Medicilon has more than 10 years of experience in recombinant protein expression and purification services. Medicilon has a variety of protein expression synergies, including prokaryotic protein expression system, yeast protein expression system, insect cell protein expression system (rod-shaped Virus) Mammalian cell protein expression system, with a variety of fusion technologies, can provide you with a variety of options in protein expression and purification. From scheme design, gene optimization, expression condition optimization to purification technology system, to improve your target protein expression level.
Medicilon protein expression system: prokaryotic protein expression system (Escherichia coli expression system), yeast protein expression system, insect cell protein expression system (baculovirus expression), mammalian cell protein expression system, Medicilon provides a variety of The recombinant protein system is for your choice, and you can choose flexibly from price, cycle, advantages and disadvantages to meet your different scientific research needs.
Protein expression and purification services include codon optimization, gene synthesis, expression vector construction, small-scale protein expression and purification, and large-scale protein expression and purification. A large number of purified recombinant proteins can be used in a series of biomedical research in drug screening, structural biology research, cell biology research, proteomics, etc. The prokaryotic protein expression system is by far the most mature and reliable protein expression system, which can quickly express and purify proteins from various sources. Medicilon has successfully constructed a complete E. coli prokaryotic and yeast cell eukaryotic protein expression technology platform, and can provide a complete set of services from protein expression plasmid construction, protein expression condition exploration and protein purification according to customer needs. .
Principles of protein purification
First, we must understand the properties of the target protein and main impurities in the sample to be purified, collect as much as possible about the source, nature (molecular size, isoelectric point) and stability (protein to temperature, extreme pH, protease, oxygen and metal) of the relevant protein Tolerance of ions, etc.), which helps design protein purification.
Secondly, before the purification begins, the use of the final product must be understood to design the protein purification process, and the quality, quantity and economy of the purified product must be considered in three aspects. How high the purity of the purified protein is, how much activity is allowed to be lost during the purification process, and how much time and cost the purification process takes are all affected by the use of the target protein. The higher the purity of the target protein, the longer the operation time and the higher the cost.
Finally, it is also important to fully understand the large amount of information of each separation and purification technology operation unit. For example, when the cell is broken, it is necessary to understand the flow rate, type of agitator, operating pressure, cell concentration and type, product release fragments and size, etc.; design analysis When adsorbing chromatography, understand the characteristics of the column, the performance of the gel or other adsorbents (binding capacity, dissociation constant, flow rate, etc.).
The selection and determination of protein purification methods should be determined according to the nature of different protein samples and the specific research purpose. Commonly used methods for initial extraction and concentration of proteins are adsorption method, ultrafiltration method, precipitation method (such as salting out, organic solvent precipitation, isoelectric point precipitation and selective precipitation, etc.), dialysis method, etc. When high resolution is required, chromatography (such as gel filtration, ion exchange, affinity chromatography, and covalent chromatography) and electrophoresis (such as isoelectric focusing, two-dimensional electrophoresis, capillary electrophoresis, and immunoelectrophoresis, etc.) are usually used. . The principle of these separation and purification methods is mainly based on the differences in solubility, chargeability, molecular weight or affinity specificity of proteins. The research and application of chromatographic technology and electrophoresis technology in protein purification are extensive and in-depth.
At present, ion exchange chromatography has become the most commonly used method in protein separation and purification. Statistics show that in protein purification schemes, ion exchange chromatography accounts for 75%, followed by affinity chromatography and gel filtration chromatography, respectively 60% and 50%. In fact, the protein purification process is often achieved by using several purification technologies in combination. In this process, it is important to choose a reasonable purification technology. However, how to use these technologies in a reasonable combination and order is also a successful separation. Must be considered.
Gel filtration chromatography
Gel filtration chromatography technology is an efficient separation and purification method in the field of protein research. On the one hand, in theory, the protein sample almost does not have any effect on the stationary phase matrix in the gel column. On the other hand, the sample eluent is a salt or pure aqueous buffer, and the eluent is The composition does not change, so this technology has many advantages in terms of easy operation, gentle elution conditions, good reproducibility, no need for organic solvents, easy sample denaturation and high recovery rate.
Main applications: for protein concentration and purification, molecular weight and distribution range determination, sample desalting and protein buffer replacement.
Main considerations: gel parameters, volume parameters, partition coefficient and effective partition coefficient,
Ion exchange chromatography
The basis of ion exchange chromatography for the separation of biomolecules is that the material to be separated is oppositely charged with the ion exchanger under certain conditions and thus can compete with it, and different molecules have different types, quantities and distribution of charges under this condition. , Showing the difference in binding strength with the ion exchanger, which is eluted and separated in the order from weak to strong in the ion exchange chromatography. Features of ion exchange chromatography:
①High resolution, with the emergence of various high-efficiency chromatographic media, choosing an appropriate ion exchanger can ensure that ion exchange chromatography has good selectivity and resolution;
② The high protein exchange capacity is conducive to enlarging the scale of separation and its application in industrial production, which is difficult to achieve by methods such as gel filtration;
③ The application is flexible, and the separation process can be optimized by selecting different ion exchange agents, controlling the composition of the buffer, pH, and ionic strength conditions;
④ The separation principle is relatively clear. The technology is based on different charges. However, for large molecules such as proteins, in addition to electrostatic effects, non-ionic interactions such as hydrophobic interactions, hydrogen bonds, and the nature of buffer ions also affect the separation behavior. ;
⑤ The operation is simple and easy. When the sample is separated on a large scale and the resolution requirement is not high, the protein adsorption and desorption can even not be performed in the chromatography column.
Affinity chromatography
Affinity chromatography uses the biological activity of protein molecules rather than their physical and chemical properties for separation. That is, the specific affinity between protein and ligand is used as the basis for separation, so it is highly selective and its degree of purification can sometimes be Up to 1000 times, so affinity chromatography is a very effective method of protein purification, mostly used to separate a small amount of specific proteins from a large number of complex solutions, and this purification method also has the effect of concentration. Sometimes only one-step separation by affinity chromatography can achieve rapid and satisfactory protein purification, which is unmatched by other purification methods.
The types of binding between proteins and ligands in affinity chromatography include: the active center or allosteric center of the enzyme binds to specific substrates, coenzymes, activators or inhibitors through secondary bonds, antigens and antibodies, hormones, etc. Combination with its receptor, biotin and avidin/streptavidin, glycoprotein and lectin.
Covalent chromatography
Chromatography techniques used to separate proteins and other biological macromolecules, such as ion exchange chromatography and hydrophobic interaction chromatography, are based on the non-covalent interaction between the molecule to be separated and the adsorbent, while covalent chromatography is The separation or covalent bond formation between the stationary phase and the solute is used to achieve separation.
The chromatographic technique commonly used for biologically active proteins requires that under mild conditions, both stable bonds can be formed and the structure of the immobilized protein can not be destroyed when the immobilized protein is released.
Electrophoresis technology
Electrophoresis is the movement of charged colloidal particles towards an electrode with opposite electrical properties under the action of an electric field. The separation principle is based on the charge density of biological macromolecules.
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The above is about the protein purification method, protein purification system, protein purification service, protein purification company, etc., from the official website of Medicilon.
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