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Application Cases and Key Points of Purifying His-tagged Proteins with Ni-based Resins


The His tag has become the most commonly used affinity tag due to its small molecular weight, strong load ability to metal ions, and the ability to maintain binding to the resin under denaturing conditions. The principle of immobilized metal ion affinity chromatography (IMAC) is that certain amino acid side chains (mainly His, with a small amount of Cys and Trp) exposed on the protein surface can undergo specific reversible interactions with transition metal ions, thereby achieving the separation and purification of proteins. The metal ions are usually Zn2+, Ni2+, Cu2+ or Co2+, and now the immobilized metal ion affinity chromatography using Ni2+ has become the standard method for purifying histidine tag proteins. The strength of the interaction between the protein/peptide and the immobilized metal ion depends on the type, number, and spatial distribution of the amino acid side chains, as well as the nature of the metal ions used.


Common Ni chelation methods:


Application Cases and Key Points of Purifying His-tagged Proteins with Ni-based Resins



Application Cases and Key Points of Purifying His-tagged Proteins with Ni-based Resins


Application of Ni resin in veterinary subunit vaccine

In genetic engineering subunit vaccine, the His-tag sequence is added to the expression vector sequence to achieve rapid chromatography purification and obtain the target protein. After fermentation and cultivation, the target protein with high purity can be obtained through one-step chromatography purification using metal chelating affinity chromatography resins material Ni Chromstar FF or Ni Chromstar Excel. Buffer exchange can be performed using Puredex G-25 M or UF/DF for formulation and seedling preparation.



Typical case: application of Bio-Link Ni resin in recombinant subunit vaccine

Cases of animal subunit vaccine


Application Cases and Key Points of Purifying His-tagged Proteins with Ni-based Resins



As shown in the figure, after the fermentation and clarification of the cell disruption, the target protein can be highly purified through one-step Ni-based affinity chromatography purification. After concentration and buffer exchange, the protein can be used for further formulation and seedling preparation.


Typical case: application of Bio-Link Ni resin in recombinant collagen

Application cases of recombinant collagen protein expressed in large intestine


Application Cases and Key Points of Purifying His-tagged Proteins with Ni-based Resins



As shown in the figure, recombinant collagen protein can be purified by one-step Ni Chromstar FF purification to obtain a highly purified protein elution solution, which is subsequently desalted and exchanged with Puredex G-25 M for ion exchange chromatography resins to obtain highly purified recombinant collagen protein.


Comparison of purification effects of different Ni chelating methods (another recombinant collagen protein)


Application Cases and Key Points of Purifying His-tagged Proteins with Ni-based Resins



1. Both Ni Chromstar FF and Ni Chromstar Excel can be used to purify recombinant collagen;

2. The elution purity of Ni Chromstar Excel is significantly higher than that of Ni Chromstar FF, with a significant improvement in the removal of impurities


In view of the above situation, Ni affinity chromatography is widely used in various recombinant protein projects, but various problems often arise during experiments. How can we solve them? Below is a summary of common problems and corresponding solutions.


01. His-tagged protein does not bind to affinity resin

① His label is not translated

Solutions:

Ø Using Western blot to detect the expression of His tag

Ø Reconstruct the target protein molecule and change the position of the inserted His tag (C-terminal or N-terminal)

② The His tag is not fully exposed

Solutions:

Ø Add a certain amount of denaturant (such as 3-8 M urea, 3-6 M guanidine hydrochloride, etc.) to the sample, and then purify it by loading it

Ø Reconstruct the target protein molecule, appropriately increase the number of histidine groups, or add a linker between the His tag and the target protein


02. Low purity of His-tagged protein after purification

① Ion exchange of the resin leads to nonspecific binding

Solutions:

Ø Add 0.5 M NaCl to the equilibration buffer, sample, and elution buffer to shield the ion exchange effect of the resin

② Miscellaneous proteins can also bind to metal ions

Solutions:

Ø Adjust the binding conditions of the sample and the resin, such as adding 10-50 mM imidazole to the sample and equilibrium buffer in advance to shield proteins that have weak binding to the resin

Ø The elution process uses a gradient elution method to explore the optimal purity conditions

Ø Try other metal ion chelating affinity chromatography resin

Ø Construct an additional protein tag on the target protein molecule, such as the GST tag, to purify the target protein in two steps, or to link other purification methods for further purification

③ His-tagged protein is degraded

Solutions:

Ø Proteases released during cell disruption may degrade the target protein, resulting in the production of small fragments with His tags, forming product-related impurities. A certain amount of protease inhibitor can be added during sample processing to prevent this.

Ø If the target protein is unstable and degrades, it is necessary to explore the optimal conditions for maintaining its stability, including temperature, buffer composition, and operating time.


03. His-tagged protein cannot be eluted

① The target protein has too strong a binding ability with the mediator

Solutions:

Ø Further increase the elution imidazole concentration, or appropriately reduce the pH value at the same time

Ø Elution is performed by shedding metal ions, which can be eluted using pH 3.5 conditions or EDTA

② The target protein precipitates in the chromatography column

Solutions:

Ø Grasp the optimal conditions for maintaining the stability of target proteins, including temperature, buffer composition, and operating time

Ø Appropriately reduce the sample size

Ø Wash with denaturant, such as 8 M urea or 6 M guanidine hydrochloride


04. Chromatography resin discoloration

① White color occurs in the resin after long-term use

Solutions:

Ø If the resin is used for a long time or a large amount of sample is used in a single use, it will cause the metal ions in the resin to fall off, resulting in the resin becoming white. This can be solved by regenerating the resin.

Ø If a large amount of target proteins is bound to the surface of the resin, it will appear slightly white during purification, and the color will return after elution, which is a normal phenomenon.

② The resin appears brown during purification

Solutions:

The resin turns brown during purification, mainly due to the influence of reducing agents in the buffer solution or sample. You can choose to remove the reducing agent component or use Ni Chromstar Excel resin.

③ The resin appears yellow during long-term use

During the use of the resin, non-specific adsorption of impurities, including proteins and pigments, will accumulate. At the same time, sedimentary proteins will also accumulate at the top of the chromatography column. After long-term use, the top of the resin will appear yellow. At this time, it is often accompanied by a decrease in resin loading, column efficiency, and column pressure. In-situ cleaning methods can be used to systematically clean the chromatography column to solve this problem and establish a reasonable maintenance method for the resin life.

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