I. Protein Purification Techniques

    Protein purification primarily involves exploiting the differences in properties among the various protein components to be separated. First, based on the mass similarity of the proteins, non-protein substances can be removed; then, by capitalizing on the differences between the proteins, the target protein can be isolated and extracted.

II. Protein Purification Methods

1. Tag-Based Purification

    Compared to other protein purification and detection methods, tag-based separation and purification is a relatively mature and easy-to-implement technique. An ideal protein tag should possess at least the following common characteristics: (1) The tag has virtually no effect on the basic structural type or biochemical activity of the target protein; (2) The tag can be easily cleaved off; (3) Ideally, a single-step purification process should yield a highly purified product; (4) The product has a broader range of applications and can be used for the detection of various biological target proteins and in gene expression diagnostic systems. The choice of purification tag should be based on specific circumstances. The table below lists some commonly used tags:

KMD Bioscience offers a wide range of tags for protein purification, including His, GST, FLAG, SUMO, and others. We provide a variety of purification products, such as resins and magnetic columns, for purifying recombinant proteins from E. coli, mammalian, yeast, and insect expression systems.

2. Separation Based on Differences in Protein Molecular Size

(1) Protein Membrane Ultrafiltration Technology for Protein Retention and Dialysis: Ultrafiltration retention technology generally refers to the use of ultra-high pressure fields or centrifugal forces to allow macromolecular hydrogel particles and a small amount of solute molecules—which are slightly smaller than other carrier particles—to pass directly through a semipermeable membrane, without the need to firmly retain macromolecular proteins on the surface of the filter membrane. allowing for the flexible selection of various filter membrane carriers with different pore sizes to retain proteins of specific molecular weights based on their respective pore sizes. Dialysis uses a semipermeable membrane to separate proteins of different molecular sizes.

(2) Gel filtration: The pore volume in a gel network is fixed, allowing only small particles of a corresponding volume to enter the interior of the gel particle network, while the majority of particles are completely excluded. As the solution flows through the chromatographic column, larger solute molecules flow inward through the gaps between the particles along with the eluent and are deposited first, while smaller solute molecules travel farther within the laminar flow of the gel network and are gradually retained by the eluent.

3. Separation Based on the Charge Properties of Proteins

(1) Electrophoresis: Under the same solution pH conditions, different types of protein samples are separated based on variations in their electrophoretic mobility within a capillary electric field, resulting from differences in molecular weight and significant variations in the distribution of their charges.

(2) Ion-Exchange Chromatography: Ion exchange resins primarily include cation exchange resins and strong anion exchange resins. When the protein buffer solution to be separated flows over the surface of an ion exchange chromatography column, proteins carrying a charge opposite to that of the ion exchange resin are adsorbed onto the resin. Subsequently, the newly adsorbed and immobilized proteins can be eluted from the surface by appropriately adjusting the pH of the eluent or modifying the ionic strength.

III. Common Protein Purification Experimental Workflows

1. Ni²⁺ Affinity Chromatography for Purification of His-Tag Proteins

Ni²⁺ can bind to fusion proteins bearing a His6 tag, as well as to imidazole. When labeled protein impurities interact with protein impurities present on the surface of the chromatography column and with Ni²⁺, causing a repulsive effect, sequentially adding at least two completely different proportions of imidazole solutions of the same concentration to the system allows for the precise elution of all these labeled protein impurities or impurities from other heterologous proteins through automatic filtration. These impurities are then precipitated and removed, thereby enabling the isolation of the target protein with relatively higher purity. The experimental procedure is as follows:

(1) Sample Preparation: After ultrasonic disruption of the sample, measure the protein content to determine the appropriate solution concentration. The sample solution must remain clear and free of fine white particles; otherwise, direct contamination may occur, leading to clogging of the sample column and shortening the normal service life of the equipment;

(2) First, use approximately 5 times the column volume of deionized water to wash out about 20% of the ethanol, then sequentially add Binding Buffer at a ratio of approximately 10 times the column volume to equilibrate the column;

(3) Determine the loading volume based on the concentration of the target protein;

(4) Dispense approximately 20 times the column volume of Washing Buffer and wash until no protein is detected in the solution; collect the eluate. Then, add approximately 20 column volumes of Elution Buffer to elute the target protein; this step can be used to set up an elution gradient;

(5) Wash away the buffer with 5 column volumes of deionized water;

(6) Store in 20% ethanol at 4°C.

2. AKTA System Gel Filtration Chromatography Procedure

(1) Preparation of Solutions and Samples: All buffers required for purification must be degassed. Concentrated proteins must be filtered through a 0.22 µm filter membrane and stored at low temperature;

(2) Power-Up and Flushing: Turn on the computer and the instrument. Once the white light on the AKTA™ main unit control panel remains steady, indicating that the self-test is complete, click the UNICORN software to enter the System Control interface. The cleaning of Pumps A and B will stop automatically upon completion;

(3) Pre-equilibration: After cleaning is complete, transfer the pump head to the equilibration/elution buffer and replace the solution in all tubing with the equilibration/elution buffer until the UV, conductivity, and pre-column pressure baselines stabilize;

(4) Column Installation: Remove the top cap containing ethanol, unscrew the system connection line, place the end dripping with solution on top of the column to inject the buffer solution, and once it is full, screw it onto the column. While tightening the top cap, loosen the bottom plug to prevent the gel from collapsing due to high column pressure. When liquid begins to drip from the bottom of the column, connect the other end of the system to the bottom of the column and tighten it;

(5) Equilibration: Replace all the solution inside the column and in the system with buffer; approximately two column volumes of solution are required;

(6) Sample loading: Wash the sample loop multiple times with ultrapure water and buffer;

(7) Protein collection: Select “Fraction Collection” → “Peak Fractionation,” then “Insert” → “Feed Tube,” enter the collection volume per tube, and select “Insert” → “Execute.” To stop collection, select “Fraction Collection” → “Stop Peak Fractionation” → “Execute.” Maintain a flow rate of 0.5–0.8 mL/min;

(8) Wash and remove the chromatography column: First, wash the column with 2 column volumes of ultrapure water, then switch to 1.5 column volumes of 20% ethanol to preserve the column. After washing, reduce the flow rate and seal the column in the order of “bottom first, top last”;

(9) Save the peak profile and raw data, then close the software, shut down the system, and finally power off the instrument.

IV. Application of Column Chromatography in the Purification of Recombinant Proteins

    The principle of column chromatography involves loading a sample onto a stationary phase within a chromatographic column. Since the stationary phases in different columns vary, and the adsorption capacities of the components differ relative to the sample’s stationary phase, when various solvents are used to selectively elute the components retained on the column, separation, adsorption, and desorption occur automatically. Components with relatively weaker adsorption capacity to the sample’s stationary phase will be adsorbed and eluted from the column first, while components with relatively stronger adsorption capacity will be separated and eluted from the column later. This ultimately achieves the combined goal of sample separation and purification.

Comparison of Chromatography Methods Used in Protein Separation and Purification:

V. Applications of Protein Purification Technologies

1. Separation, purification, and concentration of chemical substances; 2. Purification and recovery of useful substances from production wastewater; 3. Concentration and purification of marine biological extracts; 4. Concentration and purification of amino acids/proteins; 5. Product recovery during the production of ultrafine powders.

    For many years, KMD Bioscience has been dedicated to research on the expression and purification of recombinant proteins and can propose diverse protein purification solutions tailored to customers’ specific needs. Based on the properties of the protein, we design affinity purification, ion exchange, and molecular sieve-based methods to provide customized protein purification services.