Affinity chromatography
The affinity chromatography is a chromatographic separation process to isolate an analyte from a solution of various substances. The prerequisite is that a suitable ligand (binding partner) for the analyte of interest (protein) is available. It is one of the most powerful separation methods . However, the columns used are relatively expensive, so that the process is only used in special cases or on a small scale (laboratory scale).
principle
The separation usually takes place in columns, but can also be carried out in a batch process. The cleaning effect of this method is based either on the specific recognition of a protein by an antibody or, in the case of enzymes, on the use of the specific affinity of an enzyme for an inhibitor , substrate or cofactor .
The stationary phase , often a gel , e.g. B. from dextrans or cross-linked agarose (trade name Sepharose ), is coupled with a suitable ligand (z. B. antibody ) that specifically binds the analyte to be purified. In practice, care must be taken that the affinity for the analyte is not too high, as this makes elution more difficult. Conversely, a stationary phase with a protein (usually protein A , G or L) that binds certain immunoglobulin classes can also be used for the preparative purification of antibodies (immunoglobulins) .
To bind a ligand to the carrier material, it is converted into an activated form beforehand. In the example of agarose, the cyanogen bromide activation method comes into consideration, in which reactive imidocarbonate groups are generated which react with amino groups of the analyte to form a covalent bond. After a low molecular weight ligand has been fixed on the matrix (e.g. activated agarose), steric hindrances can occur if the analyte has a large molecule size. In this case the ligand can be coupled to the matrix via a bridge member (spacer). Short hydrocarbon chains are normally used as spacers, which then to a certain extent protrude from the matrix surface.
The association constant, which is derived from the equilibrium constant , is important for the binding of ligand and target protein . The larger this is, the more favorable the affinity chromatographic separation is. The value should be at least K Ass = 10 4 mol −1 .
application
Affinity chromatography is primarily used to purify and concentrate a substance from a mixture in a buffer solution or to reduce an amount of substance in a mixture. Affinity chromatography is also often used to determine which biological compounds bind to a particular substance or to purify and concentrate an enzyme solution.
execution
To carry out affinity chromatographic work, biocompatible HPLC systems are mostly used today, but these are only operated with lower pressures of up to 150 bar. So-called FPLC systems (for English fast protein liquid chromatography ) even work with significantly lower pressures, often below 10 bar. The mixture to be separated is usually applied to the column via sample loops or via sample pumps and the substance of interest is bound by the ligands. All other substances leave the column again quickly because they do not interact strongly with the ligand. After a washing step in order to remove unspecifically bound impurities, the analyte bound to the ligand is also caused to leave the column ( elution ) by changing the conditions ( buffer composition ). An acidic buffer or a solvent / water mixture is often used as the eluent. Alternatively, substances that act competitively with the target protein or an excess of free ligands can also be added. The eluate contains the purified and enriched analyte. The batch process is very similar to the procedure for the column operation explained above. An important difference, however, is the use of centrifugation and other separation methods which are independent of pressure. Depending on requirements, the two processes can also be combined with one another, in which the column operation follows the batch process.
More recent developments are based on the use of several columns connected in series. Here the expensive ligands can be used up to the maximum occupancy. This is not possible in one-column processes, as part of the product is no longer bound and would therefore be eluted and lost. In multi-column processes, this eluate is collected in another column. By alternately loading and eluting the columns, a periodic process is achieved which is often referred to as continuous chromatography. This can be efficiently implemented with two pillars; further pillars in turn reduce efficiency.
Special procedures
Affinity chromatography has special uses in the purification of nucleic acids, proteins or blood. Examples include a. the His-Tag or the Strep -Tag for the purification of recombinant proteins:
| 1. Immune affinity: Purification of antibodies from blood sera |
| 2. Immobilized metal ion affinity chromatography (IMAC): Based on the covalent binding of amino acids, especially histidine, to metals |
| 3. Recombinant Proteins |
| 4. Lectins : Proteins that bind carbohydrates to separate substances in the sample. |
Examples
Examples of ligands for protein purification:
| Ligand | Target protein |
|---|---|
|
Antigen , Protein A , Protein G, or Protein L. |
antibody |
| Substrate , cofactor | enzyme |
| Ligand | receptor |
| Lectin | Glycoprotein |
| nucleic acid | Nucleic acid binding protein |
| Streptavidin , avidin |
Biotin , protein with streptavidin peptide |
| Metal ion chelate such as Ni 2+ - NTA |
Protein with poly- histidine peptide |
literature
- Heinz Bende: Affinity Chromatography . In: Chemistry in Our Time . tape 8 , no. 1 , 1974, p. 17-25 , doi : 10.1002 / ciuz.19740080104 .
- Jerker Porath et al. (1975): Metal chelate affinity chromatography, a new approach to protein fractionation. In: Nature. Vol. 258, No. 5536, pp. 598-599. PMID 1678
See also
Individual evidence
- ↑ Daniel Baur, Monica Angarita, Thomas Müller-Späth, Fabian Steinebach, Massimo Morbidelli: Comparison of batch and continuous multi-column protein A capture processes by optimal design . In: Biotechnology Journal . tape 11 , no. 7 , July 1, 2016, p. 920-931 , doi : 10.1002 / biot.201500481 .
- ↑ Thomas GM Schmidt, Arne Skerra: The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins . In: Nature Protocols . tape 2 , no. 6 , p. 1528-1535 , doi : 10.1038 / nprot.2007.209 .
- ↑ Immobilized Lectin. gelifesciences.com, GE Healthcare Lifesciences
Web links
- Amersham: Affinity chromatography - Principles and Methods (PDF; 3.0 MB)