Prokaryotic expression, due to various intrinsic and extrinsic factors, most of the foreign protein expression process will form insoluble forms of inclusion bodies. Due to the high yield of inclusion bodies, good stability and easy purification, inclusion body proteins have become an important way to prepare protein products on a large scale. Although inclusion bodies have such good advantages, they are not biologically active. In order to achieve this important goal, various refolding mechanisms and strategies have been developed, but often there is no universal strategy, but the nature of protein expression needs to be combined. Try multiple times to be successful.
Image courtesy of MIT The Jonathan King Lab http://web.mit.edu/king-lab/
The usual step of renaturation inclusion body is washing-dissolving-refolding: after washing to remove the impurities in the inclusion body, the high-purity recombinant protein can be obtained, and then dissolved with a strong denaturing agent. At this time, most of the high-concentration is used. The urea or guanidine hydrochloride is finally renatured by various methods, and the concentration of the strong denaturant is reduced as much as possible to obtain a recombinant protein having the correct spatial structure.
There are three classic methods commonly used for renaturation: dilution renaturation (highest use rate), dialysis refolding method, and ultrafiltration renaturation method. With the development of technology, many new methods have also been developed, such as high hydrostatic renaturation (HHP), chromatography, high pressure renaturation, reverse micellar renaturation, zeolite renaturation, multi-walled carbon nanotubes. Refolding method, three-phase separation and renaturation method, etc.
Refolding is the process of using external methods to promote the correct folding of proteins. So what affects the folding of proteins in the renaturation process? Studies have shown that the main influencing factors of renaturation success are the nature of the protein, such as isoelectric point, intramolecular disulfide bond, and secondary structure of protein.
1. The isoelectric point. The pI value of the isoelectric point of the protein is equal to the pH of the solution which is such that it dissociates into cations and anions to the same extent that the molecule is electrically neutral. When the pH of the solution in which the protein is stored is pI, the protein is electrically neutral and easily forms an unstable state. In the renaturation of conventional inclusion bodies of guanidine hydrochloride/urea dissolved, proteins with a pI value of less than 6.0 are more susceptible to successful renaturation.
2. Intramolecular disulfide bonds. If the protein is renatured to form the correct disulfide bond, the success rate of renaturation will be greatly improved. Because the reducing environment of E. coli inhibits the formation of disulfide bonds and forms a large number of mismatched disulfide bonds, proteins with disulfide bonds need to be added with redox pairs (usually GSSH/GSSG) during the renaturation process. The redox potential of the renaturation environment is controlled to promote the formation of the correct disulfide bond.
3. The secondary structure of the protein. The secondary structure of the protein also has a certain degree of influence on the renaturation process, but it is not the main factor. For example, an increase in the ratio of α-helix and β-sheet will adversely affect the recovery of renaturation, and an increase in the proportion of the corner structure will promote the effect.
In addition, environmental factors have a certain impact on the renaturation process, and it is necessary to try different combinations of different proteins.
1. The concentration of the protein. The process of renaturation can be represented by the following formula, wherein D is a denatured protein, I is a folded intermediate, N is a natural conformational protein, and A is a polymer. Reaction 1 is usually very rapid, reaction 2 is slower than the rate-limiting step of the folding process, and reaction 3 is an irreversible secondary reaction. In the renaturation process, it is necessary to reduce the denatured protein concentration as much as possible, and reduce the rate of reaction 3 as much as possible to promote The correct folding of the protein.
2. Refolding buffer pH. Usually, the pH of the refolding buffer is above 7.0, which prevents the protonation of the free mercaptan from affecting the correct pairing of disulfide bonds, and is most suitable from 8.0 to 9.0;
3. Refolding temperature. The higher the reaction temperature in the renaturation process, the faster the reaction rate, which will increase the rate of protein renaturation, and the resulting disulfide bond mismatch, the easy formation of aggregates will greatly reduce the activity and renaturation of the refolding protein. Recovery rate, so the tempering temperature is generally selected at normal temperature or lower.
4, renaturation time. In contrast, the more complex the structure, the more complex the intermolecular secondary bonds, the longer the renaturation time is required to form the correct fold.
The renaturation process generally involves the addition of some conventional materials to assist in renaturation, such as redox couples (usually GSSH/GSSG), low concentrations of urea, etc. Other types such as low concentration denaturants, amino acids, sugars, surfactants, poly-polymerization Substances such as substances, protein cofactors, and molecular chaperones can also promote the renaturation of inclusion bodies.
1. Low concentration denaturing agents such as urea and guanidine hydrochloride can effectively prevent the intermolecular hydrophobic interaction of denatured proteins, thereby inhibiting the formation of aggregates.
2. L-Arg can specifically bind to mismatched disulfide bonds and incorrect folded structures, making misfolded molecules unstable and pushing them in the correct folding direction.
3, sugars such as cyclodextrin and molecular chaperone have a similar conformation, with a hydrophobic cavity, can bind to the hydrophobic site of the denatured protein polypeptide chain to inhibit its aggregation inactivation, and promote the correct folding of the protein.
4. Surfactants can capture proteins in non-natural state to form complexes, which prevent the aggregation of proteins, thereby inhibiting the aggregation of folded intermediates, such as SDS and sulfobetaine, but SDS is only suitable for certain proteins. The renaturation of certain proteins may have an adverse effect.
5. Some polymers, such as PEG, have a fatty chain structure that promotes renaturation of proteins by binding to non-polar regions of the folded intermediate to organize the aggregation of the intermediate.
6. Cofactors of proteins such as divalent ions Zn 2+ and Cu 2+ can promote protein renaturation by stably folding intermediates.
7. Molecular chaperones are proteins that promote proteolysis by prokaryotic and eukaryotic organisms. The addition or co-expression of chaperones can promote the correct folding of exogenously expressed proteins. The molecular chaperone mainly binds to the hydrophobic region of the protein. Prevent proteins from forming aggregates and mismatching.
The choice of renaturation methods for inclusion body proteins and the selection of additives should be combined with the nature of the protein itself. For example, DTE (Dithioerythritol) may be added to a protein-refolding system containing no disulfide bond, and a redox pair such as 3 mM reduced form and 1 mM oxidized glutathione may be added to the disulfide-containing protein. When renaturation of a protein that requires a metal ion in an active state, it is necessary to avoid the addition of a chelating agent such as EDTA to the system, and it is necessary to add a corresponding metal ion. In addition, the renaturation of inclusion body proteins is a very complicated process, and some external conditions need to be gradually optimized, and many attempts can be made to obtain the best refolding yield.
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