Protein purification

Protein purification (including protein purification ) is the process to enrich a complex biological mixture, or a solution containing a plurality of biomolecules, one or more proteins, and to clean. This accumulation can take place in several successive purification steps by applying different cleaning methods whose effectiveness ( the meaningful sequence ) and their efficiency ( the degree of purification ), followed by analytical methods and quantified.

Properties of the proteins

Proteins are generally zwitterionic biopolymers from amino acids with a molecular weight of between one and 350 kilodaltons (protein complexes are not included), in which most of the proteins are about 20 to 70 kilodaltons. Due to the protein folding they take up less volume than nucleic acids or polysaccharides. Also secretory proteins can be cross-linked intramolecularly by disulfide bonds.

By peptide binding proteins ultraviolet light absorbing at a wavelength of about 205 nm (190 nm to 230 nm), in addition also absorb phenylalanine, tyrosine and tryptophan UV light at wavelengths of 280 nm to 288 nm This absorption can be used for photometric quantification and determination of the purification factors are used. Unlike carbohydrates and nucleic acids come through the various amino acids contained some structural motifs in proteins only before, such as sulfhydryl -containing cysteines.

Since be purified proteins, mostly recombinant proteins, are especially vulnerable during cell disruption through inactivation, denaturation or proteolysis, protein purification is often performed rapidly at 4 ° C in the presence of protease inhibitors. Some protease inhibitors are used only if the function of the protein be purified shall not be reduced or a receipt whose function is irrelevant since they can modify the protein via a covalent bond. To avoid an undesired formation of disulfide bridges sulfhydryls such as mercaptoethanol, dithiothreitol or dithioerythritol be added often. Occasionally, a cell fractionation occurs after cell lysis and before protein purification by differential centrifugation to separate cell compartments such as nuclei, mitochondria, microsomes and cytosolic components from each other.

Separation principles

To separate proteins, one takes advantage of her due to the sequence -specific structure and different characteristics. In ion exchange chromatography, and isoelectric focusing, the isoelectric point of this is, on SDS - PAGE, the molecular weight, and post-translational modifications in the size exclusion chromatography and centrifugation, the molecular weight, and conformation. The isopycnic centrifugation separated on the basis of density. Affinity chromatography makes use of the differences in affinity to a selective ligand or in the respective dissociation constants. Said hydrophobic, reversed-phase chromatography and the separated polar upon the polarity of the various exposed polar amino acids and post-translational modifications. Precipitation with cosmotropic salts from the Hofmeister series, water-soluble organic solvents or temperature based on the changing solubilities. The extraction with an extraction agent (usually a different phase ) or polyethylene glycol based on the polarity and the different solubilities in a solvent or detergent, such as mixtures of phenol, chloroform and isoamyl alcohol (with or without chaotropic agents such as guanidinium thiocyanate ), or phase separation of one percent ( w / v ) solution of Triton X -114 at 4 ° C.

In most cases several methods are performed serially, the choice of method is guided by the properties of the protein, the respective methods - dependent impairing components for subsequent procedure and the possible concomitant denaturation. Higher levels of purification ( or purification factors ) of a method allowing a smaller number of purification steps, eg in the Tandem Affinity Purification.

Separation processes

Chromatography

• Ion exchange chromatography ( IEX )
• Size Exclusion Chromatography ( SEC)
• Affinity chromatography
• Polar chromatography
• Hydrophobic interaction chromatography ( HIC) and reverse phase chromatography (RPC)

Electrophoresis

• Electrophoretic procedures

Extraction & precipitation

• Serial extractions
• Serial precipitations

Filtration

• Dialysis
• Tangential flow filtration
• Microfiltration

Sedimentation

• Centrifugation
• Pull-down assays
• Using antibodies: MACS, bead assays and immunoprecipitation

Evaporation

• Lyophilization

Detection methods

After the individual stages of protein purification is carried out a protein characterization and quantification of the purified proteins. The efficiency of the entire purification is determined by the balance sheet, with a degree of purification ( as the inverse of the mass fraction ) of the protein mass, or - in enzymes - a degree of purification of the enzyme activities can be determined, for example, the ratio of the total mass of purified protein and the initial mass of the considered protein in the starting material or the analogous quotient formed with the activities.