Protein complexity (v) - Protein complexity.

key
are:
protein
complexity, cyclodext, molecular partnerrecombination DNA technology for large-scale production of target proteins provides a new way. But people encounter unexpected difficulties in isolating purified genetically engineered expression products: many exogenetic gene expression products that use E. coli as the host cell, such as urine kinase, human insulin, human growth
monthrogen
, lebuline-ion 6, human γ-interferon, etc., not only can not be secreted outside the cell, but in the cell gathered into a non-biologically inactive diameter of about 0.1 to 3.0 m of solid particles in-containing body. Although the first structure (i.e. amino acid sequence) of these gene expression products is correct, its stereoscopic structure is wrong, so there is no biological activity. Therefore, in order to obtain the target product of natural state, it is necessary to dissolve the inclusion after separating the recovered inclusion and try to restore the natural composition and activity of the target protein in it. This has put forward
new topic to
the protein folding mechanism of biochemistry and biochemistry..1. Protein compound mechanism researchthe operation steps of separating the containing and compounding proteins are not complicated, starting with the broken cells, then centrifuging the cells, recovering the inclusions, adding the denatured agent dissolves the inclusions, making them soluble extensions, and then removing the denaturation agent to make the expression product fold to restore natural composition and activity. However, in the actual study, it is found that when folding in vitro, the compounding efficiency is often very low due to the large number of misfolding and polymerization between protein molecules. The reason is that although the three-dimensional structure of protein is determined by its amino acid sequence, the process of stretching peptide chains into natural active structure is also affected by the surrounding environment.in order to develop the technology of auxiliary protein compounding, researchers have carried out a discussion on protein folding mechanism. At present, there are two different hypotheses: one hypothesis is that the local peptide segment in the peptide chain first forms some formation units, such as α helix, β folding, β corner and other secondary structures, and then by the combination and arrangement of the secondary structural units to form a protein three-stage structure; Although different hypotheses, many scholars believe that there is a so-called 'melting icing' intermediate state. In the molten speral state, the secondary structure of protein has been basically formed, and its overall spatial structure has begun to take shape. After that, the molecular stereostructs do some local adjustment, and finally form the correct three-dimensional structure. In summary, the specific steps of protein folding can be described below: U→I→N


i.e. the stretch state U changes from an early change to intermediate I, and then transitions from the intermediate to the final natural state N. But while folding from the intermediates into a natural state, there is another bypass, where the intermediates congreide with each other as a condensed body A (containing body). In a folding reaction, the formation from the stretch state to the intermediate is very fast and is usually done in milliseconds, but the process of transitioning from the intermediate to the natural state is slow and is a speed limit step of the reaction. When the concentration of ion strength or denaturation agent in the solution is very low, and no other auxiliary means exist, the aggregation trend dominates, resulting in the protein's spontaneous compounding efficiency is very low.it is generally believed that proteins involve two hydrophobic interactions in the complex process, one is hydrophobic interaction within the molecule and the other is the hydrophobic interaction between partially folded peptide chain molecules. The former causes the protein to fold correctly, while the latter causes the protein to gather and be inactive, which competes with each other and affects the protein's re-reactive yield. Therefore, in the compound process, inhibiting hydrophobic interaction between peptide chains to prevent aggregation is the key to improve the recovery rate..2. The initial results of protein compounding researchextensive protein compounding research was carried out in the late 1980s. For example, Carlson and others have found that monoclonal
antibody
has the effect of assisting protein compounding, Cleland, etc., has found that adding an appropriate concentration of polyglycol to diluted denatured protein solution inhibits coagulation precipitation during protein compounding, and triples the recovery rate of protein compounding, and Hagen, etc., uses anti-gel groups to extract artificially denatured proteins to remove denatured agents for compounding. The compounding rate can be up to 100% (but the extraction rate is very low due to the presence of denaturing agents), the molecular sieve action of the gel
-filtering
chromatography
medium, etc., prevents contact and condensation between denatured proteins and assists in their proper folding, and Zardeneta, etc., uses detergents and mixed gels to successfully assist in the compounding of pyrethromatase..3. Study on the complexity of cyclodextrem and straight-chain deptextrete-assisted proteins In 1995, Karuppiah and Sharma published an article describing the complexity of carbonate-assisted bicarbonase B using cyclodextide. Cyclodext is produced by starch degradation through cyclodext glucose-based
-transferase
and is a ring-shaped lysosaccharide that binds the D-glucose unit with α-1,4-glycoside bonds to form each other as a chair-like structure, and its molecules usually contain 6 to 12 mutter glucose units. It is useful to have α, β, γ-cyclodextyclin containing 6, 7, and 8 glucose units, but the α-cyclodext cavity is smaller, γ-cyclodext is expensive, and is commonly used as β-cyclodext. Cyclodextor is characterized by the able formation of envelope compounds, in which the object molecule enters its molecular cavity from the wide end. The formation of envelopes mainly depends on non-co-priced key interactions such as Van der Wali, hydrogen bonds, hydrophobic interactions, geometric shape matching, etc. The hydrophobic bits of the denatured protein
peptide
chain can be combined with the hydrophobic cavity of cyclodext, which can inhibit their mutual aggregation ineration, thus promoting the proper folding of the peptide chain into active proteins.1999, Sundari and others reported on the compounding of insulin, carbonate and egg white lysomal enzymes with straight-chain dexterin, and found that straight-chain dexterity basically simulates the role of cyclodextin in assisted protein complexity. And has some advantages: straight chain dexterity spiral structure to form a hydrophobic empty tube, can combine more protein molecules; Research4. Molecular partner-assisted protein complexityrecently, more and more research on protein folding has turned to the use of molecular partner GroE family. Some scholars have successfully used molecular partners to aid protein complexity in the body and in vitro. However, molecular partners in practical applications still have high costs and need to be separated from compound proteins and other shortcomings, so research and development of molecular partners reuse and stability is the key to achieve their application.. GroEL has the effect of binding proteins, equivalent to the affinity of denatured proteins. The fixed GroEL column (fixed bed) is equivalent to the affinity adsorption column of the denatured protein, which increases the processing volume of the sample and concentrates and purifys the protein while compounding. Its characteristics are: (1) not only can solve the problem of the reuse of molecular partners, but also because of the use of fixed beds (nearly flat-push), can improve the efficiency of molecular partners, (2) due to the use of continuous operation, raw material processing volume, the product can be concentrated, purified, (3) easy to establish relevant models, for enlarged production has an important guiding role. Teshima and others assisted in vitro with the folding complexity of amylase, carbonatease, and DNA enzyme by using a fixed molecular partner. also reported the use of "small molecular partners" to compound target proteins. "Small molecular companion" refers to a fragment of GroEL that is not GroEL's hydrolysis product, but an expression product of a gene fragment in E. coli that, after genetic recombination, includes GroEL 191-345 amino acid residue fragments (16.7kD) or 191-376 amino acid residue fragments (21kD). They can effectively promote the duplication of procyclic protein A, thiocyanate enzymes, and RNA enzymes of Bacillus spores. Because the "small molecular companion" molecule is small, more suitable for fixation, and does not need to add compound cofactors (such as GroES and ATP, etc.), it has great application prospects. . 5. Application of artificial molecular companions in protein complexity inspirated by protein molecular partner-assisted protein complexity, Rozema and Gellman studied the assisted carbonate and egg white lysase complexity of artificial molecular companion systems (detergents and cyclodext). Similar to the molecular partner GroEL-ATP-assisted compounding mechanism, the compounding process is carried out in two steps: the first stage of capture, the addition of detergents to the denatured protein solution, the detergent molecules through hydrophobic interactions with the protein's hydrophobic point binding to form a complex, inhibit the peptide chain Mutual aggregation between each other; the second stage of stripping, the addition of cyclodextrein molecules on the detergent molecules competitive adsorption, detergent molecules are stripped down, so that the peptide chain in the process properly folded into the active protein. Its reaction mechanism can be expressed as follows: U→U-dn→→→U-dn-m→→→F→N i.e. the stretching state U first forms a compound U-dn with decontamination agent d, after adding cyclodext, the small molecule desomination agent is peeled off one by one, and becomes partially folded, non-decontamination agent molecule inactive state F, and then folded into the natural active protein N. Compared with protein molecular partners such as GroE, the combined use of detergents and cyclodext as artificial molecular partners auxiliary protein compounding has obvious advantages: (1) artificial molecular partners are not proteins, not easily affected by environmental influences and insulation, operating conditions are more relaxed; . . Bio-separation Project, Beijing: Chemical Industry Press, 1998, 23-24
. Wang Keyyi. Chemistry of Life, 1999, 19 (19) (5): 233-235
, Goldberg ME ET. Biochemistry, 1991, 30:2790-2797
. Carlson JD et al. Bio/Technol, 1992, 10:86-91
Cleland JL. J Biol Chem, 1992, 267:13327-13334
Hagen AJ. Biotechnol Bioeng, 1990, 35:955-965
. Biotechnol Bioeng, 1996, 50:16-23
. J Biol Chem, 1992, 267 (9): 5811-5816
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochem Biophys Res Commun, 1995, 211 (1): 60-66
, etc. Analytical Chemistry, 1996, 24 (12): 1387-1390
. Sundari CS et al. FEBS Letters, 1999, 443:215-219
. J Biol Chem, 1995, 270 (37): 21517-21523
. J Biol Chem, 1997, 121:331-337
. Appl Microbiol Biotech, 1997, 48:41-46
. Proc Natl Acad Sci USA, 1996, 93:15024-15029
.16. taguchi H et al. J Biol Chem, 1994, 269 (11): 8529-8534
8534 rozema D et al. J Biol Chem, 1996, 271:3478-3487
18 Rozema D et al. Biochemistry, 1996, 35:15760-15771 (this article originally appeared in The Chemistry of Life No. 6 of 2000) .