I. Protein Purification Technology

The main content of protein purification refers to the use of multiple protein components to be separated between the nature of the protein differences, first of all, based on the quality of protein similarity can be further removed from other non-protein substances, and then respectively, based on the differences between the proteins can be separated and extracted out of the target protein.

II. Protein Purification Methods

1. Tag Purification Method

Tag separation and purification relative to the application of other protein purification detection methods, the technology is more mature and easier to operate, the ideal protein label should have at least the following common characteristics: (1) the tag on the separation of the basic structure of the target protease enzyme type and biochemical activity does not have an impact on the role of the label; (2) the tag is more convenient to excise; (3) the sample is best to be able to get ultrapure product after one step of purification (3) the sample should be purified in one step to obtain ultrapure product; (4) the product has a wider range of applications, which can be applied to a variety of biological target protein detection and gene expression diagnostic system respectively. Purification labels need to be selected according to the actual situation, the following table is part of the commonly used tags:

KMD provides a wide selection of tags for protein purification, including His, GST, FLAG, SUMO, and more. A range of purification products such as resins and magnetic columns are available for purification of recombinant proteins from E. coli, mammalian, yeast, and insect expression systems.

2. According to the Separation of Protein Molecular Size Difference

(1) Protein membrane ultrafiltration technology retention of protein and dialysis: ultrafiltration retention technology generally refers to the use of ultra-high pressure force field or discrete force, so that the large molecules of hydrogel particles and a smaller number of relative to the other carriers of smaller particle size of a point of solute molecules can be intercepted directly through the semi-permeable membrane without the need for large molecules of protein firmly retained in the filtration membrane surface, can be realized at will to select the use of various types of filter membrane carriers with different pore sizes and respectively retained in their different pore sizes on various molecular weight types of proteins. It is possible to choose to use various types of filter membrane carriers with different cross-sectional apertures to retain proteins of various molecular weights in their different apertures. Dialysis is the use of a semi-permeable membrane to separate proteins with different molecular sizes.

(2) Gel filtration: the volume size of the pore molecules in the gel network is certain, and only small particles of the corresponding volume size are allowed to enter into the interior of the gel particle network, and most of the particles are completely blocked. When the solution molecules flow through the chromatographic column surface, the larger solute molecules will first be deposited with the eluent together with the particles from the interstices of the particles to the inward flow, and the smaller solvent molecules will first be in the mesh structure of the laminar flow of the elution course of the length of the elution process, and then gradually be fixed by the eluent.

3. Separation of Proteins According to Their Electrically Charged Properties

(1) Electrophoresis: different kinds of protein samples in the same solution under the action of pH conditions, due to the different molecular weight of the sample size and the number of charges carried by the distribution of significant differences in the capillary electric field environment, resulting in different changes in the electron mobility of the proteins and separation of the results.

(2) lon exchange chromatography: Ion exchangers mainly include cation exchangers and strong anion exchangers, when the protein buffer to be separated flows through the surface of a certain ion exchange chromatography column, proteins with opposite charge to the ion exchanger are adsorbed on the ion exchanger, and then it can be used to appropriately change the pH value of the ions or appropriately adjust the ionic strength approach to re-adsorb the newly fixed new proteins from the surface. The immobilized new protein is eluted from the surface.

III. Commonly Used Experimental Procedures for Protein Purification

1. Ni2+ Affinity Chromatography for His-Tag Purification

Ni2+ can bind to fusion proteins with His6 tags and also to imidazole. When the tag protein impurities and the surface structure of the chromatographic column contain protein impurities and Ni2+ binding exclusion effect occurs, in which at least two completely different ratios of the same concentration of imidazole solution can be sequentially added to the automatic filtration of the tag protein impurities or impurities with other hybrid proteins are eluted accurately and fixed deposition, so that the separation of the target protein to obtain a relatively higher purity. higher purity of the target protein. The experimental procedure is as follows:

(1) Sample preparation: After the sample is broken by ultrasonic crushing, measure the protein content and determine the appropriate concentration of the solution. The sample water must be kept clear and free of white fine particles, otherwise, direct contamination may occur and block the sample column, shortening the normal service life of the equipment;

(2) Wash off about 20% of the ethanol with about 5 times the column volume of deionized water, and then add about 10 times the column volume of Binding Buffer to balance the column;

(3) Determine the sample volume according to the concentration of the target protein;

(4) Dispense about 20 times the column volume of the Washing Buffer solution and mix and wash until no protein is detected in the solution, and collect the effluent. Add about 20 times the column volume of the Elution Buffer to elute the target protein, this step can be used to set the elution gradient;

(5) Wash off the buffer with 5 times the column volume of deionized water;

(6) Store in 20% ethanol at 4°C.

2. AKTA System Gel Filtration Chromatography Experimental Steps

(1) Preparation of solutions and samples: remove the air bubbles in the working solution of all the buffers needed for purification, and filter the concentrated proteins with 0.22 µm filter membrane and store at low temperature;

(2) Power on and cleaning: Turn on the power of the computer and the instrument, wait until the white light on the control panel of the AKTATM mainframe is on, then the instrument self-test is completed, click on the UNICORN software to enter the System control interface. Cleaning A and B pump is completed automatically stops;

(3) Pre-equilibrium: After the cleaning is completed, the pump head is transferred to the equilibrium/elution buffer, and the solution used in the pipeline is replaced with the equilibrium/elution buffer until the UV, conductivity, pre-column pressure of the three baselines tends to stabilize;

(4) Loading the column: Remove the plug containing ethanol at the top, unscrew the system connection line, put the end of the drop-out solution on the top of the column to inject the buffer solution, screw it onto the column after the drop is full, and tighten the top while loosening the plug under the column to prevent the gel from collapsing due to the high column pressure. When the liquid drips from the lower end of the column, connect the other end of the system to the lower end of the column and tighten it;

(5) Equilibration: Replace all the solution in the column and system with buffer, the solution needs to go roughly 2 column volumes;

(6) Sampling: Use ultrapure water and buffer to clean the sampling ring several times;

(7) Protein collection: Choose Fraction collection→Peek fractionation, Insert→Feed tube, enter the volume to be collected per tube, Insert→Exectue, and set Fraction collection→Stop peek fractionatio→Exectue when stopping the collection. When stopping the collection, set Fraction collection→Stop peek fractionation→Exectue, and control the flow rate at 0.5~0.8 ml/min;

(8) Cleaning and unloading the chromatographic column: first use 2 column volumes of ultrapure water to clean the chromatographic column, and then switch to 1.5 column volumes of 20% ethanol to save the chromatographic column. At the end of cleaning, reduce the flow rate and seal the column in the order of "first down then up";

(9) After saving the peak graph and raw data, turn off the program, then turn off the system and finally turn off the machine.

IV. Application of Column Chromatography in Recombinant Protein Purification

The principle of column chromatography is to load the sample stationary phase into the chromatographic column, the stationary phase in the sample column is different and the adsorption capacity between the components and the sample stationary phase is different when using various solvents for selective elution of the components left on the adsorbed column, the separation adsorption and resolution of adsorption will occur automatically, and the components which have a relatively weaker adsorption bonding capacity with the sample stationary phase will be adsorbed and eluted out of the sample column first, while the components which have a relatively stronger separation and adsorption capacity will be separated and eluted out of the column in the latter phase. The components with relatively weaker adsorption binding ability with the sample stationary phase will be adsorbed and eluted out of the sample column first, while the components with relatively stronger separation and adsorption ability will be separated and eluted out of the column in the latter phase. In this way, the purpose of combining sample separation and purification is finally achieved.

Comparison of the types of chromatography used for protein separation and purification:

V. Application of Protein PurificationTechnology

1. Application to separation, purification and concentration of chemical substances;

2. Purification, recycling and utilization of useful substances in the production of recycled wastewater;

3. Concentration and purification of marine biological extracts;

4. Concentration and purification of amino acids/proteins;

5.Recycling of products in the production of ultrafine powders.

KMD has been committed to the expression and purification of recombinant proteins for many years, and can propose diversified protein purification solutions according to the different needs of customers. Affinity purification, ion exchange, molecular sieve, etc. are designed according to protein properties. Provide customized protein purification services.