When using engineered bacteria or cells to express foreign proteins, in order to obtain high-purity proteins more quickly and reduce downstream purification pressure, it is often necessary to add tag sequences on one or both ends of the expressed proteins during protein design. As the most widely used tag, histidine tags (His-Tag) have many advantages.
His-tag protein purification principle
Immobilized metal ion affinity chromatography (IMAC) is usually used to purify His tagged proteins with histidine residues on the surface by the adsorption of metal ions chelated by medium ligands. Under general or denatured conditions (such as 8M urea), it can form coordination bonds with transition metal ions such as Ni2+ and Co2+ and selectively bind to metal ions. These metal ions can be fixed to the chromatographic medium by chelating ligands. Therefore, proteins with His tag can selectively bind to the chromatographic medium equipped with metal ions. Other impurity proteins do not bind or bind only weakly. The His tag protein bound to the medium can be eluted competitively by increasing the concentration of imidazole in the buffer to obtain a higher purity of His tag protein. Purification of His tag protein by IMAC is the most commonly used method in prokaryotic protein expression purification.
Types of histidine tag
- 4~10 series of His amino acids, commonly used is 6×His.
- His-Asn-His-Asn-His-Asn-His-Asn-His-Asn-His-Asn – his – ASN is 6×HN.
- Lys-Asp-His-Leu-Ile-His-Asn-Val-His-Lys-Glu-His-Ala-His-Ala-His-Asn-Lys, that is, HAT.
- In addition to histidine, there are cysteine (Cys) and tryptophan (Trp), which can be combined with transition metal ions such as Ni2+.
- The order of binding between transition metal ions and histidine tags is: Cu2+>Ni2+>Zn2+>Co2+.
His-tag protein purification benefits
The molecular weight of the tag is small, only ~0.84KD, which generally does not affect the function of the target protein.
His tag fusion protein can be purified in the presence of non-ionic surfactants or under denaturation conditions.
His tag fusion proteins are also used in protein-protein and protein-DNA interaction studies.
The immunogenicity of His tag is relatively low, and the purified protein can be directly injected into animals for immunization to prepare antibodies.
Can be applied to a variety of expression systems, purification conditions are mild.
The His-tag can be combined with other affinity tags to construct dual affinity tags.
BOC Sciences provides enzyme labeling of proteins, biotin labeled proteins and other protein conjugation services aimed at optimizing protein performance for research, diagnostics, and therapeutic purposes.
General procedure of His tag protein purification
Column equilibration: Try to be consistent with the buffer of the crushed bacteria. 50mM Tris or 20mM phosphate buffers containing 300mM NaCl are often used. In addition, imidazole with a final concentration of 10-20mm can be added to reduce non-specific adsorption.
Sample loading: According to the protein content of the sample and the volume of the filler used to reasonably adjust the sample loading speed, generally speaking, the sample from the entry to the filler to the outflow of the filler retention time should be more than 1min.
Impurity washing: The balance buffer can be used for cleaning, and the concentration of imidazole can also be appropriately increased on the basis of the balance buffer, and the volume of washing should not be less than 5 times the volume of the filler, and increasing the volume of washing can improve the purity of the purified protein.
Elution: Linear elution can be used, generally used to explore elution conditions; Stepwise elution can also be used to optimize elution conditions. For the sample with little heterologous protein content, it can also be directly eluted in one step.
For various types of Ni chromatographic fillers, the pH of each step of the purification process should be controlled between 7 and 8.
Related chemicals at BOC Sciences
| Classification | Product Name | CAS Number |
| Detergent | SODIUM DODECYL SULFATE | 12768-45-5 |
| Zephirol Related Compound 5 | 57-09-0 | |
| Polysorbate 20 | 9005-64-5 | |
| Nonidet P-40 | 9016-45-9 | |
| Sodiumdodecylsulfate(SDS) | 151-21-3 | |
| Triton X-100 | 9002-93-1 | |
| Affinity ligands and tags | Imidazole | 288-32-4 |
| Glutathione | 70-18-8 | |
| Glutathione S-Transferase from equine liver | 50812-37-8 | |
| Streptavidin | 9013-20-1 | |
| Avidin | 1405-69-2 |
His tag protein purification common problems
The target protein does not hang on the column
Reason: The target protein is not attached to the column mainly because of the lack of binding ability between the protein and the column.
- His tag is not fully exposed: Add an appropriate amount of urea or guanidine hydrochloride and other denaturing agents to the protein to see if the protein can be hung on the column, if it can be hung on the column, it can try to purify it under denaturing conditions, if it cannot accept the protein denaturing purification, it can change the length or position of His at the upstream molecular level, so that His is exposed to the protein surface.
- Whether His tag is lost: Sometimes sequence loss occurs during the expression of foreign protein in E. coli. If the tag is lost, the protein will naturally not be able to hang on the column. To detect His tag, His antibody can be directly used for Western blot detection.
- Whether the buffer conditions are appropriate: if there are metal ion chelators such as EDTA and citric acid in the buffer, they must be removed; In addition, appropriately increasing the buffer pH, reducing the imidazole concentration, adjusting the salt ion concentration in the buffer or changing the buffer may improve the column hanging ability of His tag protein.
- The contact time between the protein and the column is too short when purified: the contact time between the protein and the column can be appropriately extended.
- When there are target proteins in both the flow solution and eluent, it may be because the protein of the sample exceeds the load of the column, and several HisTrap columns can be used in series; Or replace bulky columns.
Protein can not elute after hanging column
Reasons: Protein can not elute after hanging column mainly because the binding ability of protein and column is too strong or the elution conditions are too mild.
(1) The elution conditions are too mild: re-explore the elution conditions, increase the concentration of imidazole in the buffer or appropriately reduce the pH of the buffer to determine the best elution conditions or change the composition of the buffer (change to Tris-HCl buffer).
(2) Protein precipitation in the column: appropriately reduce the loading amount or protein concentration, linear elution with imidazole. Use additives or change the concentration of NaCl, or eluate under denatured conditions (add 4-8 M urea or 4-6 M guanidine hydrochloride).
(3) Non-specific hydrophobic or other effects: adding non-ionic additives to the eluent (e.g. 0.2% Triton X-100) or increasing the concentration of NaCl.
(4) The contact time between protein and column is too long: the contact time between protein and column can be reduced.
Eluted protein impure
(1) The target protein is partially degraded by protease: Add appropriate protease inhibitor.
(2) Contaminants have a high affinity for nickel ions: Elution with a gradient or linear imidazole concentration to determine a suitable imidazole concentration. The same concentration of imidazole was added to the sample as in the binding buffer. If the contaminant still cannot be removed, further purification using ion exchange chromatography or molecular sieve is attempted. (3) The contaminant interacts with the target protein by increasing the concentration of the additive in the eluent (e.g., to 2% Triton X-100 or 2% Tween 20) or glycerin (up to 50%) to disrupt non-specific binding.
(4) Dual tag purification: StrepII is also a tag with a small length. If the purified protein is not pure, it can be tried to add His and StrepII tags at both ends of the protein amino acid for two-step affinity purification.
(5) Optimization of metal ions: The purity is not enough mainly because some natural proteins containing His tag in the host protein are also bound to the column, which can try to replace with Co2+ with weak binding ability, so that the impurity protein can not be bound, and thus improve the purity of the protein.
Other Blogs
Magnetic Beads (MBs) for Protein Purification
SDS-PAGE: Separation of Proteins Based on Size
People also like