Protein purification (also protein purification ) describes the process of enriching and purifying one or more proteins from a complex biological mixture or a solution that contains several biomolecules . This enrichment can take place in several successive cleaning steps by using different cleaning methods , whose effectiveness (the meaningful sequence) and their efficiency (the degree of cleaning) are tracked and quantified with analytical methods. The proteins are mostly generated by overexpression with an expression vector .
Properties of Proteins
Proteins are mostly zwitterionic biopolymers made from amino acids with a molar mass between one and 350 kilodaltons ( not including protein complexes ), with most proteins being around 20 to 70 kilodaltons. Due to the protein folding , they take up less volume than nucleic acids or polysaccharides . Secretory proteins can also be intramolecularly cross-linked by disulfide bridges .
Due to the peptide bond , proteins absorb ultraviolet light at a wavelength of around 205 nm (190 nm to 230 nm), and phenylalanine , tyrosine and tryptophan also absorb UV light at wavelengths from 280 nm to 288 nm. This absorption can be used for photometric quantification and for Determination of cleaning factors to be used. In contrast to carbohydrates and nucleic acids, the various amino acids they contain mean that some structural motifs only occur in proteins, e.g. B. sulfhydryl -containing cysteines . These can be used for selective molecular labeling .
Since proteins to be purified, mostly recombinant proteins, are endangered by inactivation , denaturation or proteolysis after cell disruption , protein purification is often carried out quickly at 4 ° C. in the presence of protease inhibitors . Some protease inhibitors are only used if the function of the protein to be purified is not impaired or maintaining its function is irrelevant, since they can modify the protein via a covalent bond . To avoid the undesired formation of disulfide bridges, mild reducing agents are often added, including sulfhydryls such as mercaptoethanol , dithiothreitol or dithioerythritol and phosphines such as tris (2-carboxyethyl) phosphine . Occasionally, after cell disruption and before protein purification, cells are fractionated by differential centrifugation in order to separate cytosolic components and cell compartments such as cell nuclei , mitochondria and microsomes . For the degradation of nucleic acids is Benzonase used, thereby incidentally decreases the viscosity of the lysate.
To separate proteins, one takes advantage of their different characteristics due to the sequence and specific structure . Usually several methods are carried out one after the other, the selection of the method depends on the properties of the protein, the respective method-dependent interfering accompanying substances for subsequent procedures and any associated denaturation. Higher degrees of cleaning (or cleaning factors) of a process allow a smaller number of cleaning steps, e.g. B. with the Tandem Affinity Purification . Good buffers or TBS buffers are often used as buffers .
In ion exchange chromatography and isoelectric focusing, the separation is based on different isoelectric points .
The precipitation with cosmotropic salts from the Hofmeister range , water-soluble organic solvents or temperature is based on the changed solubilities . The extraction with an extractant (usually a different phase ) or polyethylene glycol are based on the polarity and the different solubilities in a solvent or detergent , such as. B. Mixtures of phenol, chloroform and isoamyl alcohol (with or without chaotropes such as guanidinium thiocyanate ) or the phase separation of one percent (m / V) solutions of Triton X-114 at 4 ° C.
- Ion exchange chromatography (IEX)
- Size exclusion chromatography (SEC)
- Affinity chromatography
- Polar chromatography
- Hydrophobic Interaction Chromatography ( HIC) and Reverse Phase Chromatography (RPC)
Extraction & Precipitation
- Serial extractions
- Serial precipitations , e.g. B. ammonium sulfate precipitation , PEG precipitation , TCA precipitation , ethanol precipitation , heat precipitation
- Pulldown assays
- MACS , bead assays and immunoprecipitation with the help of antibodies
After the individual stages of protein purification, protein characterization and quantification of the purified proteins takes place. The efficiency of the entire cleaning is determined by the balance, whereby a degree of cleaning (as the reciprocal of the mass fraction) can be determined from the protein mass or - in the case of enzymes - also a degree of cleaning can be determined from the enzyme activities , e.g. B. the quotient of the total mass of purified protein and the initial mass of the protein in question in the starting material or the analogously formed quotient with the activities.
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