Affinity Chromatography was introduced by Pedro cuatrecasas, Chris Afinsen and Meir Wilchek in the year 1968. This is an analytical technique used for separation of biochemical mixture. This is technique is based on Bio-recognition (ligand specificity).The technique has been seen as a powerful tool mainly to separate protein mixtures. The principle is "reversible tight binding formation of the protein of interest in the protein mixture to the ligand on the matrix ". The protein mixture is under the condition where there is a specific binding between the proteins of interest in the mixture to the ligand on the matrix. (Source: Tosoh Biosciences)
For example: protein that binds to Zn metal can be separated by adding immobilized Zn in the resin matrix, the protein of interest then binds to the Zn when the protein mixture is run through the chromatography column and the other mixture is washed away. The protein is then separated from the metal either by changing the environment like PH, ionic strength, solvent and temperature. This technique is most commonly used for purification of recombinant proteins. (Source: Methods in Molecular biology: Affinity chromatography, Vol.147)
Table 1: Biomolecules purified by affinity chromatography
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(Source: Methods in Molecular biology: Affinity chromatography, Vol.147)
Affinity chromatography technique is generally done by the following steps:
1. The ligand is first covalently annexed to a resin matrix like the agarose beads.
2. The resin matrix containing the ligand (that has specific binding with the protein of interest) is poured into a column.
3. An impure mixture containing protein of interest is added on the affinity column
4. Protein mixture passes over resin matrix that has the affinity beads and interacts with affinity ligand. Molecules do not favour binding to ligand come out from the column
5. Washing off contaminant molecules that bind to ligand loosely with a buffer.
6. Collect purified protein of interest using either a biospeciifc or nonspecific elution methods
a. Biospecific - An inhibitor is added in the mobile phase (free ligand), and the free ligand compete.
b. Nonspecific - A reagent is generally added so that it denatures the solute. Once they are denatured the solute releases the ligand .
Generally there are 3 groups of properties of the target molecule that is used in this technique.
The most common and the simplest way is the specific binding property of the protein of interest we are trying to purify. Examples of such properties are enzyme active site, receptor binding, antigen - antibodies binding site etc.
The next method is the use of prosthetic groups like polysaccharides present on the proteins.
This protocol is mainly useful in performing group separation. Example: we can bind ligand such as concanavalin A on the matric and used the interaction to perform affinity separation.
The third way is to use affinity tags that are not present naturally in the protein of interest.
Example: His-tag, Glutahione-S-Transferase(GST) etc. By doing this we can force our protein of interest to bind to the ligand on the matrix and therefore separate it from the protein. This protocol is mainly used for recombinant proteins. Here the tag is added by the method of genetic coupling. (Source: www.asdlib.org)
Fig. 2: Purification of recombinant fusion proteins (Source: www.asdlib.org)
Steps in affinity chromatography
The affinity chromatography medium is equilibrated with the binding buffer.
Fig. 3: Equilibration (Source: GE Healthcare)
The protein mixture is then applied to the affinity medium .The conditions are met in order to which the ligand on the matrix binds to the protein of interest. All other mixtures are washed off along with the buffer.
Fig. 4: Binding of target protein to matrix (Source: GE Healthcare)
The target proteins were achieved by changing the conditions which releases the ligand on the matrix.
Fig. 5: Release of ligand (Source: GE Healthcare)
Affinity medium is then re-equilibrated with binding buffer.
Fig. 6: Re-equilibration (Source: GE Healthcare)
Fig. 7: Affinity chromatogram (Source: GE Healthcare)
Advantages of using Affinity chromatography
High level of purity is obtained
The binding sites of biological molecules of interest can be studied easily
Always on Time
Marked to Standard
Disadvantages of affinity chromatography
Is a costly procedure
When it comes to large amount of sample its difficult to use
Fig. 8: Potential impact
Examples of proteins purified by affinity chromatography
PURIFICATION OF FIBRONECTIN BY AFFINITY CHROMATOGRAPHY UNDER NON-DENATURING CONDITIONS
By Matti VUENTO
Department of Biochemistry, University of Helsinki, Unioninkatu 35, SF-001 70 Helsinki 17, Finland and Antti VAHERI
Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, Finland
Fibronectin is protein with a molecular weight of (~440 KDa). It is involved in many functions likeÂ cell adhesion,Â growth, differentiation,Â and migration. This is protein in this paper is purified from human plasma.
Fibronectin has been purified by many other techniques like ion exchange, molecular-exclusion chromatography. (Mosesson & Umfleet, 1970; Mosher, 1975; Chen & Mosesson, 1977). Fibronectin has also been purified by affinity chromatography with immobilized antibody (Vuento et al.,1977) or on immobilized gelatin (Engvall & Ruos lahti, 1977; Dessau et al., 1978).
The affintity technique used had some advantages; but the problem it made was that strong denaturing elution buffers were used. It has been found that complexes between fibronectin and gelatin can be separated with arginine at pH 7.5 (Vuento & Vaheri, 1978). the paper has addressed the question using a non-denaturing affinity-chromatography method
Gelatin-agrose was used in the previous method. The problem they faced was that the fibronectin got exposed to denaturing agents as it's a large molecule; therefore agents like urea denature fibronectin. This made the study of fibronectin difficult. In this paper they used the affinity chromatography technique which uses gelatine-sepharose(1m- arginine, PH 7.5) and arginine-Sepharose (pH7.5,NaCl to elute the proetin), it was seen that the recovery of fibronectin was 70-80% from the column of the plasma, out of which 90-95% was pure and rest was made up of hetrogenous group of protein. First the Gelatin- sepharose affinity was carried then the arginine - Sepharose was carried to high yield and purity of fibronectin protein.
The purification procedure was carried at 22Â°C, except the dialysis step where the temperature is maintained was 4Â°C. The plasma sample (500 ml) was poured into column of size 8cm x 20cm)of Sepharose 4B and equilibrated with 0.05M-Tris/HCI at pH of 7.5, containing 5mM-enzamidine and 0.02% (w/v) sodium azide. Gelatin-Sepharose is firstly washed with the equilibrating buffer (1000ml) and then with NaCl and finally with 0.2M-arginine buffered with 0.05M-Tris/HCI, pH7.5 (400ml). Fibronectin was then eluted with 1m- arginine, PH 7.5. Fractions of 50ml were collected, and those having fibronectin were collected together and dialysed exclusively against 0.05M-Tris/HCI, pH7.5. After dialysis the fibronectin solution (150ml) was centrifuged for 20min to remove all precipitates, and put into the column (2.5 cm x 10cm) of arginine-Sepharose equilibrated with 0.05M-Tris/HCI, pH7.5. Washing the arginine-Sepharose column with the equilibrating buffer (250ml), fibronectin was eluted with 0.1 M-NaCl in the buffer. When this method is used multiple times, the capacity of columns (gelatin-Sepharose and arginine-Sepharose) to bind fibronectin protein gradually reduces.
Fig.9: 2-Dimensional of immunoelectrophorises of normal human plasma (a) and purified protein (b). The purified protein gives a single precipitation arc.
The result was tested by SDS/polyacrylamide-gel electrophoresis, immuno elecrotphoresis and analytical ultra centrifugation. This purification shows a high dergee of purity and high yield. The protein was obtained without exposing it to denaturating conditions. Thus the protein purification technique proposed may be useful in the structural and fuctional studies.
SINGLE-STEP AFFINITY PURIFICATION OF RECOMBINANT PROTEINS USING A SELF-EXCISING MODULE FROM NEISSERIA MENINGITIDIS
LENKA SADILKOVA, RADIM OSICKA, MIROSLAV SULC, IRENA LINHARTOVA,
PETR NOVAK, PETER SEBO
Institute of Microbiology of the Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic
(RECEIVED April 9, 2008; FINAL REVISION June 23, 2008; ACCEPTED July 20, 2008)
Production of recombinant proteins in an active and highly purified form is an important step in biomedical research, biotechnology, and the pharmaceutical industry, and producing of recombinant protein with consistency is still a difficult challenge. Many affinity-tag systems for single-step purification of recombinant proteins have been developed (Ford et al. 1991; Nilsson et al. 1997; Arnau et al. 2006). The affinity tag sequence is genetically added to N or C terminal of the target protein, thus allowing the purification of tagged proteins from the cell mixture by high affinity binding to the matrix.
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The limitations to these affinity systems are that the affinity tag should be detached before the biochemical or biological activity characterization, crystallization, or use in antibody production. A specific cleavage site should be engineered between the target protein and the affinity tag and this should be cleaved off.
But this suffers some limitations like:
Separating the target protein from the affinity tag by adding appropriate protease to cut at the site. This then generally requires a extra purification step
The protease (eg.Thrombin, enterokinase) used are not that specific and may cleave bonds into the target sequence or protein
The protease may not be able to reach the site of cleavage site in fusion protein
The cleavage of the protein with appropriate protease often require a long reaction time
Based on the above limitations the affinity tag system has been combined together with a site specific processing module for one- step purification system taking the basis of self cleaving affinity tags (Chong et al. 1997; Mao 2004). The self cleaving affinity tags consists of self- processing modules that is joined to one end of the affinity tag genetically, thus helping in doing only one step of purification and the second end is joined to the protein of interest.
After the fusion protein is bound to the matrix and all other contaminant is removed during purification, the cleavage activity of the self-processing module is carried out by a low-molecular weight compound letting the protein of interest to go.
A suitable self-processing module has to satisfy two conditions:
Only a specific peptide bond must be cleaved that is between the protein of interest and the self-processing module-affinity tag
It must cleave off only after induction in vitro and not inside of the producing organism.
Thus they are very rare to find, these self-processing module-affinity tag.
It has in this paper they have used module of FrpC to design a novel type of self-cleaving affinity tag.
Fig. 10: (A) The 198-kDa FrpC protein of N. meningitidis consists of an N-terminal part harboring the self-processing module (SPM), a C-terminal portion with the putative calcium-binding nonapeptide repeats (RTX), and the C-proximal secretion signal. (Arrowhead) Asp414-Pro415 bond autocatalytically and specifically cleaved upon activation by calcium ions (
Protein production and purification
E.coli was used to produce recombinant protein that was transformed with appropriate plasmids. Exponential 500-mL cultures are grown by shaking at 30Â°C in LB medium supplemented with 150 mg/mL ampicillin or 60 mg/mL. kanamycin were induced at OD600 Â¼ 0.6-0.8 with 1 mM IPTG and grown for an extra 4 h. All recombinant proteins are made using E. coli strain BL21 (lDE3) transformed by their appropriate plasmid. The cells were gathered by centrifugation, it was then washed twice with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl (TN buffer), 5 mM EDTA to remove Ca2+ ions associated with the cell surface, suspend in TN buffer containing 1 mM EDTA (EDTA buffer) or TN buffer alone (cells that express proteins to be purified on a Ni-NTA agarose), and disrupted by sonication at 4Â°C. The homogenates are cleared at 20,000g for 20 min and then the extracts were again purified.