Basic principle – affinity chromatography

TAG elimination

TAG elimination
Application of recombinant proteins

 Biochemical studies (to study enzymatic properties and

regulation, to identify binding partners, etc)
 Production of polyclonal and monoclonal antibodies
 Structural studies: Crystallography and NMR analysis
 Cell biology: microinjection into mammalian cells
 Biotechnology: large-scale production of growth factors and
hormones, such as interferon, insulin etc
 Pharmaceutical industry: to develop screening assays for
drug discovery programs
 Clinics: production of vaccines, diagnostic kits, etc
 Purification of recombinant proteins with GST tag
Different types of column:
Glutathione Sepharose
Glutathione−Agarose
Glutathione Magnetic Beads

Glutathione-S-transferase (GST) is a small enzyme

(depicted here by the purple icon) that binds
glutathione (a glutamate residue to which a Cys-
Gly dipeptide is attached at the carboxyl carbon of
the Glu side chain, hence the abbreviation GSH).

Three Distinct-Type Glutathione S-Transferases in E.coli !!!

 The GST tag is fused to the carboxyl terminus of the target protein by genetic engineering. The
tagged protein is expressed in host cells, and is present in the crude extract when the cells are lysed.
 The extract is subjected to chromatography on a column containing a medium with immobilized
glutathione. The GST-tagged protein binds to the glutathione, retarding its migration through the
column, while the other proteins wash through rapidly.
 The tagged protein is subsequently eluted from the column with a solution containing elevated
salt concentration or free glutathione.
 rP+GST

GST

 To avoide protein degradation use

protease-defficient expression host
 Work fast
 Magnetic beads are especially appropriate if small
amount of protein is going to be purified

Magnetic GST Affinity Beads are superparamagnetic nanoparticles coated with

Glutathione (GST). The Magnetic GST Affinity Beads specifically bind to
glutathione S-transferase and can be used to separate and purify GST-fusion
proteins from crude cell extract, which are used for subsequent experiments.
 Purification of recombinant proteins with His tag
 Recombinant proteins containing a
6xHistidine-tag can be purified by Ni-NTA
(nickel-nitrilotriacetic acid) chromatography
which is based on the interaction between a
transition Ni2+ ion immobilized on a matrix and
the histidine side chains.
 Following washing of the matrix
6xHistidine-tag fusion proteins can be eluted
by adding free imidazole or EDTA or by
reducing the pH.
 Purification under native or denaturing
conditions is possible.

 If the protein is insoluble and forms inclusion bodies after expression, it has to be solubilized with
chaotropic salts like guanidine or urea at high concentrations in order to allow affinity chromatography.
 Purification under such conditions can be excellently achieved by using the metal chelate activity of
the 6xHistidine-tag.
 Proteins can be purified and optionally be refolded on Ni-NTA columns.
 Optimize binding and elution conditions
His-tag protein purification

Lysis Buffer may contain (10 mM) imidazole to minimize binding of untagged,
histidine rich contaminating proteins and increase purity with fewer wash steps.

Higher concentration of imidazole in lysis and washing buffer will

provide higher purity but lower yeald of recombinant protein.
SDS-PAGE analysis of His-tag fusion proteins in total
bacterial lysates and after affinity purification

Affinity purified
Total proteins His-tag fusion proteins

NYBR62
NYCO58

A34
CT17
Magnetic His-tag Affinity Beads are superparamagnetic nanoparticles
coated with Ni-NTA. The 6XHis-tag fusion protein affinity beads are
used to selectively separate and purify 6XHis-fusion protein from crude
cell extracts, which are used for subsequent experiments.
Maltose-binding protein (MBP) tag

 Maltose binding protein (MBP) is a common

protein expression tag.
 Fusion of a target protein to MBP permits its
one-step purification using amylose resin.
 Additionally, in E. coli, MBP is known to
significantly enhanced the solubility of many
proteins it has been fused to.
 MBP fusion proteins can be expressed and
purified from yeast.
 To improve purity:
Affinity step + Gel filtration
 Used if after affinity chromatography truncated forms with tag are co-purified, or if
binding/interacting proteins are co-purified with taged protein.
 Subsequent gel filtration is suitable if co-purified proteins have significantly different Mw.
Dual tagging of protein for full length protein isolation

 Recombinant proteins may be partially degraded during expression which cannot

be prevented by adding protease inhibitors during downstream processing.
 Soluble degradation products still carrying the tag are co-purified and cause an
inhomogeneous protein preparation with protein fragments of different lengths.
 This problem can be solved by adding a second tag to the other protein
terminus. Performing a second purification run using the affinity of this second tag
selects for full-length proteins.
1 2 3 4

5 6 7

After two successive affinity chromatography full length protein is purified.

Protein yield and purity depend on the
tag and method used for purification
 Immunoprecipitation (IP) assay
Immunoprecipitation is a procedure which permits the purification of the protein of
interest with the use of specific antibody (poly or monoclonal)
It is used:
* To analyse the activity of immunoprecipitated proteins
* To prove the interactions between two proteins
* To isolate multienzyme complexes and to identify their components
* To analyse protein-DNA interactions (chromatin immunoprecipitation assay)

Procedure:
1. Extraction of proteins: cells or tissues are lysed in extraction buffer in the presence of proteinase
inhibitors
2. Formation of antibody/antigen complex. Specific antibodies are added to the supernatant. The
interaction between the antibody and the target protein take place.
3. Precipitation of the immune complex. Protein A or Protein G Sepharose beads are added in order to
physically separate the antibody-antigen complex from cell or tissue lysates.

Precipitated immune complexes are analysed according to the experimental design.

 IP strategies involve incubating an antibody with a protein
mixture containing the antigen, followed by capturing the
antibody:antigen complex with Protein A or G immobilized to
agarose resin. After washing, nonbound and presumably
undesired components of the sample are removed.
 Co-immunoprecipitation (Co-IP) is a popular technique to identify physiologically relevant
protein-protein interactions by using target protein-specific antibodies to indirectly capture
proteins that are bound to a specific target protein.
 The immunoprecipitation is performed with the ANTI-FLAG M2 affinity gel, which is a highly specific
monoclonal antibody covalently attached to agarose resin. The use of affinity resin allows an efficient
binding of FLAG-tagged proteins.
 The immunoprecipitated FLAG-tagged proteins can be efficiently eluted from the resin with acidic
conditions or by competition with FLAG peptide. The immunoprecipitated proteins can be detected for
their size, post-translational modifications, and interactions on gel electrophoresis, and by activity assays.
 Highly specific antibody does not cross-react with endogenous proteins.
 Dual tagging of protein to improve purity:
Tandem Affinity Purification (TAP)
protein of interest Tag

 Especially important
Calmodulin binding site Protein A (IgG-
binding domain) for protein purification
from complex systems
TEV-site

TEV
TEV Bind IgG Elute after
protease
site
beads TEV protease
IgG
CBP ProtA
beads
Protein of Wash out
interest
contaminants
Bind calmodulin
beads

Elute with
EGTA Calmodulin
beads

After Rigaut et al. (1999) Nature Biotech. 17: 1030

 TAP tag purification of proteins

 May be used to identifies

proteins that interact with
your protein of interest

Can be used in all eukaryotes

C-terminal TAP tag
N-terminal TAP tag
Major application: isolation of stable protein complexes
Limitations/problems with TAP tag purifications

The tag to be fused in frame with the ORF of interest (almost universally done at
the C-terminus) may disrupt normal protein function and consequently its
interacting partners

In any given purification, the conditions of cell lysis and subsequent manipulation
may disrupt native, physiologically relevant interactions (false negatives)

In any given purification, proteins which do not normally interact in vivo may
interact in vitro with your protein of interest (false positives)

Proteins may interact with the purification matrices (false positives)

 TAP tag strategies

Purify proteins that

interact with taged protein

Detect interacting proteins by liquid chromatography/mass spectrometry

Identified proteins that differ are possible interacting partners

 Determining the functional
P1
interconnections of proteins

• On average, one protein interacts with five others.

• Some protein–protein interactions are short-lived, others form
stable multicomponent complexes.
• At a higher level of cellular organization, complexes interact with
one another.
• In eukaryotes, there are many more proteins than genes due to
alternative splicing, posttranscriptional modifications to RNA
(RNA editing), and posttranslation modifications
 Protein-protein interaction studies
 The biochemical interactions between the various proteins of the cell are just as
important as the information in the genes themselves, and the ultimate aim of cell and
molecular biology is to understand cells fully at the molecular level.
 Protein complex assembly can result in the formation of homo-oligomeric or hetero-
oligomeric complexes.
 In addition to the conventional complexes, as enzyme-inhibitor and antibody-antigen,
interactions can also be established between domain-domain and domain-peptide.
 Interactions can be classified into stable or transient, and also according to the nature of
the chemical bonds established between proteins.
 Stable interactions involve proteins that interact for a long time, taking part of permanent
complexes as subunits, in order to carry out structural or functional roles.
 Transient interactions are brief and reversible interactions of proteins that happened in
only certain cellular contexts – cell type, cell cycle stage, external factors, presence of other
binding proteins.
 Protein-protein interaction databases
Prediction databases include many protein-protein interactions that are predicted:
 Known and Predicted Protein-Protein Interactions (STRING)
http://string-db.org
 Protein-protein interaction databases
BioGRID: Biological General Repository for Interaction Datasets http://thebiogrid.org
IntAct Molecular Interaction Database http://www.ebi.ac.uk/intact
 Pull down – in vitro assay for protein-protein interactions

 "Pull-down" is a small-scale affinity co-purification technique.

 A specific affinity chromatography method where a molecule of interest (bait) is bound to a
column, often via an affinity tag expressed as a protein fusion (GST, HIS tag and others) or
chemically linked to the bait molecule.
 The molecule may be expressed or synthesised and purified first, often in an heterologous
system, bound to the matrix at high concentration and then challenged with a solution or cellular
extract containing the candidate partner molecules.
 Useful for both confirming the existence of a protein-protein interaction predicted by other
research techniques (e.g., co-immunoprecipitation, Y2H, Tap-tag), and as an initial screening assay
for identifying previously unknown protein-protein interactions.
 The fusion-tagged protein acts as the "bait" to capture a putative binding partner (the "prey").
 Pull down
 one on one

GST His
? His
GST

Purified protein1 Purified protein2

Are both proteins purified by
glutathione or Ni-NTA agarose?

YES: proteins No: proteins do

interact with not interact
each other with each other
 Pull down
 coexpression assay

GST His
? His
GST

Tagged proteins are

Are both proteins purified by
coexpresed in the same cell
glutathione or Ni-NTA agarose?

YES: proteins No: proteins do

interact with not interact
each other with each other
 Pull down
 coexpression assay
 one on two
His

GST His
? His
GST

Taged proteins are

Are all taged proteins purified
coexpresed in the same cell
by glutathione agarose?

YES: proteins No: proteins do

interact with not interact
each other with each other
 Pull down
 one on cell extract
His

OR Cell ?
+ protein
GST

GST extract

Purify proteins by glutathione

(for GST tagged bait) or Ni-NTA
Purified protein agarose (for His tagged bait)

Identify proteins that interact with

your protein (antibodies or LQ/MS)
 Pull-down
In a typical pull-down assay the immobilized bait
protein is incubated with a cell lysate.

After the washing steps, the

'interactors" are selectively eluted for
analysis in-gel or by Western blot.
Possible purification of stable protein
complexes.
GST pull-down assay for descovery of interactions
SH3
GST domains

pGEX

express GST- Prepare protein

fusions in E.coli extracts from mouse brain

Incubate GST or GST-SH3 domains coupled to

Glutatione Sepharose with tissue extracts

Remove non-specific interactions by extensive washing

Separate bound proteins by SDS-PAGE

Cut-out the bands of interest from the gel

Identification of binding partners by Mass spectrometry

 Pull down
 CONTROLE

GST His
? His
GST

Purified tag Purified protein

Is GST tag purified by
Ni-NTA agarose?

YES: you could No: you could

not examine examine
protein-protein protein-protein
interaction by interaction by
pull down assay pull down assay
PULL DOWN
Does the
proteins
interact?
 Mutagenesis and pull down:
To answer the question: Which domain or residue
is important for the interaction of two proteins?
DNA-Protein Pull-down
 RNA end-labeled with
RNA-Protein Pull-Down biotin and streptavidin
magnetic beads

 Direct enrichment of the

protein-RNA interactions
 Provides an alternative to
antibody capture of protein-
RNA complexes.
 Chromatin Immunoprecipitation (ChIP)
 Used to investigate the interaction between proteins and DNA in the cell.
 Determine whether specific proteins are associated with specific genomic regions.
 Protein and associated chromatin in a living cells or tissues are temporarily bonded
 The DNA-protein complexes are sheared into ~500 bp DNA fragments
 DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from
the cell debris using appropriate antibody along with cross-linked DNA fragments,
 The associated DNA fragments are purified, (amplified) and their sequence is determined.

Cross-linked ChIP: The agent for reversible cross-linking is formaldehyde or UV light. Then the cross-
linked chromatin is usually sheared by sonication, providing fragments of 300 – 1000 bp in length.
Native ChIP (NChIP): Mainly suited for mapping the DNA target of histone modifiers. Histones wrap
around DNA to form nucleosomes, they are naturally linked. Then the chromatin is sheared by
micrococcal nuclease digestion, which cuts DNA at the length of the linker, leaving nucleosomes intact
and providing DNA fragments of one nucleosome (200 bp) to five nucleosomes (1000bp) in length.
 Chromatin Immunoprecipitation (ChIP)
Invaluable method for studying interactions between specific proteins or
modified forms of proteins and a genomic DNA region.
 Yeast two-hybrid screening
Binary in vivo method for study of protein-protein interaction

 Yeast cells are transfected with two

plasmids: the bait (fused with the DNA-
binding domain of a yeast transcription
factor, like Gal4), and the prey (linked to
the activation domain of the transcription
factor).
 Transcription of reporter genes does
not occur unless bait and prey interact
with each other and form a functional
transcription factor.
 The interaction between proteins can
be inferred by the presence of the
products resultant of the reporter gene
expression.
 Two-hybrid assay for detecting pairwise
protein interactions

The DNA-binding domain of a transcription factor binds to a specific

sequence in the regulatory region of a gene, which orients and
localizes the activation domain that is required for the initiation of
transcription of the gene by RNA polymerase.
The coding sequence for the activation domain is fused to the DNA for protein
Z (DNA Z) and co-transformed into a cell containing the DNA binding domain–
DNA X fusion construct.
The proteins encoded by the cDNAs of the hybrid genes interact, and the
activation domain is properly oriented to initiate transcription of the reporter
gene, demonstrating a specific protein–protein interaction.
The coding sequences for the DNA-binding domain and the activation domain are
fused to DNA X and DNA Y, respectively.
Both constructs (hybrid genes) are co-transformed into a cell.
After translation, the DNA-binding domain–protein X fusion protein binds to the
regulatory sequence (promoter) of a reporter gene.
However, protein Y (prey) does not interact with protein X (bait), and the reporter
gene is not transcribed because the activation domain does not, on its own,
associate with RNA polymerase.
 For a two-hybrid system, the coding regions of the DNA-binding
and activation domains of a specific transcription factor are isolated
and cloned into separate vectors.
 Gal4 transcriptional factor from Saccharomyces cerevisiae or the
bacterial LexA transcription factor is used.

 CONTROLS are necessary !!!

Protein of interest mast not interact with Activation domain or

binding domain!!

Many false positives

In yeast, proteins of interest mast enter the nucleus to induce
reporter gene transcription !!
Many false negatives
 One to one Y2H

-Leu, -Trp -Leu, -Trp, -His

 LARGE-SCALE PROTEIN–PROTEIN INTERACTION STUDY
 Library is prepared, that contain thousands of cDNAs generated from
total cellular mRNA. The cDNAs are cloned into a vector containing the
sequence for the activation domain to form the prey library.
>Many different prey clones can be screened simultaniously
 The cDNA of the protein of interest is cloned into the vector adjacent
to the DNA sequence for the DNA-binding domain of the transcription
factor Gal4 to form a hybrid bait gene.

Yeasts able to grow on

medium without Leu,
Trp and His synthesize
proteins that interact
 How to identified proteins that interact
with target protein
 Prepare bait: target protein fused to
DNA binding domain (GAL4)
 Transform vector into yeast: stable cell
line is commonly used
 Prepare fusion protein library with an
activation domain
 cDNA library: all cDNAs are fused
with activation domain
Transfect library into cells and either select for
survival or activation of reporter gene.
Purify and characterize positive clones.

What is key factor required for success?

No activation domain in bait or prey!