What is the principle and application strategy of His-tagged protein purification technology?

I. What is a His-tag and its advantages in protein purification?

The His-tag is a short peptide sequence composed of 6-10 consecutive histidine residues that can be fused to the N- or C-terminus of a target protein through genetic engineering. This tag enables efficient capture and purification of fusion proteins through the specific coordination between histidine residue side chains and divalent metal ions (e.g., Ni²⁺, Co²⁺). Its core advantages are reflected in three aspects:

1. Operational simplicity: Single-step purification can be achieved under denaturing or non-denaturing conditions

2. High versatility: Suitable for various expression systems (prokaryotic, eukaryotic) and protein types

3. Excellent compatibility: Seamless integration with downstream functional studies such as enzymatic cleavage and crystallization

Notably, the introduction of a His-tag typically does not affect the biological activity or structural integrity of the target protein, which lays the foundation for its widespread application in structural biology and functional research.

II. What is the technical principle of His-tagged protein purification?

This technology is based on immobilized metal ion affinity chromatography. Its core mechanism includes:

- Coordination chemistry basis: The imidazole groups of histidine residues form coordination bonds with transition metal ions

- Chromatographic matrix composition: Consists of three parts—solid-phase carrier, chelating ligand, and metal ion

- Binding specificity: Under neutral or weakly alkaline conditions, the His-tag specifically binds to metal ions, and reversible elution is achieved by increasing imidazole concentration or lowering pH

The choice of different metal ions affects purification performance: Ni²⁺ offers higher binding capacity, Co²⁺ demonstrates better selectivity, while Cu²⁺ provides the strongest binding affinity.

III. What are the characteristics and selection criteria for major purification resins?

Based on metal ion chelation methods and matrix properties, commonly used purification resins can be categorized as follows:

Pre-chelated resins

- Nickel-based agarose resins: Employ a 90µm highly cross-linked agarose matrix pre-loaded with Ni²⁺ ions

- High-performance models: Feature alkali resistance (tolerates 1M NaOH) and EDTA tolerance (100mM), supporting direct cleaning

- Fast-flow models: Balance binding capacity and flow rate requirements, suitable for manual purification operations

- High-resolution models: 34µm particle size provides narrower peak widths and higher purity

- Cobalt-based selective resins: Utilize Co²⁺ ion chelation, exhibiting higher selectivity for His-tags and effectively reducing non-specific adsorption

Self-chelating resins

- IDA-coordinated resins: Metal ions form 6 coordination bonds, with 3 used for protein binding, offering higher capacity but relatively lower stability

- Tetradentate-coordinated resins: Metal ions form 4 coordination bonds, providing better binding stability, suitable for harsh purification conditions

Resin selection should comprehensively consider target protein characteristics, purity requirements, scale, and cost-effectiveness.

IV. How to optimize the His-tagged protein purification process?

A complete purification process should include the following key steps:

Sample preparation phase

- Lysis buffer should contain 20-50mM imidazole to reduce non-specific binding

- Add protease inhibitors to prevent protein degradation

- Optimize pH to 7.0-8.0 to maintain metal ion stability

Chromatography purification phase

- Equilibration: Use binding buffer containing 20-50mM imidazole

- Loading: Control flow rate to ensure sufficient contact time

- Washing: Gradually increase imidazole concentration to 50-100mM

- Elution: Use elution buffer containing 150-500mM imidazole

Post-processing phase

- Desalting to remove high-concentration imidazole

- Enzymatic cleavage to remove His-tag (e.g., using TEV protease)

- Gel filtration chromatography for final polishing

V. What are the typical applications of His-tagged protein purification in biomedical research?

Using novel coronavirus research as an example, its specific application value is demonstrated:

Spike protein RBD domain purification

- Expression optimization: Adding arginine and proline to the medium significantly increases expression yield

- Purification strategy: Sequential nickel affinity chromatography and gel filtration chromatography

- Result validation: Obtains high-purity monomeric RBD protein suitable for structural resolution and functional studies

Antibody fragment preparation

- Expression system: Uses Brevibacillus expression system

- Purification scheme: Combines nickel affinity chromatography with size-exclusion chromatography

- Quality assessment: SDS-PAGE shows high-purity Fab fragments obtained

These application cases demonstrate that His-tag purification technology has become a key platform technology for recombinant protein production, playing an irreplaceable role in vaccine development, antibody engineering, and structural biology.

VI. What are the future technological trends?

With the increasing demand for precision medicine, His-tag purification technology is developing in the following directions:

- Intelligentization: Integrating online monitoring and automatic control to achieve precise regulation of purification processes

- High-throughput: Developing microcolumn arrays and parallel processing systems to meet omics research needs

- Greenification: Developing reusable resins and environmentally friendly elution solutions

- Integration: Combining with downstream analytical technologies to build complete protein research platforms

Through continuous technological innovation and method optimization, His-tag purification technology will demonstrate greater application value in biomedical research.

VII. Which manufacturers provide His-tag antibodies?

Hangzhou Start Biotech Co., Ltd. has independently developed the "His-tag Recombinant Rabbit Monoclonal Antibody" (product name: His tag Recombinant Rabbit mAb (S-1398-151), catalog number: S0B1838), a recombinant protein detection and purification tool with high specificity, high affinity, and excellent stability. This product was developed using recombinant rabbit monoclonal antibody technology and has been rigorously validated across multiple technical platforms including Western Blot (WB), immunofluorescence (IF), immunohistochemistry (IHC), and ELISA. It has broad application value in recombinant protein identification, protein expression analysis, and protein purification.

Professional Technical Support: We provide detailed product technical documentation, including validation data across multiple application platforms, recommended experimental conditions, and professional technical support, fully assisting customers in obtaining accurate and reliable results in protein science research.

Hangzhou Start Biotech Co., Ltd. is committed to providing high-quality, high-value biological reagents and solutions to global innovative pharmaceutical companies and research institutions. For more information about the "His-tag Recombinant Rabbit Monoclonal Antibody" (catalog number S0B1838) or to request sample testing, please contact us.

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