Protein purification experiments
["Collaborating Expert | M.S. Hanyin Sun", "Biology and Pharmaceutical Sciences University of Chinese Academy of Sciences"], ["Reviewed by | Dr. Xu Kong", "Biology Xiamen University"]
Summary
His-Tag is the most common tag used in the purification of recombinant protein expression and can be used for initial purification of most proteins (soluble or inclusion bodies).
Principle
Ni2+ is able to chelate the ligand to fill and immobilize the chromatography medium. Histidine (His) carries an imidazole group, which is able to form a ligand bond with Ni2+ and selectively binds to it, while proteins that do not carry His residues are unable to form a ligand bond with Ni2+ and bind to it, which effectively differentiates the target proteins (proteins that have a His-Tag) from the nondestructive proteins.
Subsequently, the target protein is eluted and collected by a high concentration of imidazole buffer, which competes with the His-Tag for the binding of Ni2+, thus obtaining a pure target protein.
Appliance
Purification of His-tagged target proteins.
Operation method
Protein purification through nickel columns
Principle
Ni2+ can chelate the ligand to fill and immobilize on the chromatography medium. Histidine (His) carries an imidazole group, which can form a ligand bond with Ni2+ and bind selectively, while proteins without His residue cannot form a ligand bond with Ni2+ and bind to Ni2+, which can effectively differentiate the target proteins (proteins with His-Tag) from the non-target proteins. Subsequently, the target protein is eluted and collected by a high concentration of imidazole buffer, which competes with the His-Tag for Ni2+ binding, thus obtaining a pure target protein.
Materials and Instruments
Ni2+ affinity column, 0.2 M NiSO4 solution, 100 mM EDTA solution, 0.2 M NaOH solution, ddH2O, 0.22 μm filter membrane
Binding buffer: low concentration of imidazole buffer.
Elution buffer: high-concentration imidazole buffer, etc.
Move
1、Rebirth of Ni2+ Affinity Columns
1) Connect the Ni2+ affinity column to the pump head and flush 5 column volumes with ddH2O.
(2) Subsequently replaced by EDTA solution flushing 5 column volumes, using ddH2O flushing 5 column volumes.
3) Flush 5 column volumes with 0.2 M NaOH solution and flush 10 column volumes with ddH2O.
4) Fill the column with NiSO4 solution, rinse 5 column volumes, and flush 5 column volumes with ddH2O.
The above Ni2+ affinity column regeneration is completed.
2、Equilibration of Ni2+ affinity column
Connect the Ni2+ affinity column to the pump head and rinse 5 column volumes with low concentration imidazolium salt buffer.
3、Binding of target protein with His-Tag to Ni2+ affinity column
(1) Put the protein-expressing bacterial liquid into a high-speed centrifuge and centrifuge it at 5000 rpm, 4 °C for 10 minutes, collect the bacterial mass, and freeze it at -20 ℃ or -80 ℃ for spare use.
2) Resuspend the clusters with low concentration of imidazole buffer.
3) Under the condition of ice bath, use pressure crusher or ultrasonic crusher to release the expressed proteins, the ultrasonic crusher is different and needs to optimize the experimental conditions. If the bacterial liquid is still turbid after sonication, the power should be increased appropriately and the sonication time should be prolonged.
4) Place the suspension in an ultra-high-speed centrifuge at 4 °C, 18000 rpm for 25 minutes, and separate the upper layer of protein clear liquid and precipitate.
5) SDS-PAGE electrophoresis: Determine whether the protein is soluble (supernatant) or inclusion body (precipitate) expression.
6) A Ni2+ affinity column is attached to the pump head and the protein lysate is passed through the Ni2+ affinity column at 4 °C at a slow and constant rate.
7) Use a low concentration of imidazole (10 mM imidazole) to flow 2~5 mL/min through the Ni2+ affinity column, about 5 column volumes.
8) Stage elution was performed with 50, 100, 200, 300, 400 mM imidazole buffer at a flow rate of 2~5 mL/min, and the elution peaks of each stage were collected, with each concentration of imidazole buffer flowing through 2 column volumes.
9) Collect the protein samples and test the molecular weight and purity of the fusion protein by SDS-PAGE.
10) Flush the Ni2+ affinity column with ddH2O for 5 column volumes, and then rinse the Ni2+ affinity column with 20% ethanol for 3 column volumes at a flow rate of 2~5 mL/min, and the column was always kept in a low temperature environment.
(11) Concentrate the high purity protein obtained by SDS-PAGE, and carry out the buffer replacement and the next purification experiment in a short time.
Caveat
1. When designing recombinant proteins, it is usually recommended to add 6 His-Tag at the C-terminal or N-terminal to ensure that the expression plasmid is successfully constructed and the concentration of IPTG induced recombinant expression is appropriate, so that we can carry out the formal experiment after designing the pre-experiment judgment.
2. The molecular weight of His-Tag is so small that it hardly affects the high-level structure of the target protein.
3. His-Tag is often designed at the N-terminus, so that it is more compatible with the transcription mechanism of bacteria.
4. ÄKTA can be used if the laboratory conditions are good.™ÄKTA TM system can be used for automatic protein purification if laboratory conditions are favorable. 5.
5. All buffers used should be passed through a 0.22 μm membrane. 6.
6. Proteins stored in high concentrations of imidazolium salts tend to become distorted and the next step should be carried out as soon as possible or a buffer change should be performed.
7. Various buffer formulations are available, but PBS pH 7.4 with various concentrations of imidazolium salts is commonly used.
8. If you do not have an ultra-high speed centrifuge in your lab, you can use a 0.45 μm membrane to filter the expressed protein suspension from the Pressure Bacteriostat or Ultrasonic Bacteriostat.
9. When the column is not used for a long period of time, the internal Ni2+The columns should be eluted and stored in a 20% ethanol solution to prevent microbial growth. 10.
10. Each time the column is filled with Ni2+column should be regenerated after three uses to improve purification efficiency. 11.
11. The design should be pre-determined to determine whether the protein is soluble (supernatant) or inclusion body (precipitate) expressed.
Common Problems
1. Protein does not hang on the column
(1) First of all, determine whether the target protein has good water solubility, if the target protein has poor water solubility, it is recommended to add MBP label to increase the water solubility of protein.
(2) Maybe the protein is not fully released in the process of bacterial cleavage, you can increase the pressure, ultrasonic power or time to break the bacteria and add the right amount of lysozyme.
(3) The upper layer of protein clear liquid should be flowed through the Ni2+ affinity column at a slower flow rate, and the column can be looped 2~3 times.
(4) His-Tag is not fully exposed, 1% glycerol or 2~5 mM DTT/β-mercaptoethanol can be added during the lysis process to fully expose His-Tag.
2. Failure to elute the target protein
Increase the concentration of Ni2+ in the eluent, or increase the concentration of NaCl and nonionic detergent. Note that high concentrations of NaCl may cause denaturation of the protein.
3. More heterogeneous protein bands are found during SDS-PAGE.
1) Increase the imidazole concentration of the low concentration imidazole buffer.
2) Use multi-tag multi-step purification, such as adding MBP-Tag tag can be further purified using MBP affinity column. Or, according to the isoelectric point of the protein, use HP/Q column for purification, and finally, the molecular weight of the protein can be differentiated by the molecular weight of the protein using SEC columns.
4. Air in the column during flushing.
Hang the column upside down on the pump head and flush 10-20 column volumes with binding buffer or ddH2O at a fast flow rate to evacuate all the gas.
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