Solubilization and replication experiments of inclusion body proteins
Solubilization and replication experiments of inclusion body proteins
Although inclusion bodies are mixtures of unnatural forms of expressed proteins, the peptide chain itself is intact, or the primary structure is correct. One advantage of purifying expressed proteins from inclusion bodies is that inclusion bodies are easily separated from other components of the cell. Normal aqueous solutions have difficulty solubilizing inclusion bodies, and they only dissolve well in solutions of denaturants (e.g., guanidine hydrochloride, urea). At this point, the solubilized inclusion body protein obtains complete denaturation, that is, except for the primary structure and covalent bonds are retained, all the hydrogen bonds and hydrophobic bonds are destroyed, and the hydrophobic side chains are completely exposed. Then, under certain conditions, the denaturant is removed to induce the product protein to complete the correct protein folding process and obtain the natural structure, which is called denaturation. Protein denaturation is a very complex process, there are two main methods currently used. One is to dilute the solution, so that the concentration of denaturant in the solution is gradually reduced, and the protein begins to revert. This method is simple and easy to implement, but the operation of the volume of liquid volume is sometimes too large, but also reduces the concentration of protein. Another is dialysis, ultrafiltration or electrodialysis to remove denaturant. Dialysis is often used in the laboratory, the solution to water or buffer dialysis, denaturant diffusion through the membrane concentration gradually decreased, the protein to obtain the restitution. The total volume of liquid and protein concentration do not change much with this method, but it takes longer and sometimes a protein precipitate is formed. Source Handbook of Protein Technology
Operation method
basic program
Principle
The cause of inclusion body formation is complex and is still not fully understood. Studies have shown that its formation is not caused by the concentration of the expressed protein exceeding the solubility, and the mismatch of disulfide bonds within the chain is not the main reason for its formation. The dominant view is that the expressed exogenous protein lacks certain cofactors or because the local microenvironment is not suitable, it cannot form secondary bonds correctly and continuously, and finally forms a natural three-dimensional structure through multiple intermediate folders. Inclusion bodies are probably the insoluble material formed by the accumulation of inappropriate intermediate folding bodies of the expressed protein in high concentration. They cannot be further folded correctly into a natural structure, but rather are a mixture of unnatural forms of the expressed protein. Inclusion bodies can sometimes be directly observed under a phase contrast microscope and are located at the ends of E. coli and appear as dark-colored refractive dots of about 0.5 to 1 um. They are easy to observe and identify by electron microscopy.
Materials and Instruments
Cell Move 1. Inclusion determination Firstly, it is necessary to check whether there is a high level of expression of product proteins in the total cell extract; secondly, it is necessary to check whether the high level of expression of product proteins exists mainly in the insoluble form; finally, it is necessary to determine whether the product proteins accumulate to form inclusion bodies. Frequently used methods include electrophoresis, immunoblotting, phase contrast microscopy or electron microscopy observation. 2. Cell lysis 2.1 Select the working buffer for cell lysis, the common common buffer in the laboratory is: 0.05 mol/L, pH 7.0 phosphate buffer, containing 0.05 mol/L NaCl and 1 mol/L EDTA. Suspend the cells in the appropriate amount of buffer. 2.2 Lysis of cells by sonication, glass bead milling, or lysozyme treatment is optional. It is recommended that a protease inhibitor be added to this step as appropriate. 3. Inclusion body isolation 3.1 Centrifuge the cell lysate at 12 000 × g 4°C for 5 min. 3.2 Suspend the precipitate in a 9-fold volume of working buffer containing 0.5% Triton X-100. Incubate for 5 min at room temperature. 3.3 Centrifuge again at 12000 × g 4°C for 5 min. 4. 4. Inclusion body lysis 4.1 Suspend each gram of precipitate in 9 ml of working buffer containing 8 mol/L urea, 2 mmol/L reduced glutathione and 0.2 mmol/L oxidized glutathione. Please note that the protein concentration should not exceed 2.5 mg/ml, as high concentrations are detrimental to the formation of natural structures. 4.2 Keep the solution at room temperature for 60 min. 5. Rehabilitation 5.1 Slowly add 9 ml of working buffer containing 2 mmol/L reduced glutathione and 0.2 mmol/L oxidized glutathione per ml of urea-protein solution. 5.2 Incubate for 2-4 h at room temperature. Caveat 1. Always test for soluble product proteins in the supernatant of the lysate. Sometimes, despite the formation of inclusion bodies, a certain amount of soluble product proteins are still present. 2. To monitor the extent of cell lysis, the lysate should be examined by phase contrast microscopy. Further lysis is required if intact cells will precipitate with the inclusion bodies, thereby reducing the purity of the inclusion body proteins. 3. 6 mmol/L guanidine hydrochloride is often used instead of 8 mol/L urea to lyse inclusion bodies. Occasionally, 1% SDS is used to dissolve the inclusion bodies, but this is more difficult to remove in later steps. 4. The transformation of disulfide bonds during denaturation can also be accomplished by dissolving the protein at alkaline conditions (pH 10.7) and titrating to pH 8.0 with hydrochloric acid without the use of reducing agents such as glutathione, oxidizing agents, etc. 5. Restoration can also be achieved by adding chaperone molecules, ligands, substrates or cofactors, etc., which may promote close association with the natural structure. They may promote the stabilization of folding intermediates that are closely related to the natural structure. 6. Different inclusion body proteins have different optimal complexation pathways and sometimes chemical modification of the side chain or addition of protein precipitating agents can also help to complete the complexation. For more product details, please visit Aladdin Scientific website.
Cell lysate Working buffer
Sonicator or glass bead mill