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Protein Separation and Purification Technology Service Company

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Medicilon Protein Separation and Purification Technology Service

Sample preparation: According to customer requirements, help customers get more than 98% of samples;

Preparation process design: According to customer requirements, provide protein separation and purification services from small trials to large-scale production, and customize suitable processes and systems to customers according to process requirements and product characteristics;

Protein separation service: According to customer requirements, to undertake the purification and separation of compounds from mg to kg;

Impurity enrichment/separation: assist customers to separate trace and trace impurities in APIs or other compounds, cooperate with Shanghai Zhangjiang Pharmaceutical Valley Public Service Platform to provide a full set of compound separation and structure identification reports

Common methods of protein separation and purification

Precipitation; salting-out method, organic solvent precipitation method, protein precipitation agent, polyethylene glycol precipitation, selective precipitation method

Electrophoresis: The protein is charged in the solution above or below its isoelectric point, and can move to the positive or negative electrode of the electric field in the electric field. Depending on the support, there are thin film electrophoresis, gel electrophoresis, etc.

Dialysis: A method of separating large molecular proteins from small molecular compounds using dialysis bags.

Chromatography: a. Ion exchange chromatography, which utilizes the amphoteric free nature of proteins. At a certain pH, the charge and properties of each protein are different, so it can be separated by ion exchange chromatography. Like anion exchange chromatography, proteins with a small amount of negative charge are eluted first. b. Molecular sieve, also known as gel filtration. Small molecular proteins enter the pores for a long time, and large molecular proteins cannot enter the pores and flow out.

Ultracentrifugation: It can be used to separate and purify proteins or to determine the molecular weight of proteins. Different proteins have different densities and morphologies and are separated.

Scope of protein separation and purification services

Separation, purification and concentration of chemical substances;

Concentration and desalination of dyes and dye intermediates

Product recovery during the production of ultrafine powder;

Purification and reuse of useful substances in production wastewater;

Concentration and purification of marine biological extracts;

Concentration and purification of amino acids and proteins.

protein separation and purification services

Protein separation and purification

Protein purification tag His-Tag

Only choose the appropriate purification method to obtain high-purity recombinant protein. Many purified recombinant proteins can be used for drug screening, structural biology research, cell biology research, proteomics, and various biomedical research fields.His tag is based on the side chain of histidine residues on the protein surface, which can interact with immobilized metal ions (such as Ni ions, Zn ions, Co ions, etc.) under neutral and weakly primary conditions to purify proteins.

And it is one of the standard protein purification tags nowadays.

One 、What is His-tag

His-tag is a more commonly used tag for recombinant protein expression, whether the expressed protein is soluble or inclusion bodies can be purified by immobilized metal ion affinity chromatography (IMAC).

Metal chelation affinity chromatography, also known as Immobilized metal ion affinity chromatography (IMAC), is a novel separation technique developed in the last 30 years. It was first proposed by Paroth et al. This method uses the principle that some amino acids on the surface of proteins, such as histidine, tryptophan, and cysteine, can interact with metal ions in a specific way to separate proteins. These interactions include ligand bonding, electrostatic adsorption, and covalent bonding, among which ligand bonding is the main, and the 6-histidine tag (His-Tag) is the most widely used.

His-Tag purification tags combined with metal chelate affinity chromatography provide a powerful tool for isolating and purifying recombinant proteins. Since metal chelate affinity chromatography is characterized by simple ligands, large adsorption capacity, mild separation conditions, and high versatility, the loading conditions can be selected in a wide range. Under the high salt-specific concentration of denaturant and detergent conditions, the proteins with 6 His purification tag can specifically bind to the affinity packing, which gradually becomes one of the most effective techniques for separating and purifying proteins from other bioengineered products.

Two 、His-tag is the preferred tag for protein purification.

(1) His-Tag is very small and has no effect on protein structure after fusion protein crystallization.

(2) The N-terminal His-Tag is compatible with the bacterial transcription-translation mechanism, which facilitates protein expression.

(3) The purification of His-Tag fusion protein by IMAC (immobilized metal ion affinity chromatography) is easier to operate.

(4) His-Tag has almost no effect on the properties of the target protein itself and does not form dimers.

(5) His-Tag immunogenicity is relatively low, and the purified protein can be injected directly into animals for immunization and antibody preparation.

(6) It is constructed with other affinity tags to form bi-affinity tags and can be applied to various expression systems.

Three 、His-tag protein purification principle

His-tag can interact with various metal ions in a unique way, including Ca2+, Mg2+, Ni2+, Cu2+, Fe3+, etc. The principle is to use the properties of the protein surface so that it is adsorbed on the gel column to separate and purify the protein. Histidine (His) residues with an imidazole group can form coordination bonds with Ni2+, Co2+, and other transition metal ions and selectively bind to the metal ions, which can be fixed on the chromatography medium with chelate ligands.

So the protein with His tag can selectively bind to the medium when it passes through the chromatography medium assembled with metal ions. In contrast, other impurity proteins cannot bind or can only weakly bind. The His-tagged proteins bound to the medium can be competitively eluted by increasing the concentration of imidazole in the buffer, resulting in higher-purity His-tagged proteins.

Ni2+ is most widely used in affinity purification experiments. According to the different binding groups, Ni2+ affinity chromatography columns can be divided into Ni-IDA and Ni-NTA. NTA has a uniform and smaller particle sizes, and chelated nickel is more stable. It can tolerate higher reducing agents, making the packing more stable and the nickel ions less likely to be shed, so Ni-NTA affinity chromatography columns are chosen for protein purification.

Four 、Frequently asked questions about His-tagged protein purification.

(1) His-tagged proteins do not bind to the medium

1) Possible causes: inappropriate ultrasound power (too large, protein carbonization, too small, protein not fully released). Solution: Change the ultrasound power or other methods to break the cells.

2) The sample or buffer is not suitable. Solution: Ensure the concentration of the chelating agent, reducing agent, and imidazole in the buffer is not very high.

3) Incomplete exposure of His label.

Solution: Add denaturant (4-8M urea, 4-6M guanidine hydrochloride) to the buffer and then purify with IMAC media.

4) His label is lost. Solution: Increase the number of His, ensure correct expression, and reduce loading speed to ensure sufficient incubation time.

(2) Difficulty in elution of His-tagged protein binding on the medium

1) Possible cause: Elution conditions are too mild. Solution: Increase the concentration of eluting imidazole or lower the pH.

2) Possible causes: Protein deposition on the medium. Solution: Reduce the sample volume and optimize the chromatographic conditions.

3) Possible cause: Non-specific binding. Solution: Add 2% Triton X-100 and NaCl to the buffer.

(3) Elution peak with many impurities

Possible causes: non-specific binding, incomplete cleaning, degradation, etc. Solution: add protein inhibitor to prevent degradation during purification, wash thoroughly after sampling, and add a certain amount of NaCl and imidazole to reduce non-specific binding.

(4) use a few times, reducing the media bonding efficiency, and the column efficiency is reduced.

Possible causes: many impurities deposited on the medium, etc. Solution: thorough cleaning, IDA/IMAC de-nickel regeneration process.

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