp is 10 x KD (i.e. the free competing ligand binds more weakly) then the concentration of competing

ligand will need to be 10 x higher to get effective elution

121125AntiHer2Ni30ml005:10_UV1_280nm 121125AntiHer2Ni30ml005:10_UV2_260nm 121125AntiHer2Ni30ml005:10_Conc 121125AntiHer2Ni30ml005:10_Fractions 121125AntiHer2Ni30ml005:10_Logbook

0

100

200

300

400

500

mAU

200 300 400 500 600 700 800 ml

F3 F4 1 2 3 4 5 6 7 8 9 10 11121314 15161718 192021 23 28 33 38 43 48 53 58 63 68 71 74 77 80 83 86

121014AntiHer2Ni16ml004:10_UV1_280nm 121014AntiHer2Ni16ml004:10_UV2_260nm 121014AntiHer2Ni16ml004:10_Conc 121014AntiHer2Ni16ml004:10_Fractions 121014AntiHer2Ni16ml004:10_Logbook

0

100

200

300

400

500

600

700

800

mAU

200 250 300 350 400 450 ml

F4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 50

Load lysed pellet of 2L culture on 16ml Ni-SepharoseFF used many times

Load lysed pellet of 4L culture on 30ml Ni-Sepharose6B used a few times

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Kinetics of adsorption and desorption Unexpected results

Slow binding

Some of the target elutes under binding conditions as a broad, low peak

Slow down binding. Stopped flow binding to bind the target in portions

Slow desorption

Target elutes as a long, low 'peak'

Stopped flow elution to elute the target in pulses. Change elution scheme

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Synthetically “mimicking” and enhancing the natural molecular affinity of binding ligands toward

targeted biomolecules

Mimetic Ligand adsorbents are biologically inert, very durable, have excellent liquid flow properties,

and are resistant to most chemical treatments.

Stable in the pH range 2 - 14, can be treated with denaturants (8M urea), detergents (Triton, SDS)

and can be sterilized by autoclaving.

Capacities can exceed 50mg protein per ml of packed gel. Low Ligand Leakage

Alkali-stable adsorbents - Enables cleaning, sterilization and depyrogenation with 1M sodium

hydroxide, ensuring long operational lifetimes.

Albumin • Proteases • Blood Proteins • Oxidases • Dehydrogenases • Ligases • Kinases • Antibodies

• Nucleases • Cytokines

Custom Media Development Programs

Chemical Combinatorial Library™, + with a rational ligand design approach

Mimetic Ligand™ Affinity Chromatography www.prometic.com

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BAC (BioAffinity Company) Capture Select®

Camelid derived single domain antibody fragments

http://www.bacbv.com/

Custom designed ligands (12-15kDa)

Produced in Saccharomyces cerevisiae, enabling high level production with minimal purification

effort as well as easy scale-up.

Stability, affinity, and selectivity

Reduction of process steps: higher yields, reduced costs

Selectivity: high purity in single step / feed stock independent

Mild elution conditions: retaining biological activity of target

Efficient clearance of HCP, DNA, virus: high selectivity in capture step

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IgG Select

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Scil Proteins: Affilin™ Techology

Novel human binding proteins for therapy and applications in chromatography.

Affilin™ molecules are small non-immunoglobulin proteins which are designed for specific

binding of proteins and small molecules. New Affilin™ molecules can be quickly selected

from libraries which are based on two different human derived scaffold proteins.

Gamma crystalline, a human structural eye lens protein and ubiquitin one of the highest

conserved proteins known today. Both human scaffolds are small, show high temperature

stability and are almost resistant to pH changes and denaturing agents.

By engineering the protein surfaces through a locally defined randomization of solvent

exposed amino acids, completely new binding sites are created. Former non-binding

proteins are thereby transformed into specific binding proteins that can be easily

produced in the cytoplasm of E. coli

Affinity Chromatography (AC) - Hebrew University of...

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