Protein compounding (II) - in vitro complexity of gene recombined proteins.

summary
gene recombinant protein is expressed in E. coli, it is often formed as a non-biologically active encapsulation due to the high amount of expression. The encapsulation must go through the process of denaturation and complexity in order to obtain an active recombinant protein. How to improve the compound
of
recombination and protein protein is a hot research topic in bioengineering technology. This paper reviews the compounding methods of recombined proteins in recent years and provides a basis for the study of protein folding and the further application of compounding techniques.Key words
recombinative proteins contain complex desulfur bonds
So far, people have expressed more than 4000 kinds of recombined proteins, of which more than 90% are expressed with E.coli, although gene recombination technology for large-scale production of target proteins provides a new way, but people are separating Purification encountered unexpected difficulties, that is, the vast majority of these proteins in E.coli in the form of encapsulation, recombinant proteins not only can not be secreted outside the cell, but in the cells gathered into a non-biologically inactive diameter of about 0.1 to 3.0 m solid particles. Since the application of E. coli system to express genetic engineering products, people have been expecting high-activity, high-yield recombinant proteins. Insoluble, non-biologically active inclusions must be denatured and compounded in order to obtain natural structure and biological activity, so a suitable compounding process should be selected to achieve the correct folding of proteins, to obtain biological activity, recent studies can make complex hydrophobic proteins, multistructive proteins, oligopolybin, desulfur-containing bonds protein in vitro successful compounding.the causes of the formation of the encapsulation when recombined proteins are expressed at a high level in the host system, both the primary or the ceuteral expression system and even the higher ceuteral expression systems, the encapsulations are formed. Mainly because of the expression process of recombinant proteins, the lack of certain protein folding process needed enzymes and cofactors, or environmental discomfort, can not form the correct secondary bonds and other reasons for the formation of .1, the amount of expression is too high, the study found that in low expression rarely formed an inclusive body, the higher the amount of expression, the easier it is to form an inclusive body. The reason may be that the synthesis is so fast that there is not enough time to fold, the disulfur bonds are not properly paired, the nonse specific binding between too many proteins, the proteins are not soluble enough, etc.2, recombinant protein amino acid composition, generally speaking, the more sulfur amino acids more easily form an inclusion.3, recombination protein environment: fermentation temperature is high or intracellal pH close to the isoelectrine of the protein is easy to form an inclusive body.4, recombinant protein is the isogenetic protein of E. coli, due to the lack of translation and modification in the etonymical organisms required enzymes, resulting in a large number of intermediate accumulation, easy to form inclusive precipitation.5, there are reports that rich
culture
is conducive to the expression of active proteins, when the culture conditions are not good, easy to form inclusives.Strategies to reduce the formation of encapsulants 1, reduce the growth temperature of recombinant bacteria, reduce the culture temperature is the most commonly used method to reduce the formation of inclusives, lower growth temperature reduces the rate of formation of inactive aggregates and hydrophobic interactions, thereby reducing the formation of encapsulants.2, the addition of growth additives that promote the soluble expression of recombinant proteins, the addition of high concentrations of polyols, sucrose or non-metabolic sugars when cultured E.coli can prevent the secretion to the pertinate protein aggregation reaction, and the addition of these additives in the most appropriate concentration range will not affect cell growth, protein synthesis Or transport, other growth additives that promote the soluble expression of recombinant proteins are ethanol (inducing the expression of thermos shock protein), low molecular weight -based or desulfur compounds (affecting the reduced state of cellular peritonum, thereby affecting the formation of the desulfur bond) and NaCl.3, the supply of
culture
, to create the best conditions for cultivation, such as oxygen supply, pH and so on.Seconding and dissolving the encapsulation is also beneficial to the biopharmaceutical industry, not only to obtain high expression, high purity recombinant proteins, but also to avoid the destruction of recombinant proteins by cell hydrolyzed enzymes. Since the encumeration is a dense particle of protein aggregation, the first step of separation is to break the cells collected in culture, the more effective method is to combine the high pressure homogenization lysomal enzyme treatment, and then 5000 to 20000g centrifugation, can make most of the enumeration precipitation, separation from soluble protein, and then, the culvert precipitation needs to be decontamination This step is important when the agent (Triton X-100 or sodium deoxycholicate) and the low concentration denaturation agent (2mol/L urea, etc.) are washed to remove lipids and membrane proteins, which otherwise lead to degradation of recombinant proteins during the dissolution and compounding of the encapsulating body.the dissolution of the encumeration must be increased by a strong denaturation agent, such as 8mol/L urea, 6 to 8mol/L hydrochloride, which destroys the hydrogen bonds between the enshrined proteins through ion interaction. Among them, the solubility effect of urea is slightly worse, isocyanate is the strongest, detergents, such as SDS, can destroy the hydrophobic bonds in the protein, can increase the dissolution of almost all proteins, but can not be completely removed and not allowed to use in the pharmaceutical industry; For proteins containing cysteine, a reducing agent (e.g. DTT, GSH, β-ME) should be added to the dissolution to open all the two sulfur bonds in the protein, and sometimes a reducing agent should be used for proteins that do not have a desulfurization bond for the target protein. The hemoglobin of the sulfur bond affects the dissolution of the enumerating body and should also be added to the metal chelating agent, such as EDTA or EGTA, to chelate metal ions such as Cu2, Fe3 and the -base oxidation reaction in the reduced state.. The folding mechanism of proteininclusive protein under the action of denaturation agent, as soluble stretch state, when the denaturation agent is removed or the concentration is reduced, it will spontaneously change from the denatured thermal instability state to the thermodynamic stability state, forming a biologically active natural structure. However, while removing denatured agents, recombinant proteins fold in vitro, there are a large number of misfolding and polymerization between molecules, compound efficiency is often very low, the efficiency of the folding complexity of the encapsulating body protein actually depends on the competition between the correct folding process and the aggregation process. There are many different hypotheses about the folding mechanism of proteins, but many scholars believe that there is a "melting icing state" of the intermediate state, in the "melting icing state", the secondary structure of the protein has been basically formed, its spatial structure is also beginning to take shape, and then do some local adjustment can form the correct stereostructive structure, in short, the specific steps of the protein can be described in the following way. , 14): stretching state→ intermediate →second intermediate →nogeneous state→ aggregate In the folding reaction, the speed from the stretch state to the intermediate is very fast, only a few milliseconds, but the process of transitioning from intermediate to natural state is slow, is a speed limit process. The aggregation process and the compound process compete with each other, so we should try our best to avoid the aggregation. It is generally believed that the protein involves two hydrophobic effects in the compounding process, one is the hydrophobic interaction within the molecule, which can promote the correct folding of the protein, and the other is the hydrophobic interaction between the partially folded peptide chain molecules, and in the compounding process, the hydrophobic clusters of partially folded intermediates are exposed, and the hydrophobic interaction between the molecules leads to protein aggregation. Although the three-dimensional structure of protein is determined by the order of its amino acids, the process of folding the extension peptide chain into a natural active structure is also influenced by the surrounding environment, such as temperature, pH, ion strength, compound time and other factors.Methods to improve the folding complexity of recombinant proteins An effective and ideal folding compounding method should have the following characteristics: high recovery rate of active proteins, easy separation of the product of correct compounding from misfolded proteins, high concentration of protein products after folding complexity, easy amplification of folding compounding method, and less time-consuming complex process.1, dialysis, dilution and ultrafiltration compounding: these three methods are the most traditional and widely used protein folding compounding methods, compound activity recovery rate is low, and difficult to separate from hemoglobin. Dialysis is time-consuming and easy to form inactive protein aggregates, ultrafiltration is aggregation degeneration on the membrane, which is easy to cause membrane contamination, and dilution treatment is too large to be conducive to industrial amplification. 2, high protein concentration compound method: a successful compounding process is to be able to achieve a high compound rate at high protein concentration. One approach is to slowly or continuously add denatured proteins to the complex fluid. Between protein additions, there should be sufficient time intervals for the protein to fold through the easy-to-aggregate intermediate stage. This is because fully folded proteins do not usually gather with the folding proteins. The second method is to use a temperature-jumping strategy. Denatured proteins fold at low temperatures to reduce aggregation until most of the easily aggregable intermediates are converted into late intermediates that are not easily aggregation, and the temperature rises rapidly to promote the rapid folding of late intermediates into the natural composition of the protein. The third method is compounding at a moderate denaturant concentration, which should be high enough to effectively prevent aggregation, and must be low enough to trigger correct compounding. 3. Compounding method of adding promoter: The promotion effect of the compounding promoter of the inclusive protein folding compound can be divided into: stabilizing the natural structure of the correct folding protein, changing the stability of the misfolded protein, increasing the solubility of the folding compound intermediate, and increasing the solubility of the non-folding protein. Additives commonly used are: a, cosolfects: e.g. PEG6000 to 20000, through the formation of non-aggregation compounds specific to intermediates, can prevent the protein molecules from colliding with each other, reduce protein aggregation;
Surfactants
: Such as Trition X-100, CHAPs, phospholipids, sulfonate beetroits, etc. promote protein complexity, but they bind to proteins and are difficult to remove; The protein of sulfur bond should be added to the redox system in the compound process, such as GSH/GSSG, DTT/GSSG, DTE/GSSG, etc., and the redox system improves the correct pairing by promoting the rapid exchange reaction of incorrectly formed sulfur bonds. The yield of the dithione bonds, ;d small molecule additives: such as hydrochloric acid or urea, alkyl sulfonate, carbonateamide, etc., can prevent protein aggregation, their role may be: stable protein activity, Reduce the stability of non-correct folding, increase the stability of folding intermediates, and increase the stability of the defolding state. e, 0.4 to 0.6M L-Arg:L-Arg can make the incorrectly folded protein structure and the incorrectly connected desulfur bonds unstable, so that the folding in the right direction, can greatly improve the folding efficiency of the inclusive protein. f. Addition of molecular companions and folding enzymes: Molecular partners are a class of proteins that bind and stabilize the unstable composition of another protein and promote the
folding, assembly or degradation of polymers, and cross
-membrane transport of cytogenetic proteins through controlled binding and release. Folding enzymes have disulfur bond isomerase, proline isomerase, etc. Both molecular partners and folding enzymes regulate the correct folding of proteins in-body, improving the efficiency of protein synthesis. However, these proteins are removed after folding complexity and are very expensive, so it is good to use recyclable methods such as fixation. g, artificial partner: a method developed to imitate the molecular partner: First, the denatured protein is captured by the detergent in the compound solution, forming a protein-defragmenter complex, the formation of the complex inhibits protein aggregation, and then add cyclodextin to remove the detergent from the complex, so that the protein folds correctly. h, monoclonal
antibody
: antibodies to be folded compound protein can effectively assist in compounding, but only this protein can obtain obvious folding effect. I other: polyion compounds such as heparin can promote protein complexity, has the role of stabilizing natural proteins, glycerin can increase viscosity, reduce the chance of molecular collision, reduce mismatch to improve compound efficiency, moderate salt concentration can reduce the refractory between certain charged groups, is conducive to protein folding, cofactors, short-chain alcohol, high seepage, etc. can effectively reduce the formation of aggregates, protein has a stable effect. jklmn 4, liquid phase
chromatography
(LC) complex method: liquid chromatography is one of the most effective methods of purifying proteins, has become an indispensable means of gene recombinant protein purification, existing reports, hydrophobic interaction chromatography (hydrophobic interaction chromatography) HIC), Ion Exchange Chromatography (IEC), Gel Drain Chromatography (SEC), Affinity Chromatography (AFC), and Denatured Proteins have been successfully compounded. Compared with traditional dilution and dialysis methods, the advantages of liquid chromatography complexity are that denaturing agents can be removed quickly after sample, and adsorption of relatively denatured proteins due to chromatography fixation can be significantly reduced, or even completely eliminated after denaturing protein molecules are removed from the denaturing agent environment. Molecular aggregation, thus avoiding the production of precipitation, improve the quality of protein compounding and activity recovery rate, at the same time, protein complexity, can make the target protein and hemoglobin separation to achieve the purpose of purification, so that compounding and purification at the same time, easy to recover denaturing agents, reduce the cost of wastewater treatment. 4 chromatography, SEC separation effect is the worst in LC, hydrochloric acid will be retained on the IEC column, with the protein washed away, AFC use range is narrow, long time required, expensive, HIC is more ideal. The compounding of denatured proteins in HIC is as follows: When proteins, denaturants and hemagnetic proteins enter the HIC system, because the denaturing agent has a weak effect on the column, the denatured protein has a strong force, the denatured agent is first separated from the denatured protein, flowing out of the
column along with the flow.