Affinity

Chromatography
(AC)

1
 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
2
 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
3
 What is it used for?

 Monoclonal and polyclonal antibodies

 Fusion proteins

 Enzymes

 DNA-binding proteins . . . . . .

 ANY protein where we have a binding partner!!

4
 Purifying monoclonal antibodies

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%

SDS-PAGE reduced
Starting
AU
material
2.0
Eluate
1.5
94
43
1.0
30
0.5

0.0

0 200 400 600 Volume (ml)

5
Designing and preparing an affinity gel

 Choosing the matrix

 Designing the ligand - Spacer arms

 Coupling methods

6
 Ligand Immobilization

Ligand
+ Activating
agent +

Matrix Activated Immobilised

matrix ligand

7
 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

8
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

Spacer arm adsorption to the spacer!

9
 Design of spacer arms

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

Hydrophilic chain
Risk of unspecific interactions greatly
O reduced
H

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. 10
 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
Cyanogen bromide activation

Gel
O
H
+ C N
Gel
O
C N + H Br
Br
Sepharose Cyanogen Cyanate ester
bromide formed

Cyanogen bromide coupling

H
O O N
Gel C N + Lig NH2 Gel C Lig
CNBr-activated NH
Protein ligand N-substituted isourea
Sepharose

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

Multiple bonds formed with protein ligands. No spacer arm present.
13
WARNING!: Cyanogen bromide is dangerous. Consult safety data before start working
 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

14
 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.

15
 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.
16
 Keep in storage buffer (Na Azide , Thimerosol 0.02% or ethanol if possible)
 G Protein Receptor Kinase
Technique Pig brain homogenate Purification Comment
factor

Ppt Ammonium sulfate 7

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

HIC Butyl Sepharose Fast 20

Flow

AIEX RESOURCE Q

CIEX RESOURCE S 2408

AC HiTrap Heparin 18647 • 10 mg homogenous protein

17
 Membrane Protein

Technique Placental extract in Purification Comment

1.5% Triton X-100 factor

AC Blue Sepharose 3 • Concentration and capture

AIEX DEAE Sephacel 4

T. White et al. (1995)
J. Biol. Chem. 270, 24156-24165
CIEX SP Sepharose 6

AC Muc2 Sepharose 242 • Main purification step

• Final polishing and purity

CIEX Mono S 1442 check, 20 mg obtained

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 DNA-binding Protein

Technique Purification Comment

HeLa cell nuclear
extract factor
J. Berthelsen et al. (1996)
J. Biol. Chem. 271, 3822-3830
CIEX SP Sepharose High 5
Performance

AC Heparin Sepharose 8 • General AC step for DNA-

Fast Flow binding proteins

AC DNA-1 Sepharose 9 • Removal step, non-specific DNA

binding activity removed

AC DNA-2 Sepharose 2447 • Main purification step

CIEX Mono S 4943 • Final polishing, 20 mg

obtained
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 Advantages and Disadvantages of affinity
chromatography
 Easy to achieve otherwise difficult  Economics (scale-up)
separations  Buffer / elution limitations

 Often high purity in one step  Sometimes needs harsh or

(but not always) denaturizing elution conditions

 Fast separations (depends of the

 Do not resolve all the problems
kinetics of binding and elution)

 Can be use to remove specific

20
contaminants
 The main stages in affinity chromatography

Sample application Elution

of the target

Equilibration Wash Re-equilibration

Of gel and sample contaminants Of the gel to binding conditions
to binding conditions Or keep in storage buffer (20%
Ethanol or 0.02%NaAzide in
Buffer)

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 Columns and equipment

Tips
 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 22
 Sample preparation

Tips
 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) 23
 Sample application

Tips
 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
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 Elution and re equilibration

Tips
 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 25

 Column Regeneration

Tips
 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 26
 Column Storage

Tips
 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

27
 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
28
 Type of Affinities
Mono-specific ligands Group-specific ligands

• Specific for a single substance • Specific for a group of structurally

or functionally similar substances:
Antigen antibody
Lectins glycoproteins
Hormone receptor
Protein G IgG antibodies
• Usually home-made gels
Dye-stuffs enzymes
• Elution scheme must be worked
• Often ready-made gels
out for each case:
• Known elution schemes
• Often general elution
• Standard tested elution protocols
• Little help from the literature 29
• Often competitive elution
 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

30
 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

31
(Co, Ni, Zn, Cu, Fe) 6-12 Histidines
 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) 32

 General elution conditions

Changing buffer conditions

Usually decrease pH or increase ionic strength

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

Reconstituting
+
buffer

Denaturing buffer
Usually extremes of pH or chaotropic agents

There are no guaranteed general elution methods in affinity chromatography

33
 Low pH, e.g. glycine HCl, pH 2.8, is the closest approach
 General elution conditions
• pH
• 100 mM glycine•HCl, pH 2.5-3.0
• 100 mM citric acid, pH 3.0 Elution conditions are intended to break
• 50-100 mM triethylamine or triethanolamine, pH 11.5
• 150 mM ammonium hydroxide, pH 10.5
the ionic, hydrophobic and hydrogen bonds
that hold the ligand and the target together
• Ionic strength and/or chaotropic effects
• 3.5-4.0 M magnesium chloride, pH 7.0 in 10 mM Tris
 Successful eluting conditions will be
• 5 M lithium chloride in 10 mM phosphate buffer, pH 7.2
• 3.0 M Potassium chloride dependent upon the specific ligand-target
• 2.5 M sodium iodide, pH 7.5
• 0.2-3.0 sodium thiocyanate interaction that is occurring.

• Denaturing Ideally, an elution condition effectively

• 2-6 M guanidine•HCl
• 2-8 M urea releases the target without causing
• 1% deoxycholate
permanent damage, but all eluting
• 1 % SDS
conditions result in some loss of functionality
• Organic
• 10% dioxane
Empirical evidence is needed to determine
• 50% ethylene glycol, pH 8-11.5 (also chaotropic)
which elution condition is the best.
• Competitor 34
• >0.1 M counter ligand or analog
 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.

35
 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

36
 Specific eluents

Competing ligand in solution

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

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

 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) 37
 Lectins

Lectins are proteins Lectin Specificity

Concanavalin A
which bind well-defined
Glycine Max (soybean)
sugar residues in
Ulex Europaeus I
polysaccharides,
Wheat germ lectin
glycoproteins, etc Peanut lectin
α-mannose, α-glucose
N-Acetyl Galactosamine
α-L-fucose
N-Acetyl Glucosamine & NeuNAc
α-mannose

Ricinus Communis, RCA 120 ß-Galactose

Elution with competing Arachis hypogaea ß-Gal(1->3)GalNAc

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

free binding substance:
Maackia amurensis Sialic acid
sugar
Lymulus polyphemus NeuNAc
38
 Dye-stuffs

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

Elution with High Salt

Dye-stuff Sites recognised

Cibacron Blue 3G-A NADH-binding and similar
Procion Red NADPH-binding and similar

The structural analogies are not exact.

Related structures are also recognized.
Blue Sepharose also binds serum albumin. 39
 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
40
• Future Considerations
 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
41
 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.

42
 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
43
 Advantages / disadvantages of fusion proteins

In several cases But in other cases

 facilitate protein purification  Target is not soluble after cleavage

 facilitate protein refolding /  change in protein conformation

increase solubility  lower protein yields
 improve protein yield (high  alteration in biological activity
expression)  undesired flexibility in structural studies
 prevent proteolysis  Toxicity

 Extra work if you need to cleave

44
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)45
 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

46
 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)
47
Alternatives: EDTA, Histidine
 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. 48
 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

49
 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

50
required for the second purification step: Strep-Tactin resin
 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

51
 Gluthathione S-transferase or GST-fusion proteins
Matrix Specific ligand Affinity tag Target protein

r-Protein

Cleavage site
• Column: Glutathione Sepharose 4B, pre-packed
• Binding: PBS (+ 1% Triton X-100)
• Elution: 5-10 mM Glutathione, 50 mM Tris.HCl, pH 8.0

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.
52
Useful for dimerization
 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
53
protein
 The Mechanism of Solubility Enhancement by MBP
Sreejith Raran-Kurussi and David S. Waugh PLOS ONE (2012) 7 (11): e49589 - doi:10.1371
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

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 54
 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)

55 11/19/2014
 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

56
 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.

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

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

57
 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 58
 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

59
 Comparison of Affinity tag technologies
According to J.J. Lichty et al. / Protein Expression and Puriffication 41 (2005) 98–105

Tag Size(aa) Resin Eluting agent Source Capacity Cost Cost/ 10mg
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

60
 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

61
 Purification of Fusion Proteins
Linker sequence
Ligand
Gel L Affinity
+ tail r-Protein

Gel Affinity
L r-Protein
tail

Desorption

Affinity r-Protein
tail Proteolytic cleavage

Proteolytic cleavage

Affinity
+
tail r-Protein
62
 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
63
Uses self-cleavable affinity tags. Even after cleavage un-natural termini are present on the protein of interest.
Incubation of HLT-435aaCtermBP2 with TEV-protease

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

Shajar Rotem,
Assaf Friedler

64
 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

65 A)
 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 67
 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
68
 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.
69
 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
70
 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

71
 Change elution conditions, consider elution under denaturating conditions
 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

72
 Optimization during binding pDest 17 (His tag) cc-stop of ARNO
 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

Binding and Elution 300 mM Imidazole

washing- x mM
imidazol 30 20 10 0
Each well contains 10

Step 1
Step 1
Step 2

Step 1

Step 2

Step 2
Step 2

Step 1

Elution marker micro liter of the

desired step (from 40
microliters )+ 5 micro
liter sample buffer.

20kDa
His6-cc
14 kDa
Tamar Shultz
6.5 kDA Altschuler lab

73
Each of the marker proteins is 2 micro-grams.
 P38 CAPTURE -Optimization
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].

 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.
74 11/19/2014
P38 CAPTURE - Affinity Chromatography

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

Pool P38
61 OD280nm

100gr cells (6L culture). Cell disruption with Mountain Goulin in 900ml lysis buffer. Batch binding to 15ml Ni-NTA 90min 4°C in
75 11/19/2014
the presence of 20mM Imidazol. Wash with 20mM Imidazol buffer and elution with 5cv gradient 20-250mM Imidazol.
P38 INTERMEDIATE PURIFICATION: Anion Exchange

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

P38
35 OD280

60 OD 280nm P38 after Ni-NTA and dialysis ON vs buffer A. Load on Resource Q-30 6ml column. Wash with 10cv 50mM
76 11/19/2014
NaCl buffer. Elute with gradient 5cv 50-300mM + 40cv 300-750mM + 5cv 750-1000mM NaCl
 P38 FINAL POLISHING: SEC
MW 5 7 9 10 11 12 13

P38

P38 30
OD280nm

35 OD280nm P38 after Ni-NTA, Res.Q & ultrafiltration cut-off 10000. Load on Superdex 75 60x1.6cm column (2 runnings).
77 11/19/2014
Elution 1ml/min
 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

AbrBHiTrapNiHP1mlb001:1_UV1_280nm AbrBHiTrapNiHP1mlb001:1_UV2_260nm AbrBHiTrapNiHP1mlb001:1_Conc AbrBHiTrapNiHP1mlb001:1_Fractions

AbrBHiTrapNiHP1mlb001:1_Inject AbrBHiTrapNiHP1mlb001:1_Logbook

34 mAU

26
AbrB
17 400

10
300

200

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

0
0.0
1
10.0
2
20.0
3
30.0
4 5
40.0
6
50.0
7 8 9 10 11
60.0
12 13 14 15 16
70.0
78
17 18 19 20 21
ml
 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

mAU

2500

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

2000

1500
POOL 17-22: 3.5OD x 35ml ~ 276mg

1000

500

0
79 11/19/2014
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
200 250 300 350 400 450 ml
 HLT-p53CT- Cation
Exchange after TEV
protease cleavage
HLTp53CTHiTrapSP5mlml005:1_UV1_280nm HLTp53CTHiTrapSP5mlml005:1_UV2_260nm HLTp53CTHiTrapSP5mlml005:1_Cond HLTp53CTHiTrapSP5mlml005:1_Conc

mAU
HLTp53CTHiTrapSP5mlml005:1_Fractions HLTp53CTHiTrapSP5mlml005:1_Inject HLTp53CTHiTrapSP5mlml005:1_Logbook
ON 4ºC
150
SP-Sepharose FF 5ml

100

50

0
80 11/19/2014
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
500 550 600 650 700 ml
 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

mAU

150

POOL 7-14

100

50

0
81 11/19/2014
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
60 80 100 120 140 160 180 200 220 min
 Binding equilibrium

At equilibrium
 L T
KD 
 LT
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

82
 Binding equilibrium

It can be shown* that

Bound target L
 KD  L0
0

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

For good binding, KD should be at least two orders of

magnitude less than L0, i.e. 10-6 - 10-4 M

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

83
 Binding equilibria
We can change KD
by changing pH, temperature, salt concentration etc.

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

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

Elution

Target elutes as a sharp peak

84
 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
85
Use ligand with lower affinity
 Competitive elution

Binding
+

Release

Elution +

C+T CT
86
Binding equilibrium for competing ligand

At equilibrium
 C T
KDComp 
 CT
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
87
 Competitive elution
121014AntiHer2Ni16ml004:10_UV1_280nm 121014AntiHer2Ni16ml004:10_UV2_260nm 121014AntiHer2Ni16ml004:10_Conc 121125AntiHer2Ni30ml005:10_UV1_280nm 121125AntiHer2Ni30ml005:10_UV2_260nm 121125AntiHer2Ni30ml005:10_Conc
121014AntiHer2Ni16ml004:10_Fractions 121014AntiHer2Ni16ml004:10_Logbook 121125AntiHer2Ni30ml005:10_Fractions 121125AntiHer2Ni30ml005:10_Logbook

mAU mAU

800

500

700

600 400

500

300

400

300 200

200

100

100

0 F3 F4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 28 33 38 43 48 53 58 63 68 71 74 77 80 83 86
0 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
200 300 400 500 600 700 800 ml
200 250 300 350 400 450 ml

Load lysed pellet of 2L culture on 16ml Load lysed pellet of 4L culture on 30ml
Ni-SepharoseFF used many times Ni-Sepharose6B used a few times

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 88
 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

89
 Mimetic Ligand™ Affinity Chromatography
www.prometic.com
 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

90
 Chemical Combinatorial Library™, + with a rational ligand design approach
 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 91

IgG Select

92
93
 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 94