96 results about "Protein Renaturation" patented technology

The invention relates to silk property-variable non-woven cotton with sericin and relative application. Said silk property-variable non-woven cotton via selection-modifying-blanching, and washing-carding-needling processes can be made into the silk property-variable non-woven cotton which will not lose silk. Said non-woven cotton can recover water-solubility and biological activity via protein recover treatment to supply the health function of silk protein, and apply the medical, hairdressing and daily consumption areas. Since said invention can keep a lot of sericin protein, therefore, it has lower cost, better fiber flexibility and thermal insulation than traditional silk non-woven cotton, and it can avoid de-gumming process to reduce environment pollution with better economic, social and zoology benefits

The invention discloses a method for desalting salted egg white. The byproduct salted egg white in the production of salted egg yolk is taken as a raw material. The method comprises the following steps of: filtering, performing pasteurization, regulating pH value to reach the isoelectric point of protein, heating for condensation, washing, centrifugally desalting, regulating the pH value to promote the renaturation and dissolution of the protein, and sterilizing to obtain the desalted salted egg white. By using the principle of reversible denaturation and renaturation of the protein of eggs, the desalted salted egg white of which the desalting rate is not less than 92 percent and the recovery rate of the protein is not less than 80 percent is obtained, the problems of high-quality proteinresource waste and environmental pollution caused by the abandonment of the salted egg white are solved, a new path is created for the utilization and development of the salted egg white, and considerable economic benefit and great social benefit are brought to enterprise production.

The invention discloses a production process of fusion expression recombinant chicken interferon alpha. The process includes the steps of: S1, according to the preference of Escherichia coli codon, conducting codon optimization on a chicken interferon alpha gene sequence published in Genebank, and artificially synthesizing the chicken interferon alpha gene; S2, according to the codon optimized chicken interferon alpha gene, designing three specific primers; S3, constructing recombinant chicken interferon alpha plasmid containing ProS2 dissolution promoting label; S4, transforming and identifying the recombinant expression plasmid; S5, conducting inducible expression of recombinant chicken interferon alpha; S6, extracting an expression product and conducting protein renaturation purification: S61, inclusion body extraction and treatment; S62, inclusion body denaturation; S63, denaturation solution renaturation; and S64, nickel column affinity purification. By means of cell cytopathic inhibition, the invention detects that the interferon has the activity of inhibiting vesicular stomatitis virus proliferation, and the activity unit reaches 7.32*10<7>UI / mg.

The invention relates to methods for preparing a multiple environment-responding type hairy polymer micro-spheres and photo-initiation RAFT (Reversible Addition-Fragmentation chain Transfer) polymerization thereof. The hairy structure of the surface of the hairy polymer micro-spheres prepared by the invention is a polyN-isopropyl acrylamide, polyN-vinyl pyrrolidone, polyacrylic acid homopolymerization-type, diblock-type or triblock-type polymer brush. The hairy polymer micro-spheres have the advantages of controllable structure, temperature sensitivity and pH sensitivity, and can be used for protein renaturation, chemical valves, drug encapsulation and catalyzer loading and other aspects.

The invention relates to a recombinant protein extraction and purification method, comprising engineering bacteria fermentation, engineering bacteria fragmentation, inclusion body washing, recombinant protein denaturation and purification, second recombinant protein denaturation and purification, denaturized recombinant protein renaturation, renaturated recombinant protein placing, ion exchange chromatography and molecular sieve chromatography, wherein, the renaturation recombinant protein is placed for 5 to 8 days under the temperature of 2 to 8 DEG C. The recombinant protein extraction and purification method improves the total yield of the recombinant protein, correspondingly improves the purity and efficiency of the final products, reduces the production cost and is beneficial to large scale industrial production.

This invention relates to protein renaturation. It is used for large inclusion body for recombination of tPA derivative and is carried out by: dissolving inclusion body of target protein, centrifuging to obtain supernatant containing it, diluting it with first renaturation liquid containing oxidation reducing pairs, settling a period, diluting it with second renaturation liquid without oxidation reducing pairs, settling a period, filtrating and concentrating to obtain primary concentrated liquid, filtrating and diluting with buffering liquid to regulate pH value 4.0-5.5, filtrating and concentrating to obtain secondary liquid containing renatured target protein.

The invention relates to silk property-variable non-woven cotton with sericin and relative application. Said silk property-variable non-woven cotton via selection-modifying-blanching, and washing-carding-needling processes can be made into the silk property-variable non-woven cotton which will not lose silk. Said non-woven cotton can recover water-solubility and biological activity via protein recover treatment to supply the health function of silk protein, and apply the medical, hairdressing and daily consumption areas. Since said invention can keep a lot of sericin protein, therefore, it has lower cost, better fiber flexibility and thermal insulation than traditional silk non-woven cotton, and it can avoid de-gumming process to reduce environment pollution with better economic, social and zoology benefits

The invention provides a Zika virus antigen and application thereof, and belongs to the technical field of protein engineering. DNA sequences corresponding to a protein sequence determined through DNA analysis and protein structure analysis are synthesized, Nde I and Xho I cleavage sites are introduced, a commercialized pET30a plasmid serves as a basic carrier, pZE400 is built, the protein sequence is as shown in SEQ ID NO.1, and the DNA sequences are as shown in SEQ ID NO.2. A protein C end is labeled with a protein band HIS expressed by the expression carrier and Zika virus protein is obtained through affinity chromatography purifying and protein renaturation. The protein is good in immunogenicity and high in specificity, can be used for preparing a Zika virus detection test strip or kit, can also be used for preparing Zika virus vaccines, and is remarkable in market value.

A process for renaturating and purifying the inclusion body protein includes such steps as dissolving the inclusion body protein in acidic / alkaline flowing phase or other solution, loading it in the ion-exchange chromatographic column, reversible adsorbing it by the chromatographic medium, and modifier concentration gradient elution. Its advantages are high concentration and activity of recovered protein and high purity.

The invention relates to a metal chelating nano medium, a preparation method and a method applied to strengthen inclusion body protein renaturation and integrated purification, wherein connection polymers P (GMA-IDA) of iminodiacetic acid and glycidyl methacrylate are grafted on the surfaces of silicon dioxide nanoparticles which are 20-30nm in mean grain sizes, and the electric density of medium is 2520-5680Mu mol / g. Extremely high electric density enhances electrostatic repulsion between the medium and homocharge protein molecules, thereby more effectively restraining gathering of proteins. A large number of metal chelating groups which are arranged on the surface of the nano medium guarantee recombinant target proteins which are marked through poly-L-histidine to be effectively adsorbed and separated after completing renaturation. The metal chelating nano medium, the preparation method and the method applied to strengthen inclusion body protein renaturation and integrated purification do not need expensive large equipment such as chromatographic columns or chromatographic systems and the like, complete integration of inclusion body protein renaturation and purification outside the chromatographic columns, are simple, convenient and easy to operate and low in cost, simplify refinement and purification method, and are more suitable for large scale industrialization production.

The invention refers to a protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier. The invention uses the specificity affinity relation between rFGF with His or SUMO labels and metal ion chelate affinity chromatography column, attaches the denatured and reduced His-rFGF or SUMO-rFGF to the metal ion chelate affinity chromatography column, replaces denatured buffer solution with refolding buffer solution by gradually changing the composition of mobile phase, so as to prevent the refolding protein folding intermediates from gathering together, and obtain highly effective protein refold while finishing the purification of target protein. Compared to the traditional refolding by dialysis, the method not only can greatly reduce the time needed for protein refolding, but also can make the denatured His-rFGF or SUMO-rFGF obtain refolding protein with high purity and activity in the high concentration(1-10mg / mL), further providing a new process for accomplishing highly effective protein refolding and purifying.

The invention relates to a freeze-dried protein renaturation method based on a natural phenomenon that solute is naturally separated from solvent in the solution freezing process. The solubility of solute salt in a solution is influenced by temperature, denaturation salt (urea, guanidine hydrochloride, and the like) is gradually crystallized and separated from the solution because the solubility of the denaturation salt lowers at a low temperature, and two vital denaturation factors, namely molecular heat movement and the denaturation salt concentration of the solution, are reduced. The crystallization speed can be controlled by the temperature drop speed so as to establish a wonderful condition for protein renaturation, namely the low temperature as well as a smooth downward gradient of the denaturation salt concentration. Meanwhile, a renaturation solution is aided with antifreezing agents (DMSO, fucose, glucose, and the like) with a certain concentration, renaturation activators (cyclodextrin, glycin, molecular chaperones, and the like), reductant-oxidant, buffer salts, and the like for preventing freezing damage to the protein activity and improving the renaturation rate. The frozen dry protein is dried by a freeze drier to obtain solid particles which are suitably vibrated to break by utilizing the difference between the denaturation salt and dry protein powder in the crystallization proportion and the crystallization form and separated by adopting the methods of pneumatic separation, sieving, electrostatic adherence and the like to obtain target protein.

The invention discloses a method and a reagent for preparing soluble interleukin recombinant protein from inclusion body. The reagent comprises a washing liquid, a thalli lysate, an inclusion body dissolving liquid, a protein renaturation liquid I and a protein renaturation liquid II. The employed inclusion body dissolving liquid is capable of substantially improving the yield of soluble interleukin recombinant protein from inclusion body, thereby solving the problem existed in a process of using an escherichia coli expression system to produce interleukin. Compared with the prior art, the reagent does not contain any protein denaturant, thereby facilitating subsequent protein renaturation. The method is simple in operation, is capable of improving the yield of soluble interleukin recombinant protein by 90% or more, has obvious advantage compared with a routine method which employs a protein denaturant and only has the yield of 5-20%, and has value of popularization and application.

The invention relates to the field of vaccine preparation, in particular to a preparation method of a recombinant novel coronavirus subunit vaccine. The method comprises the following steps: transfecting plasmids into which a nucleic acid fragment corresponding to RBD-TT recombinant protein is inserted into Escherichia coli; culturing the Escherichia coli to enable the Escherichia coli to expressthe RBD-TT fusion protein, and breaking thalli and separating an inclusion body crude extract of the RBD-TT recombinant protein; dissolving the inclusion body crude extract with a urea-containing denaturing solution, and then performing purifying through anion exchange chromatography to obtain an RBD-TT recombinant protein crude pure sample; diluting the RBD-TT recombinant protein crude pure sample with a diluent; performing filtering, and performing renaturation with a renaturation solution to obtain renaturation protein; and purifying the recombinant protein renaturation protein through anion exchange chromatography.

The invention relates to a preparation method for a decellularized tissue membrane. The method comprises the following steps: 1) removing adipose tissue on the tissue membrane, washing and cleaning bloodstain; 2) placing the neutralized and washed tissue in surfactant solution and soaking, and adequately removing impure proteins; 3) placing in protein renaturation solution and soaking; 4) placing in an organic solvent and soaking, and separating out grease; 5) placing in alkaline solution and soaking, neutralizing, and preparing pores on the tissue membrane; 6) placing the tissue membrane in solution containing a crosslinking agent and soaking, crosslinking; and 7) executing the radiation sterilization to the tissue membrane obtained in the steps in the wet state; or executing the radiation sterilization or EO sterilization in the freeze-drying mode. The method is capable of removing antigens, such as cells, lipids and impure proteins, in the human source or animal source tissue membrane, keeping and extracellular matrix and generating the suitable pores. The prepared decellularized tissue membrane can be used as a tissue engineering bent, a patching material for tissue membrane damage and a wound surface dressing.

The invention discloses a gene of sika deer IGF (Insulin-like Growth Factor)-1 mature peptide. An EcoR I restriction enzyme cutting site and a base sequence of code Asn are added at the end 5 of a forward primer; a Hind III restriction enzyme cutting site and a sequence of a termination codon are added at the end 5 of a reverse primer; a base sequence 234bp gene for expressing the sika deer IGF-1 mature peptide is cloned; an expression vector, namely pET-32a-IGF-1 is constructed and is induced and expressed in Escherichia coli Rosetta to obtain the sika deer IGF-1 mature peptide. The EcoR I restriction enzyme cutting site and the Hind III restriction enzyme cutting site are introduced and an Asn is added in front of natural IGF-1, so that a specific cracking part of hydroxylamine is formed, and further the cutting cost of protein is reduced, the influence on protein renaturation caused by extra amino acid sequence is reduced and the state of the protein in a natural state is kept. The pET-32a as an expression vector is expressed in the Escherichia coli Rosetta, so that the protein content can reach over 50 percent of soluble protein of thallus; and through the detection by adopting an MTT (Methyl Thiazolyl Tetrazolium) method, the multiplication rate of N1H3T3 cells can be increased.

A method for renaturation of proteins comprising adding to a solution of denatured, chemically modified or reduced proteins a refolding buffer containing a primary, secondary or tertiary amine. Said method has been applied, for example, to interleukin-4 and bovine pancreatic trypsin inhibitor (BPTI), wich were previously (i) solubilized in the presence of guanidinium hydrochloride as chaotronic agent, and (ii) subjected to sulfitolysis.

Existing commercial fish nervous necrosis virus vaccine must be vaccinated by injection. The invention relates to a gene recombinant fusion protein MCP-ompN1 of a major coat protein (MCP) of nervous necrosis virus and ompN1, having a transmembrane effect, of edwarsiella ictaluri as well as expression and a preparation method of the fusion protein. The MCP and ompN1 protein are flexibly linked by asequence composed of four amino acids ''GGGS''". A nucleotide sequence encoding MCP-ompN1 is first optimized according to the codon preference of a BL21-DE3 engineering strain, then an expression plasmid is constructed and expressed in the BL21-DE3 strain, and then the MCP-ompN1 fusion protein is separated and purified through bacterial disruption, inclusion body treatment, protein renaturation,nickel column purification, removal of histone tags with biotin-labeled thrombin, removal of thrombin with avidin resin and other steps. The gene recombinant MCP-ompN1 fusion protein can be used as avaccine antigen of nervous necrosis virus, or a vaccine antigen of nervous necrosis virus and edwarsiella ictalurid, and the ompN1 protein in the MCP-ompN1 fusion protein mediates mucosal immunity.

The invention discloses a method for inclusion body protein dilution and dialysis renaturation. A mild degeneration method is adopted for dissolving inclusion body protein, an existing secondary structure in the inclusion body protein is protected against damage so that inclusion body protein renaturation is greatly facilitated; the method integrates dilution and dialysis for performing protein renaturation, the concentration of a degeneration agent is lowered by first-time dilution, protein renaturation is promoted, the concentration of an additive is lowered by second-time dilution, the protein is more easily suitable for the environment of a basic buffer solution, finally, dialysis is performed to remove various additives, and the renaturated protein with the high renaturation efficiency and the high concentration can be obtained.

The invention relates to a production method of recombinant full-length human liver regeneration enhancing factor and its use for treating severe liver diseases, and belongs to the field of production methods and uses of biological products. On the basis of obtaining the full-length human enhancer regeneration factor gene sequence and successfully constructing high-efficiency expression strains, a process of prokaryotic expression of recombinant full-length enhancer of human liver regeneration factor, separation and lysis of inclusion bodies, protein purification and renaturation was established, and finally the recombinant whole-body gene was obtained. A pure product of long-term human liver regeneration enhancing factor. The latter can promote DNA synthesis of the hepatic cell line HepG2 in vitro, promote liver regeneration in rats after partial hepatectomy in vivo, and promote CCl 4 DNA synthesis and repair in rat hepatocytes after poisoning, increase in CCl 4 The survival rate of rats after poisoning indicates that the recombinant full-length human liver regeneration enhancing factor has the utility of treating severe liver diseases.

The invention relates to a method for promoting protein renaturation by increasing the charge density of a microspherical medium through secondary ligand modification, belonging to a protein renaturation technology in the technical field of biology. A ligand of the microspherical medium is secondarily modified on the basis of primary modification of the microspherical medium, so that the charge density of the microspherical medium with epoxy groups on the surface is increased. The electrostatic repulsion action between the microspherical medium and a protein molecule with the same charge number is further enhanced through modifying the microspherical medium again by using the ligand with higher charge density, so that the aggregation of proteins is effectively inhibited, and the renaturation yield is greatly increased. Compared with a primarily-modified microspherical medium assisted protein renaturation method, the method not only has the characteristics of low medium consumption and high protein renaturation yield, but also has better tolerance for environments such as high salinity and the like.

The invention discloses a preparation method of abyss sea cucumber sourced superoxide dismutase Cu, ZnSOD and application thereof. The method includes: extracting total RNA of Paelopatides sp., reversely transcribing into cDNA through RT-PCR, acquiring a gene sequence PaSOD coding superoxide dismutase through PCR amplification, establishing prokaryotic expression recombinant plasmid in pCold II vector, and guiding into chaperone Competent Cell pG-KJE8 / BL21 for recombinant protein soluble expression. Recombinant protein prepared through the method overcomes the defects that raw material sourcesare difficult to obtain and protein renaturation recycling process is complex and troublesome, purified protein is high in purity and vitality, wide in temperature suiting range and capable of resisting digestion by high-concentration digestive enzyme, and a foundation is laid for widely applying the protein in the field of biology, food, medicine and cosmetology.

The invention relates to a method for purifying a recombinant protein of the gene engineering and aims to provide a method for purifying a recombinant VP1 antigen of enterovirus type 71 viruses. The method comprises the following steps of: expressing and producing a recombinant VP1 protein of the enterovirus type 71 viruses by Escherichia coli obtain an inclusion body of the protein; dissolving the inclusion body in solubilizing liquid; and dropwise adding the solution of the inclusion body into renaturation solution under the conditions of water bath and magnetic stirring and then carrying out stirring renaturation on the mixture for 48 hours. The invention discloses the method, which can make the protein existing in a form of the inclusion body become a soluble protein by protein renaturation so as to greatly improve recovery rate of an expression product. The method adopts affinity chromatography for purification in one step. The purity is over 95 percent. The method for purifying the recombinant VP1 antigen of the enterovirus type 71 viruses has simple, convenient and time-saving operation, is favorable for large-scale industrial production, has the advantages of low cost, simple and convenient process, high purity of the purified protein and the like and can provide the diagnostic antigen for development of an EV type 71 diagnostic reagent and seroepidemiological survey.

The invention relates to a renaturation method of restructured human insulin prokaryotic-fusion protein and belongs to the field of protein purification. The renaturation method includes steps of 1) smashing escherichia coli expressing restructured human insulin prokaryotic-fusion protein, and collecting inclusion bodies; 2) washing the inclusion bodies, dissolving the inclusion bodies and modifying the fusion protein; 3) renaturating protein. The renaturation method is needless of protein purification in advance, directly performs protein renaturation, and finally obtains high-concentration restructured insulin prokaryotic-fusion protein of natural structure. By the renaturation method, the defect of low protein concentration after protein renaturation and resultantly easy protein accumulation and precipitation is overcome, renaturation efficiency is up to 80-90%, production efficiency is improved, and the renaturation method is applicable to industrial production.

The invention discloses a method for dissolving inclusion body proteins. The method comprises the following steps: centrifugally collecting thalli for expressing the inclusion body proteins, re-suspending the centrifugal thalli by using a PBS (Phosphate Buffer Solution), adding lysozyme, keeping at the room temperature for 1-3 hours, performing ultrasonic smashing, and centrifugally removing supernatant to obtain inclusion body protein precipitate; re-suspending the inclusion body protein precipitate in IBbuffer, centrifuging, removing the supernatant, and washing the inclusion body proteins for 1-6 times repeatedly; re-suspending the washed inclusion body proteins by using a urea-containing PBS; refrigerating an inclusion body protein suspension; putting the refrigerated inclusion body protein suspension at the room temperature, slowly dissolving, and centrifuging to obtain supernatant, namely, the inclusion body proteins. The method is easy to operate, an obtained inclusion body protein solution contains a low-concentration denaturant, a renaturation step is simplified, time is saved, and the protein renaturation efficiency is increased.

The invention provides a method for separating and purifying inclusion body-type HIV-1 (human immunodeficiency virus-1) protease from a prokaryotic system. In the method, a low-concentration detergent and ultrasonic disruption are combined to separate impurity protein from target protein, and an Amicon stirring type ultrafiltration device is used in an operation process, so that the purifying time of the inclusion body protein is effectively shortened. Moreover, the protein renaturation in the method is implemented by dialysis renaturation, the dialysis process is mild, and the activity and purity of the renatured protease are relatively high. The protein crystallization growth and enzyme kinetics measurement can be performed through simple ultrafiltration concentration, and the method is applied to the study of crystallography and enzymology.

The invention discloses a method for assisting the in vitro renaturation of lysozyme by using granular poly (N-isopropylacrylamide-sodium acrylate) copolymer gel, the specific steps are as follows: 1) the granular poly (N-isopropylacrylamide-sodium acrylate) copolymer gel is prepared by using the inverse suspension copolymerization; 2) the granular poly (N-isopropylacrylamide-sodium acrylate) copolymer gel is used for assisting the in vitro renaturation of the lysozyme. The granular poly (N-isopropylacrylamide-sodium acrylate) copolymer gel which is developed by the method is taken as a novel protein in vitro renaturation additive, thereby having the following advantages: (1) the activity recovery rate of a target protein can be significantly improved under the condition of high denatured protein concentration; (2) the separation and the recovery are convenient by utilizing the thermosensitive characteristic of the gel after the completion of the renaturation, and the gel can be repeatedly utilized; (3) the hydrophobic performance of the gel can be regulated by controlling the content of the co-monomer sodium acrylate, thereby changing the low critical dissolution temperature and the largest swelling ratio and being applicable to the different target proteins. Therefore, the gel has better application prospect in the field of the protein renaturation.

The invention discloses a high-performance hydrophobic interaction chromatographic medium taking benzene methanamine as a ligand. The structural formula of the medium is as shown in the specification.The invention further discloses a preparation method of the high-performance hydrophobic interaction chromatographic medium. The preparation method includes the steps: (1) silica gel activation; (2)epoxy silica gel preparation; (3) epoxide ring-opening preparation of the high-performance hydrophobic interaction chromatographic medium. Besides, the invention further discloses an application of the high-performance hydrophobic interaction chromatographic medium to recombinant streptavidin renaturation and purification. Renaturation efficiency and active recovery rate are high, and recombinantstreptavidin after renaturation is high in purity.

The invention discloses a production and purification method of a storage protein type alpha-amylase inhibitor with a Cupin structure function domain, comprising the steps of: 1, construction of a rokaryotic expression system and bacterium solution cultivation inducible expression; 2, prokaryotic expression of soluble protein purification; and / or 3, prokaryotic expression of inclusion body protein purification and protein renaturation. At present, multiple repeated experiments test that, the protein obtained through producing the alpha-amylase inhibitor by using the technical process provided by the invention is high in yield and purity, the production process is simple and convenient, the production cycle is short, expensive devices are not needed, and thereby the production and purification method is an excellent bioprocess technology for producing and purifying the alpha-amylase inhibitor.