Protein purification

Protein purification is a complex of methods aimed at obtaining a pure preparation of a particular protein from a complex mixture, such as a cellular extract. It is possible to separate different proteins based on differences in their properties such as molecular weight, size of molecules, specificity of binding to certain substances, as well as solubility, charge, which depend on temperature, pH, ionic strength of the solution and other factors. Protein purification is necessary both for the study of their properties and functions, and for use in industry, medicine and laboratory practice. Samples of tissues or biomass of cultured cells serve as the source for the isolation and purification of proteins. The first stage of protein purification is the preparation of lysates or cell extracts by destroying the plasma membranes of cells, and in the case of not animal cells, also cell walls. The lysates can then be subjected to differential centrifugation, which allows the preparation to be enriched in the desired subcellular fractions (eg, nuclei, mitochondria, microsomes, etc.). For concentration and fractionation of proteins, salting can be used, and it is possible to get rid of low molecular weight impurities by dialysis. The final purification is by the use of different types of chromatography, gel electrophoresis, isoelectric focusing or a combination of these.
1 History
2 Protein source
3 Protein conversion
4 Protein stabilization 5 Separation Methods
6 Notes
7 Sources
Until the 1920s, the nature of protein molecules remained rather mysterious. Their large molecular weight was explained by colloidal aggregation of smaller, and unknown, molecules. This idea changed when James Sumner was able to crystallize the urease enzyme in 1926. After that, it became clear that the isolation of individual proteins is not an impossible task. The first techniques were very rough by modern standards. In order to obtain a preparation of a certain protein of satisfactory quality, several years and huge quantities of starting material were rarely spent. Despite the complexity of the process, up to the 1940s, about 20 proteins were obtained in pure form.
Protein purification methods are constantly being refined and allow to obtain more pure drugs in greater quantities and in less time. Using modern techniques we can separate proteins as similar in properties to several years ago as one pure substance [1].
Protein Source
Recombinant Bacteria Culture
Proteins make up a significant proportion of the dry mass of all living organisms, however, the concentration of each individual protein may be significantly different, for example, hemoglobin significantly outweighs all other compounds in erythrocytes, while there may be only a few lac repressor molecules per cell of Escherichia coli. Usually the same protein can be isolated from different sources, in particular, many metabolism enzymes are common to all life forms. However, the properties of proteins derived from different organisms, as well as from different tissues of the same organism, may differ. Typically, the selection of sources for protein isolation and purification is guided by such criteria as the ease of obtaining the desired tissue (cells) in sufficient quantity, the concentration of the desired compound in it, as well as the criteria specific to each protein that can improve its isolation and stabilization. Most commonly used are tissues of domestic (pigs, cows, chickens) or laboratory (rat) animals, as well as cultures of microorganisms such as Escherichia coli and baker's yeast (Saccharomyces cerevisiae). However, with the development and proliferation of molecular cloning methods, proteins are increasingly derived from micro-organisms, which produced the desired gene. In this case, the cloned protein can make up about 30% of all protein byproduct, which greatly facilitates its isolation and purification [2]. Conversion of proteins to solution
Duns glass homogenizers with Teflon pistons
After selecting a protein source in the first stage purification is the transfer of it into solution, this step is unnecessary in the case of separation of proteins from biological fluids, such as blood plasma. There are many methods of destroying cell membranes to release cytosol, and the choice of one depends on the mechanical properties of the protein source as well as its location. The cells are placed in a hypotonic environment where, due to osmotic phenomena, they swell and crack. This method can be used for animal cells that do not have a cell wall, but for plant, bacterial and fungal it is mostly ineffective. The cell wall of bacteria is often destroyed by the enzyme lysozyme, also by sonication (ultrasound). Detergents (Triton X-100, Tergitol NP-40, etc.) or organic solvents (acetone, toluene) are often used in lysing solutions, but this is only permissible when such substances will not denature the desired protein.
Samples cells and tissues can also be homogenized using freeze / thaw cycles, grinding with sand or glass balls, high-speed blenders, homogenizers (consisting of sleeves and tightly fitting glass or Teflon pistons, are mechanical and manual presses) (cells are destroyed by pressing through a small, high-pressure hole).
The resulting lysate is filtered or centrifuged to deposit large fragments of cells. If the desired protein is not localized in the cytosol, but in some subcellular fraction, such as the plasma membrane, nucleus, or mitochondria, then its purification involves isolation of this fraction by differential centrifugation: first, heavy cell components are precipitated, then supernatant is selected and centrifuged selected the necessary fraction passes into the precipitate. Membrane proteins are separated from lipids by detergents or organic solvents [2]. Protein Stabilization
Once a protein has been removed from its natural conditions, it can be destroyed by many factors. In particular, proteins are denatured when pH is changed, so lysis solutions and subsequent purification steps contain buffer systems that maintain a hydrogen index in the range within which the desired molecules will remain stable.
Most proteins are subject to slow denaturation at temperatures greater than 25 ° C. . Because of this, all procedures with them are most often performed when cooled to 0-4 ° C (on ice). Some proteins retain stability beyond living cells only at temperatures below −100 ° C. On the other hand, there are also cold-sensitive proteins that cannot be cooled down.
During cell lysis, proteases, enzymes that hydrolyze proteins, are released. They can cause degradation of the desired protein. Although cell extracts maintain a pH at which such enzymes are inactive, they also preferably use chemical protease inhibitors such as PMSF. Another potential cause of protein degradation may be microorganisms, so during long-term storage, a small amount of toxic compounds are added to the protein solutions, which do not affect the structure of the proteins themselves, such as sodium azide. Many proteins may be unstable at the liquid-air interface , and at low concentrations much of their molecules are lost due to sorption to the surfaces. Because of this, during the separation of proteins they try to avoid the formation of foam and bubbles, and keep the solutions concentrated.
If the desired protein is particularly resistant to any of the following factors that can destroy other proteins, this property can be used for purification. For example, if it is very heat-resistant, the cell extract is heated to high temperature for a short period of time, resulting in most of the impurities denaturing and settling, and the necessary protein remains intact. Protease-resistant proteins can be isolated using cell autolysis, that is, to maintain the lysate under conditions in which proteolytic enzymes are active and destroy most other cellular proteins. [3]
Separation Methods
br> Methods - Solubility - Salting - Layering - Charge in ionized form - Ion exchange chromatography - Electrophoresis - Isoelectric focusing - Polarity - Adsorption chromatography
Chromatography on papers
Reverse-phase chromatography
Hydrophobic x romatography
Molecule size - Dialysis and ultrafiltration - Gel electrophoresis - Gel filtration - Ultracentrifugation - Binding specificity - Affinity chromatography
Differences in their properties can be used to separate proteins such as charge in ionized form, solubility, molecular weight, polarity, ability to specifically bind to certain compounds.
↑ Voet et al, 2011, p. 132
Vo a b Voet et al, 2011, p. 130
↑ Voet et al, 2011, p. 130-131 - Sources - Berg JM, Tymoczko JL, Stryer L (2007). Biochemistry (vol. 6th). W.H. Freeman and Company. with. 67—75. ISBN 0-7167-8724-5.
Nelson D.L., Cox M.M. (2008). Lehninger Principles of Biochemistry (vol. 5th). W. H. Freeman. with. 85—92. ISBN 978-0-7167-7108-1.
Voet D., Voet J.G. (2011). Biochemistry (vol. 4th). Wiley. with. 129—156. ISBN 978-0470-57095-1.

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