Affinity Chromatography

• Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatography matrix.

• Metal-Chelate Affinity Chromatography (MCAC), also known as Immobilized Metal Affinity Chromatography (IMAC), was first successfully demonstrated in 1975 by Porath and collaborators for human serum proteins.

• In this method, the advantage is made of the ability of the ions (Cu, Zn, Ni) to form stable co-ordinated complexes with reactive imidazoyl & cisteinyl residues of the surface of the proteins

• Most popular chelating agent used is IDA (Iminodiacetic acid) coupled to agarose via oxirane spacer.

• IDA-SepharoseB forms stable but reversible complexes with metal ions and is suitable for purifying many proteins.

• TED-SepharoseB (tricarboxy methyl ethylenediamine)

• Immobilized metal affinity chromatography (IMAC) is most frequently used for the purification of polyhistidine (His-) tagged proteins.

• This technique is based on the interaction between certain exposed protein residues (preferentially histidines) with transition metal cations (Cu2+, Ni2+, Zn2+, Co2+).

• The transition metal itself is immobilized to a cross-linked agarose matrix via a chelating group such as iminodiacetic acid (IDA)

• Successful purification experiments of His-tagged proteins strongly depend on the particular amino acid sequence, the protein conformation and the microenvironment and location of the His-tag (C- or N-terminal).

• Furthermore, undesired co-purification of non-specific host cell proteins is often observed, especially in the case of E. coli expression systems.

Preparation of the column

• The solid support (bead matrix) is a gel loaded into an elution column.

• Sepharose, agarose and cellulose are the most commonly used solid support, because the hydroxyl groups on the sugar residues can be easily manipulated to accept a ligand.

• The ligand is then selected according to the desired isolate. • For eg. to isolate antibodies specific for antigen A from an

antiserum, antigen A can be used as ligand.

Matrix

• Should have a high surface area• Water insoluble, physically rigid,• Permeable, macroporous• Should contain reactive functional groups to derivatize and

bind ligand molecules covalently• Porous glasses, silica, cellulose, polyacrylamide, agarose,

dextran

Ligands

• Choice depends on:• Availability of chemically modifyable groups on the ligand to

facilitate attachment to matrix while retaining binding capability towardscounter ligand

• Affinity towards the counter ligand • Functional groups usually used are: • Amino, carboxyl, aldehyde, thiol & hydroxyl• Classified into 2 groups: Monospecific & group specific

• Mono & group specific:• low molecular weight & macromolecular ligands• Monospecific low molecular weight: vitamins, hormones &

enzyme inhibitors• Eg: lysine which binds only plasminogen from blood plasma;

Vitamin B12 which binds to its transport protein, biotin which binds avidin

• Group specific low molecular weight: Enzyme cofactors & analogues

• Eg., 5’AMP which binds NAD dependent dehydrogenae

• Mono specific macromolecular ligands are involved in specific interactions between proteins

• Eg; Gelatin specifically binds to fibronectin, thrombin binds to antithrombin, Immuno affinity with antibody

• Group specific macomolecular : ConA & lectins; Protein A for IgG purification,

Coupling of ligand

• Three steps are involved in coupling:• Chemical activation of the matrix• Immobilization of the ligand via the chosen functional group• Blocking or deactivating the residual active groups (activation

& coupling may leave some activated groups on the matrix which may lead to non specific binding. Hence they are deactivated by reacting with ethanolamine or mercaptoethanol)

• Different coupling reagents are used to in binding ligands to a matrix

• Epichlorohydrin activation:• It activates the polysacharide matrices by introducing three

carbon atom propane -2-ol hydrophilic spacer arm containing oxirane groups.

• This epoxy activated matrices reacts with sulphydryl groups of thiol ligands or amine groups of amino ligands.

• Cyanogen bromide activation:• Used for activating polysacharide matrices like agarose beads

or cross-linked dextran.• It activates the polysacharides and give rise to reactive

cyanate ester in the matrix which reacts with primary amines or through e-lysine groups of the proteins to form isourea derivative.

• The isourea substituent is positively charged & may introduce anionic exchange character to matrix.

• This activated matrix when treated with aqueous solution of protein ligand, couple with ligand covalently to yield affinity adsorbent

• Sometimes flexible spacer-arm is attached between ligand and solid support to render better flexibility to ligand.

• For example NHS-activated Sepharose (agarose beads with 10-atom spacer arms (6-aminohexanoic acid) attached by epichlorohydrin and activated by N-hydroxysuccinimide) is used for protein ligand immobilization

Steps of affinity purification

• Loading of solution containing the substance to be isolated:• The solution is usually a protein rich mixture such as

antiserum, which is poured into the elution column and allowed to run through the gel, at a controlled rate.

• Proteins with specific affinity for immobilized ligand shall bind and other proteins will go in flow through.

• This follows washing of column with buffer to remove all unbound protein

• Since purification of a protein can be a complex and time-consuming process so that the expression vectors are designed for higher level of expression of recombinant proteins with tags to facilitate the further purification.

• The DNA sequence codes for the protein are cloned in expression vectors at multiple cloning sites in continuation with tags either at N-terminal or C-terminal.

• These tags are also DNA sequences which code a small peptide or even a small protein to facilitate the purification of the recombinant protein. e.g. 6x His Tag or GST Tag are commonly used for purifying recombinant proteins.

His-Tag for Purification of Recombinant Proteins:• A hexa-His sequence is called a His-Tag. • An amino acid sequence consisting of 6 or more His residues

in a row will also act as a metal binding site for a recombinant protein.

• The 6xHis affinity tag facilitates binding to immobilized Nickel.• 6xHis coding sequence can be placed at the C- or N-terminus

of the protein of interest and recombinant protein with 6xHis residues can be expressed.

• In most cases, the 6xHis tag does not interfere with the structure or function of the purified protein as demonstrated for a wide variety of proteins, including enzymes, transcription factors, and vaccines.

• Sometime, a protease cleavage site is inserted between protein sequence and His-tag.

• After His-tag affinity purification, the purified protein is treated with the specific protease to remove His-tag.

• A very common example is His-tagged Proteins with Thrombin, a protease, cleavage site.

• Thrombin recognizes the consensus sequence Leu-Val-Pro-Arg-Gly-Ser, cleaving the peptide bond between Arg and Gly.

• This is utilised in many vector systems which encode such a protease cleavage site allowing removal of an upstream domain

His-tagged protein with thrombin cleavage site

Protein binding:• Proteins containing one or more 6xHis affinity tags, located at

either the amino and/or carboxyl terminus of the protein, can bind to the Ni-NTA groups on the matrix with an affinity far greater than that of antibody–antigen or enzyme–substrate interactions.

• Binding of the 6xHis tag does not depend on the three-dimensional structure of the protein.

• Even when the tag is not completely accessible it will bind as long as more than two histidine residues are available to interact with the nickel ion; in general, the smaller the number of accessible histidine residues, the weaker the binding will be.

• Untagged proteins that have histidine residues in close proximity on their surface may also bind to Ni-NTA, but his interaction will be much weaker than binding of the 6xHis tag.

• Any host proteins that bind nons-pecifically to the NTA resin itself can be easily washed away under relatively stringent conditions that do not affect the binding of 6xHis-tagged proteins.

• Binding can be carried out in a batch or column mode. If the concentration of 6xHis-tagged proteins is low, the proteins should be bound to Ni-NTA in a batch procedure.

• Wash: Endogenous proteins with histidine residues that interact with the Ni-NTA groups can be washed out of the matrix with stringent conditions achieved by adding imidazole at a 10–50 mM concentration

• Protein elution : • The histidine residues in the 6xHis tag have a pKa of

approximately 6.0 and will become protonated if the pH is reduced (pH 4.5–5.3).

• Under these conditions the 6xHis-tagged protein can no longer bind to the nickel ions and will dissociate from the Ni-NTA resin.

• Similarly, if the imidazole concentration is increased to 100–250 mM, the 6xHis-tagged proteins will also dissociate because they can no longer compete for binding sites on the Ni-NTA resin. Elution conditions are highly reproducible, but must be determined for each 6xHis-tagged protein being purified.

• IDA has only 3 metal-chelating sites and cannot tightly bind metal ions.

• Weak binding leads to ion leaching upon loading with strongly chelating proteins and peptides or during wash steps.

• This results in low yields, and metal-ion contamination of isolated proteins

• NTA occupies four of the six ligand binding sites in the coordination sphere of the nickel ion, leaving two sites free to interact with the 6xHis tag.

• NTA binds metal ions far more strongly than other available chelating resins and retains the ions under a wide variety of conditions.

• The NTA matrices can therefore bind 6xHis-tagged proteins more tightly than IDA matrices, allowing the purification of proteins from less than 1% of the total protein preparation to more than 95% homogeneity in just one step.

Glutathione S-transferase for purification of recombinant proteins:

• For easy purification of recombinant proteins they are generally tagged with GST (Glutathione S-transferase) protein to create fusion proteins (protein sequence attached with GST sequence).

• The tag has the size of 220 amino acids (roughly 26 kDa), which is quite big compared to other tags like the myc- or FLAG-tag.

• It generally helps the recombinant protein for soluble expression and further purification.

• A thrombin (a protease) recognition sequence is included in between the GST tag and protein sequence which helps in removal of GST tag by cleavage after purification of fusion protein.

• The GST part of fusion proteins has affinity for glutathione as glutathione

• The GST part of fusion proteins has affinity for glutathione as glutathione is the substrate for GST.

• This enzyme (GST) substrate (glutathione) affinity is used for purification of fusion protein.

• Agarose or other polymer beads can be coated with glutathione, and such glutathione-agarose beads bind GST-proteins.

• The crude cell lysate can be loaded on a column packed with glutathione-agarose matrix, and washed extensively.

• The fusion protein will bind with glutathione coated agarose beads through GST, while other proteins will wash off.

• The elution of bind fusion protein was performed by free glutathione solution.

• Due to higher concentration of glutathione in solution, fusion protein leaves the glutathione coated beads and comes in solution

• The eluted affinity purified fusion protein will now be subjected to thrombin cleavage for removal of GST tag.

• Amount of thrombin used for cleave is very minute and generally removal of thrombin is not required (or an immobilized thrombin may be used which may be removed by simple centrifugation after completion of cleavage reaction).

• The thrombin treated protein was again loaded on regenerated column.

• This time the cleaved GST tag will bind with beads and recombinant protein will come in un-bound fraction.

• Maltose-Binding Protein (MBP) for purification of recombinant proteins:

• In this technique the gene of interest is cloned into pMAL vector creating MBP-encoding malE gene and factor Xa (a protease) cleavage site.

• This gene can be expressed in E. coli producing MBP fusion protein (MBP fused with protein of interest containing factor Xa cleavage sequence between MBP and protein of interest).

• This MBP-fusion protein is purified using amylase column, MBP has affinity for the amylase ligand and finally fusion protein can be eluted using maltose gradient.

• Finally MBP tag can be cleaved from fusion protein using factor Xa protease (as there is factor Xa cleavage site between MBP and protein of interest).

• Generally factor Xa protease used for the cleavage is in minute amount and removal is not required (or an immobilized factor Xa protease may be used which may be removed by simple centrifugation after completion of cleavage reaction).

• Cleaved MBP tag can be separated from the protein of interest by loading it again to amylase column.

• This time cleaved MBP tag will bind to column but protein of interest will go in unbound fraction.

Antibody immobilisations

• Immobilized or purified antibodies, also termed immunoglobulins, are of major importance both for

• immunochemical techniques in basic research (e.g. Immunoprecipitation) and for diagnostic applications.

• One of the most successful immobilization and purification technique is based on the high affinity and

• binding specificity of Protein A, a surface protein of Staphylococcus aureus, towards the Fc region of a broad

• range of mammalian immunoglobulins (Ig).• Immunoglobulins from different species and different isotypes

within a species (IgA, IgG, IgM, IgE, IgD) differ• in their affinity to Protein A

• The principle of affinity chromatography is that the stationary phase consists of a support medium (e.g. cellulose beads) on which the substrate (or sometimes a coenzyme) has been bound covalently, in such a way that the reactive groups that are essential for enzyme binding are exposed.

• As the crude mixture of proteins is passed through the chromatography column, proteins with binding site for the immobilized substrate will bind to the stationary phase, while all other proteins will be eluted in the void volume of the column.

• Once the other proteins have all been eluted, the bound enzyme(s) can be eluted in various ways