CN101363018A - Method for assisting lysozyme renaturation in vitro by granular poly(N-isopropylacrylicamide-sodium acrylate) copolymer hydrogel - Google Patents
Method for assisting lysozyme renaturation in vitro by granular poly(N-isopropylacrylicamide-sodium acrylate) copolymer hydrogel Download PDFInfo
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Abstract
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.
Description
Technical field
The present invention relates to a kind of method of granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer assisting lysozyme in vitro refolding by means.
Background technology
Genetic engineering technique is the main body of modern biotechnology, and its fast development makes proteinic allosome express becomes the important channel of industry of development biological medicine and acquisition speciality chemical.But often face such difficult problem in the engineered protein industrialization process: recombination expression product normally exists with the active inclusion body form of lifeless matter.Inclusion body refolding is efficiently become have native conformation and bioactive protein, be that can this engineered protein of decision industrialization and be the human key of being utilized, thereby the outer renaturation technical field of aleuroplast become one of hot research in recent years.But, also be difficult to find have universality and highly efficient renaturation method cheaply because the complicacy of diversity, structure and the folding process of protein itself makes people not to be transplanted to a kind of refolding method in the multiple proteins renaturation system simply.Therefore must each factor that influence renaturation be optimized one by one at the characteristics of every kind of target protein in the actual mechanical process, could finally determine to be applicable to the proteinic refolding method of this kind.
Up to the present, the many problems in the protein renaturation process are still perplexing people, and the protein concn that, renaturation initial sum lower as activity recovery ends is on the low side and last handling process is loaded down with trivial details etc.Along with going deep into of research, having developed some can assist protein to carry out the method for external renaturation under the high density condition, mainly contain: the chromatogram renaturation, this method renaturation rate of recovery height, easy and simple to handle quick, but often influenced its large-scale application in industrial production because equipment and material input cost are high; The protein renaturation of molecular chaperones and folding enzymes mediation, this method is the inspiration that is subjected to body internal protein folding process, can improve the renaturation yield to a certain extent, but its source is limited, the purchase price costliness, even prepare voluntarily by microbial fermentation, also can there be problems such as the higher and easy inactivation of product of fermentation costs, generally only be used for the research of renaturation mechanism; Conventional protein renaturation additive such as polyoxyethylene glycol, L-arginine, stain remover and tensio-active agent and artificial molecule companion etc., though it is not high to equipment requirements, but assist the renaturation effect limited, and renaturation process is difficult to separate with target protein after finishing.In this case, develop a kind of novel, efficient, be easy to Separation and Recovery and cheaply the outer renaturation additive of aleuroplast just seem particularly important.Developing rapidly along with cross discipline in recent years, intelligent polymkeric substance more and more causes people's attention as the outer renaturation additive of a kind of novel aleuroplast, and its major advantage is to influence that the parameter of renaturation process is less, easy to operate, plant and instrument requires lower and be easy to Separation and Recovery.
This seminar has synthesized linear poly N-isopropyl acrylamide (PNIPAAm) polymkeric substance of various molecular weight and has been applied to the lysozyme in vitro refolding by means process (referring to the method for linear poly N-isopropyl acrylamide assisting lysozyme in vitro refolding by means, application for a patent for invention number: 200810062428.3), the result shows that the effect of the PNIPAAm polymkeric substance assisting lysozyme renaturation that molecular weight is moderate is fine, when protein concentration was 600 μ g/mL, the adding of PNIPAAm can make the activity recovery of enzyme bring up to about 80% from 10% of dilution refolding.But adopt linear polymer as the renaturation additive, after finishing, renaturation needs by the mode that heats PNIPAAm to be separated out from the aqueous solution earlier, could realize separating of target protein and PNIPAAm water-soluble polymers after the renaturation through a step high speed centrifugation again, operate relatively bothering; In addition because the polymerization degree of this polymkeric substance is wayward, can cause on the one hand it to assist renaturation effect that bigger fluctuation is arranged, on the other hand the less PNIPAAm molecule of those polymerization degree since with renaturation solution in the molecular weight difference of target protein not quite be difficult to remove fully by centrifugal.The effect that this seminar had also once synthesized granular poly N-isopropyl acrylamide and investigated its assisting lysozyme renaturation is (referring to the method for temperature sensitive type poly N-isopropyl acrylamide gel assisting lysozyme renaturation, application for a patent for invention number: ZL 200410073406.9, authorize day: on August 2nd, 2006), this polymkeric substance is a kind of cross-linked network shape gel, remarkable swelling in water, but can not dissolve.Because this gel has the hydrophobic property that linear PNIPAAm polymkeric substance is had, thereby can play the effect of assisting the outer renaturation of aleuroplast; And can utilize its temperature response characteristics after renaturation process finishes, by in solution repeatedly the method for swelling (being lower than its Kraft point LCST) and shrink (being higher than LCST) make gel separate apace, reclaim and reuse.But the physical strength of this granulated gel a little less than, and the variable range of transformation temperature is narrower, thereby its application is restricted.The present invention by introduce monomer sodium acrylate (SA) altogether in polymerization process, prepares granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer on the basis of previous work.On the one hand, this gel copolymer has the hydrophobic grouping sec.-propyl, can suppress metaprotein and folding intermediate thereof because of assembling the precipitation that causes mutually by hydrophobic interaction, thereby assist protein renaturation; On the other hand, the introducing of comonomer SA makes the physical strength of gel obviously strengthen, thereby improves its water-retaining capacity significantly, has increased the maximum swelling multiplying power of gel greatly; At last, the introducing of comonomer SA makes the transformation temperature of this gel rise to some extent, and can regulate its transformation temperature easily by the add-on that changes SA, to be applicable to different target proteins.
Summary of the invention
The objective of the invention is overcoming the prior art deficiency, a kind of method of granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer assisting lysozyme in vitro refolding by means is provided.
The step of method is as follows:
1) get the NaOH solution that 40～60mL concentration is 0.2～0.4mol/L, add excessive acrylic acid aqueous solution, behind stirring reaction 4～6h, solvent removed by evaporation at reduced pressure and remaining vinylformic acid can be collected and obtain the sodium acrylate solid;
2) the tensio-active agent tween-80 is dissolved in 75～100mL organic phase whiteruss, making its concentration is 1～1.5% (g/mL), add reactor, feed nitrogen, stir 20～30min, tensio-active agent is fully disperseed, the aqueous solution of raw material that contains various reactive components that with volume is organic phase volume 1/3 then slowly is added dropwise in the reactor with liquid-transfering gun, its aqueous solution of raw material consists of: mass percent concentration is the main monomer of 14% (g/g), degree of crosslinking is 10% (g/g), the monomer N-N-isopropylacrylamide mol ratio of monomer sodium acrylate together is 19:1～9:1, concentration is the oxygenant ammonium persulphate of 0.5～0.8% (g/mL), under nitrogen protection, after stirring 30～40min, slowly add initiator N, N, N ', N '-Tetramethyl Ethylene Diamine 2～3 μ L, polymerization temperature is controlled at 20～25 ℃, stirring velocity is controlled at 250～280rpm, reaction 3～4h discharging, product dehydrated alcohol/water washing by soaking repeatedly, in vacuum drying oven, dry, promptly obtain granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer; Wherein, main monomer comprises monomer N-N-isopropylacrylamide and linking agent N, N '-methylene-bisacrylamide, and degree of crosslinking is meant linking agent N, N '-methylene-bisacrylamide shared mass percent in main monomer;
3) N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, be mixed with the solution that concentration is 10mg/mL, place 37 ℃, 100～120rpm shaking table to handle 90～120min solution, obtain the unfolding attitude lysozyme soln of non-activity, get the unfolding attitude lysozyme soln of 0.1～0.5mL non-activity, slowly add renaturation buffer, mixing obtains 10mL renaturation system solution;
4) in the renaturation system solution, add granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer, gel quality affects concentration is 160～560 times of N,O-Diacetylmuramidase mass concentration in the renaturation system solution, and solution is that 20～40 ℃, rotating speed are renaturation 16～24h in the shaking table of 100～140rpm in temperature;
Described N-N-isopropylacrylamide adopts the normal hexane recrystallization.The sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), and pH 8.5.Renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.Gel after lysozyme renaturation is finished by elevated temperature to the gel transformation temperature, thereby make gel shrink discharge protein solution in the gel network, carry out centrifuging again and gel Separation and Recovery from renaturation solution can be reused.
The present invention adopts the inverse suspension polymerization method on preparation technology, the product for preparing is spherical granulated gel, and the median size of gel is about 0.3mm, and narrow distribution.This gel copolymer has 38～43 ℃ of suitable lower critical solution temperature (LCST) scopes, and the maximum swelling multiplying power is 8.5～13.Simultaneously its surface arrangement has the hole of diameter 1～2 μ m, thereby has temperature response characteristics faster, reaches swelling and the shrink balance only needs 15min.When assisting lysozyme renaturation, unfolding attitude N,O-Diacetylmuramidase can be free and apace with the gel copolymer side chain on hydrophobic grouping sec.-propyl generation hydrophobic interaction, thereby the mutual gathering between the arrestin matter, when concentration of treatment is 500 μ g/mL, the activity recovery of N,O-Diacetylmuramidase is brought up to about 60% from 30% of dilution refolding.The activity recovery that gel reuses N,O-Diacetylmuramidase after 6 times still improves more than 13% than dilution refolding.Therefore, granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer of being developed can be used as the additive of assisting the outer renaturation of aleuroplast.The invention has the advantages that: 1) preparation technology is simple, is easy to control and amplification; 2) granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer of preparation abundant swollen while in water, also have certain hydrophobic performance, thus the aggregation tendency in can arrestin matter renaturation process.During this kind gel assisting lysozyme renaturation, the ratio work and the activity recovery of N,O-Diacetylmuramidase all are greatly improved than dilution refolding; 3) this method renaturation manipulation is simple, and the parameter that influences renaturation process is few, to instrument and equipment require low; 4) with linear PNIPAAm polymer phase ratio, after renaturation process is finished, utilize the temperature-sensing property of this gel, Separation and Recovery and repeated use easily, thus reduce production costs; 5) compare with general granulated gel, can regulate the hydrophobic performance of this gel by the content of controlling common monomer sodium acrylate, and then change its lower critical solution temperature and maximum swelling multiplying power, make it be applicable to different target proteins.
Description of drawings
Fig. 1 is the changing conditions of granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer (P (NIPA-co-SA)) of the present invention and granular poly N-isopropyl acrylamide gel (PNIPA) swelling multiplying power under differing temps.Among the figure: the main monomer concentration of two kinds of gels is 14% (g/g), and degree of crosslinking is 10% (g/g), in P (NIPA-co-SA) gel copolymer monomer N-N-isopropylacrylamide together the mol ratio of monomer sodium acrylate be 15.7:1;
Fig. 2 is under different N,O-Diacetylmuramidase starting point concentrations, and granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer assisting lysozyme in vitro refolding by means of the present invention and dilution refolding effect are relatively.Among the figure: the add-on of gel copolymer is 480 times of lysozyme concentration in the renaturation solution, and the renaturation temperature is 30 ℃.
Fig. 3 is the influence of granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer recycling number of times of the present invention to its assisting lysozyme renaturation effect.Among the figure: the main monomer concentration of gel is 14% (g/g), degree of crosslinking is 10% (g/g), and the monomer N-N-isopropylacrylamide mol ratio of monomer sodium acrylate together is 15.7:1, and initial lysozyme concentration is 250 μ g/mL, the add-on of gel copolymer is 120mg/mL, and the renaturation temperature is 30 ℃.
Embodiment
The present invention adopts granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer (P (NIPA-co-SA)) assisting lysozyme in vitro refolding by means.Step comprises the preparation and the assisting lysozyme in vitro refolding by means process thereof of granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer.For realizing this process, at first, with NaOH and excessive vinylformic acid reaction, reduction vaporization removed and anhydrates and remaining vinylformic acid after reaction was finished, and can obtain common monomer sodium acrylate.Prepare reactant aqueous solution then, with the monomer N-N-isopropylacrylamide of suitable proportion, common monomer sodium acrylate, linking agent N, N '-methylene-bisacrylamide and oxygenant ammonium persulphate are dissolved in the deionized water, with micro-filtrate membrane filtration solution to remove undissolved impurity; Adopt inverse suspension method to prepare temperature sensitive type P (NIPA-co-SA) gel copolymer then, in container, add whiteruss and tween-80, fully disperse the back to add the aqueous solution of reactant, form homogeneous, stable water-in-oil anti-phase suspension dispersion system, slowly add N, N, N ', N '-Tetramethyl Ethylene Diamine initiated polymerization, with the granulated gel that obtains with a certain proportion of dehydrated alcohol/water repetitive scrubbing after, in vacuum drying oven, dry, obtain being particulate state, dispersing property P (NIPA-co-SA) gel copolymer preferably; Secondly, carry out P (NIPA-co-SA) gel copolymer assisting lysozyme in vitro refolding by means, N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, obtain non-activity unfolding attitude lysozyme soln.The renaturation buffer that slowly adds different volumes again according to different extension rates adds granular P (NIPA-co-SA) gel copolymer in solution.After fully concussion is uniformly dispersed it, carry out renaturation process; At last, the recovery of temperature sensitive type P (NIPA-co-SA) gel copolymer.Need after renaturation process finishes solution is warming up on the transformation temperature LCST of P (NIPA-co-SA) gel copolymer, it is fully shunk, realize separating of target protein after gel and the renaturation, gel is reclaimed, so that recycling.In the refolding method of the present invention; the add-on of tensio-active agent tween-80 is 1～1.5% (g/mL) of organic phase; in the aqueous solution of raw material monomer N-N-isopropylacrylamide together the mol ratio of monomer sodium acrylate be 19:1～9:1; the concentration of oxygenant ammonium persulphate is 0.5～0.8% (g/mL); main monomer (comprises monomer N-N-isopropylacrylamide and linking agent N; N '-methylene-bisacrylamide) concentration is 14% (g/g); the degree of crosslinking ratio of main monomer quality (quality of linking agent with) is 10% (g/g); temperature of reaction is controlled at 20～25 ℃; stirring velocity is 250～280rpm; nitrogen protection is reaction 3～4h down; products therefrom dehydrated alcohol/water washing by soaking repeatedly, the oven dry back is stand-by.Protein concn is 10mg/mL in the sex change liquid, places 37 ℃, 100～120rpm shaking table to handle 90～120min solution.The add-on of sex change lysozyme soln is 0.1～0.5mL in the renaturation solution, the final volume of renaturation system is 10mL, in the renaturation system, add granular P (NIPA-co-SA) gel copolymer, the mass concentration of gel is 160～560 times of N,O-Diacetylmuramidase mass concentration, the renaturation temperature is 20～40 ℃, shaking speed is 100～140rpm, and the renaturation time is 16～24h.
Monomer N-N-isopropylacrylamide (99%, polymerization-grade, available from Beijing lark waffle company limited that learns a skill), linking agent N, N '-methylene-bisacrylamide (chemical pure, available from Chinese Medicine Shanghai reagent company), initiator N, N, N ', N '-Tetramethyl Ethylene Diamine (TEMED), N,O-Diacetylmuramidase, dithiothreitol (DTT) (DTT), sodium ethylene diamine tetracetate (EDTA), reduction and Sleep-promoting factor B (GSH/GSSG), three (methylol) aminomethane (Tris) and Xylene Brilliant Cyanine G are all given birth to worker bio-engineering corporation available from Shanghai, and substrate micrococci Micrococcus lysodeikticus ATCC4698 is available from Sigma company, and other reagent is commercially available analytical pure.The sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), pH8.5; Renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.N,O-Diacetylmuramidase is than F.I.P. method that live to measure the employing standard, and lysozyme concentration adopts the Xylene Brilliant Cyanine G method to measure, and the activity recovery of N,O-Diacetylmuramidase is the ratio of antalzyme activity before the vigor of N,O-Diacetylmuramidase and the sex change in the solution after the renaturation.
The invention will be further described by the following examples:
Embodiment 1
1) get the NaOH solution that 40mL concentration is 0.2mol/L, add the excessive propene aqueous acid, behind the stirring reaction 4h, solvent removed by evaporation at reduced pressure and remaining vinylformic acid can be collected and obtain the sodium acrylate solid;
2) the tensio-active agent tween-80 is dissolved in to make its concentration in the 75mL organic phase whiteruss be 1% (g/mL), adds reactor, feed nitrogen, stir 20min, tensio-active agent is fully disperseed.The aqueous solution of raw material that contains various reactive components that with volume is organic phase volume 1/3 then slowly is added dropwise in the reactor with liquid-transfering gun; aqueous solution of raw material consists of main monomer concentration 14% (g/g); degree of crosslinking is 10% (g/g); the monomer N-N-isopropylacrylamide mol ratio of monomer sodium acrylate together is 15.7:1; the concentration of oxygenant ammonium persulphate is 0.5% (g/mL); under nitrogen protection; after stirring 30min; slowly add initiator N, N, N '; N '-Tetramethyl Ethylene Diamine 2 μ L; 25 ℃ of polymerization temperatures, stirring velocity 250rpm, reaction 3h discharging.Product dehydrated alcohol/water washing by soaking is repeatedly dried in loft drier.Granular P (NIPA-co-SA) the gel copolymer maximum swelling multiplying power that obtains is 9, its lower critical solution temperature (LCST) is 39 ℃, increase significantly than granular PNIPA gel under the corresponding conditions (maximum swelling multiplying power 6.3,33 ℃ of lower critical solution temperatures), the result as shown in Figure 1.The median size of gel is 0.3mm, is distributed in the narrower zone of 0.2～0.5mm, good dispersion property, and the gel particles surface arrangement some amount diameter is arranged is the hole of 1～2 μ m.This granulated gel has temperature sensitivity preferably, reaches swelling and the shrink balance only needs about 15min in the aqueous solution.
3) N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, be mixed with the solution that concentration is 10mg/mL, place 37 ℃, 100rpm shaking table to handle 120min solution, obtain the unfolding attitude lysozyme soln of non-activity, get the unfolding attitude lysozyme soln of 0.25mL non-activity, slowly join in the renaturation buffer, mixing, the final volume of renaturation system is 10mL, wherein, the add-on of granular P (NIPA-co-SA) gel copolymer is 40mg/mL (for 160 times of lysozyme concentration), and solution is that 30 ℃, rotating speed are renaturation 16h in the 120rpm shaking table in temperature.The antalzyme activity rate of recovery reaches 56.5% after the renaturation.The sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), pH8.5.Renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.
Embodiment 2
1) get the NaOH solution that 50mL concentration is 0.2mol/L, add the excessive propene aqueous acid, behind the stirring reaction 4h, solvent removed by evaporation at reduced pressure and remaining vinylformic acid can be collected and obtain the sodium acrylate solid;
2) the tensio-active agent tween-80 is dissolved in to make its concentration in the 75mL organic phase whiteruss be 1% (g/mL), adds reactor, feed nitrogen, stir 30min, tensio-active agent is fully disperseed.The aqueous solution of raw material that contains various reactive components that with volume is organic phase volume 1/3 then slowly is added dropwise in the reactor with liquid-transfering gun; aqueous solution of raw material consists of main monomer concentration 14% (g/g); degree of crosslinking is 10% (g/g); monomer N-N-isopropylacrylamide is 19:1 with the mol ratio that self-control is total to the monomer sodium acrylate; the concentration of oxygenant ammonium persulphate is 0.5% (g/mL); under nitrogen protection; after stirring 40min; slowly add initiator N, N, N '; N '-Tetramethyl Ethylene Diamine 2 μ L; 25 ℃ of polymerization temperatures, stirring velocity 250rpm, reaction 3h discharging.Product dehydrated alcohol/water washing by soaking is repeatedly dried in loft drier.Granular P (NIPA-co-SA) the gel copolymer maximum swelling multiplying power that obtains is 8.5, and its lower critical solution temperature (LCST) is 38 ℃, increases significantly than granular PNIPA gel under the corresponding conditions (maximum swelling multiplying power 6.3,33 ℃ of lower critical solution temperatures).The median size of gel is 0.3mm, is distributed in the narrower zone of 0.2～0.5mm, good dispersion property, and the gel particles surface arrangement some amount diameter is arranged is the hole of 1～2 μ m.This granulated gel has temperature sensitivity preferably, reaches swelling and the shrink balance only needs about 15min in the aqueous solution.
3) N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, be mixed with the solution that concentration is 10mg/mL, place 37 ℃, 120rpm shaking table to handle 90min solution, obtain the unfolding attitude lysozyme soln of non-activity, get the unfolding attitude lysozyme soln of 0.25mL non-activity, slowly join in the renaturation buffer, mixing, the final volume of renaturation system is 10mL, wherein, the add-on of granular P (NIPA-co-SA) gel copolymer is 120mg/mL (for 480 times of lysozyme concentration), and solution is that 30 ℃, rotating speed are renaturation 16h in the 120rpm shaking table in temperature.The antalzyme activity rate of recovery reaches 71.9% after the renaturation.The sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), and pH 8.5.Renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.
Embodiment 3
1) get the NaOH solution that 60mL concentration is 0.4mol/L, add the excessive propene aqueous acid, behind the stirring reaction 6h, solvent removed by evaporation at reduced pressure and remaining vinylformic acid can be collected and obtain the sodium acrylate solid;
2) the tensio-active agent tween-80 is dissolved in to make its concentration in the 100mL organic phase whiteruss be 1.5% (g/mL), adds reactor, feed nitrogen, stir 30min, tensio-active agent is fully disperseed.The aqueous solution of raw material that contains various reactive components that with volume is organic phase volume 1/3 then slowly is added dropwise in the reactor with liquid-transfering gun; aqueous solution of raw material consists of main monomer concentration 14% (g/g); degree of crosslinking is 10% (g/g); monomer N-N-isopropylacrylamide is 9:1 with the mol ratio that self-control is total to the monomer sodium acrylate; the concentration of oxygenant ammonium persulphate is 0.5% (g/mL); under nitrogen protection; after stirring 40min; slowly add initiator N, N, N '; N '-Tetramethyl Ethylene Diamine 2 μ L; 25 ℃ of polymerization temperatures, stirring velocity 280rpm, reaction 3h discharging.Product dehydrated alcohol/water washing by soaking is repeatedly dried in loft drier.Granular P (NIPA-co-SA) the gel copolymer maximum swelling multiplying power that obtains is 13, and its lower critical solution temperature (LCST) is 43 ℃, increases significantly than granular PNIPA gel under the corresponding conditions (maximum swelling multiplying power 6.3,33 ℃ of lower critical solution temperatures).The median size of gel is 0.3mm, is distributed in the narrower zone of 0.2～0.5mm, good dispersion property, and the gel particles surface arrangement some amount diameter is arranged is the hole of 1～2 μ m.This granulated gel has temperature sensitivity preferably, reaches swelling and the shrink balance only needs about 15min in the aqueous solution.
3) N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, be mixed with the solution that concentration is 10mg/mL, place 37 ℃, 100rpm shaking table to handle 90min solution, obtain the unfolding attitude lysozyme soln of non-activity, get the unfolding attitude lysozyme soln of 0.25mL non-activity, slowly join in the renaturation buffer, mixing, the final volume of renaturation system is 10mL, wherein, the add-on of P (NIPA-co-SA) gel copolymer is 140mg/mL (for 560 times of lysozyme concentration), and solution is that 30 ℃, rotating speed are renaturation 16h in the 120rpm shaking table in temperature.The antalzyme activity rate of recovery reaches 67.1% after the renaturation.The sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), and pH 8.5.Renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.
Embodiment 4
1) get the NaOH solution that 60mL concentration is 0.3mol/L, add the excessive propene aqueous acid, behind the stirring reaction 6h, solvent removed by evaporation at reduced pressure and remaining vinylformic acid can be collected and obtain the sodium acrylate solid;
2) the tensio-active agent tween-80 is dissolved in to make its concentration in the 75mL organic phase whiteruss be 1.5% (g/mL), adds reactor, feed nitrogen, stir 20min, tensio-active agent is fully disperseed.The aqueous solution of raw material that contains various reactive components that with volume is organic phase volume 1/3 then slowly is added dropwise in the reactor with liquid-transfering gun; aqueous solution of raw material consists of main monomer concentration 14% (g/g); degree of crosslinking is 10% (g/g); monomer N-N-isopropylacrylamide is 15.7:1 with the mol ratio that self-control is total to the monomer sodium acrylate; the concentration of oxygenant ammonium persulphate is 0.8% (g/mL); under nitrogen protection; after stirring 30min; slowly add initiator N, N, N '; N '-Tetramethyl Ethylene Diamine 3 μ L; 20 ℃ of polymerization temperatures, mechanical stirring speed 280rpm, reaction 4h discharging.Product dehydrated alcohol/water washing by soaking is repeatedly dried in loft drier.Granular P (NIPA-co-SA) the gel copolymer maximum swelling multiplying power that obtains is 9, its lower critical solution temperature (LCST) is 39 ℃, increase significantly than granular PNIPA gel under the corresponding conditions (maximum swelling multiplying power 6.3,33 ℃ of lower critical solution temperatures), the result as shown in Figure 1.The median size of gel is 0.3mm, is distributed in the narrower zone of 0.2～0.5mm, good dispersion property, and the gel particles surface arrangement some amount diameter is arranged is the hole of 1～2 μ m.This granulated gel has temperature sensitivity preferably, reaches swelling and the shrink balance only needs about 15min in the aqueous solution.
3) N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, be mixed with the solution that concentration is 10mg/mL, place 37 ℃, 100rpm shaking table to handle 120min solution, obtain the unfolding attitude lysozyme soln of non-activity, get the unfolding attitude lysozyme soln of 0.25mL non-activity, slowly join in the renaturation buffer, mixing, the final volume of renaturation system is 10mL, wherein, the add-on of granular P (NIPA-co-SA) gel copolymer is 120mg/mL (for 480 times of lysozyme concentration), and solution is that 20 ℃, rotating speed are renaturation 24h in the 100rpm shaking table in temperature.The antalzyme activity rate of recovery reaches 60.1% after the renaturation.The sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), and pH 8.5.Renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.
Embodiment 5
1) get the NaOH solution that 40mL concentration is 0.2mol/L, add the excessive propene aqueous acid, behind the stirring reaction 5h, solvent removed by evaporation at reduced pressure and remaining vinylformic acid can be collected and obtain the sodium acrylate solid;
2) the tensio-active agent tween-80 is dissolved in to make its concentration in the 80mL organic phase whiteruss be 1.5% (g/mL), adds reactor, feed nitrogen, stir 30min, tensio-active agent is fully disperseed.The aqueous solution of raw material that contains various reactive components that with volume is organic phase volume 1/3 then slowly is added dropwise in the reactor with liquid-transfering gun; aqueous solution of raw material consists of main monomer concentration 14% (g/g); degree of crosslinking is 10% (g/g); monomer N-N-isopropylacrylamide is 15.7:1 with the mol ratio that self-control is total to the monomer sodium acrylate; the concentration of oxygenant ammonium persulphate is 0.8% (g/mL); under nitrogen protection; after stirring 30min; slowly add initiator N, N, N '; N '-Tetramethyl Ethylene Diamine 3 μ L; 20 ℃ of polymerization temperatures, stirring velocity 280rpm, reaction 4h discharging.Product dehydrated alcohol/water washing by soaking is repeatedly dried in loft drier.Granular P (NIPA-co-SA) the gel copolymer maximum swelling multiplying power that obtains is 9, its lower critical solution temperature (LCST) is 39 ℃, increase significantly than granular PNIPA gel under the corresponding conditions (maximum swelling multiplying power 6.3,33 ℃ of lower critical solution temperatures), the result as shown in Figure 1.The median size of gel is 0.3mm, is distributed in the narrower zone of 0.2～0.5mm, good dispersion property, and the gel particles surface arrangement some amount diameter is arranged is the hole of 1～2 μ m.This granulated gel has temperature sensitivity preferably, reaches swelling and the shrink balance only needs about 15min in the aqueous solution.
3) N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, be mixed with the solution that concentration is 10mg/mL, place 37 ℃, 100rpm shaking table to handle 120min solution, obtain the unfolding attitude lysozyme soln of non-activity, get the unfolding attitude lysozyme soln of 0.25mL non-activity, slowly join in the renaturation buffer, mixing, the final volume of renaturation system is 10mL, wherein, the add-on of granular P (NIPA-co-SA) gel copolymer is 120mg/mL (for 480 times of lysozyme concentration), and solution is that 40 ℃, rotating speed are renaturation 24h in the 100rpm shaking table in temperature.The antalzyme activity rate of recovery reaches 57.2% after the renaturation.The sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), and pH 8.5.Renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.
Embodiment 6
1) get the NaOH solution that 60mL concentration is 0.4mol/L, add the excessive propene aqueous acid, behind the stirring reaction 4h, solvent removed by evaporation at reduced pressure and remaining vinylformic acid can be collected and obtain the sodium acrylate solid;
2) the tensio-active agent tween-80 is dissolved in to make its concentration in the 100mL organic phase whiteruss be 1% (g/mL), adds reactor, feed nitrogen, stir 25min, tensio-active agent is fully disperseed.The aqueous solution of raw material that contains various reactive components that with volume is organic phase volume 1/3 then slowly is added dropwise in the reactor with liquid-transfering gun; aqueous solution of raw material consists of main monomer concentration 14% (g/g); degree of crosslinking is 10% (g/g); monomer N-N-isopropylacrylamide is 15.7:1 with the mol ratio that self-control is total to the monomer sodium acrylate; the concentration of oxygenant ammonium persulphate is 0.5% (g/mL); under nitrogen protection; after stirring 40min; slowly add initiator N, N, N '; N '-Tetramethyl Ethylene Diamine 2 μ L; 20 ℃ of polymerization temperatures, mechanical stirring speed 280rpm, reaction 3h discharging.Product dehydrated alcohol/water washing by soaking is repeatedly dried in loft drier.Granular P (NIPA-co-SA) the gel copolymer maximum swelling multiplying power that obtains is 9, its lower critical solution temperature (LCST) is 39 ℃, increase significantly than granular PNIPA gel under the corresponding conditions (maximum swelling multiplying power 6.3,33 ℃ of lower critical solution temperatures), the result as shown in Figure 1.The median size of gel is 0.3mm, is distributed in the narrower zone of 0.2～0.5mm, good dispersion property, and the gel particles surface arrangement some amount diameter is arranged is the hole of 1～2 μ m.This granulated gel has temperature sensitivity preferably, reaches swelling and the shrink balance only needs about 15min in the aqueous solution.
3) N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, be mixed with the solution that concentration is 10mg/mL, place 37 ℃, 120rpm shaking table to handle 90min solution, obtain the unfolding attitude lysozyme soln of non-activity, get the unfolding attitude lysozyme soln of 0.5mL non-activity, slowly join in the renaturation buffer, mixing, the final volume of renaturation system is 10mL, wherein, the add-on of granular P (NIPA-co-SA) gel copolymer is 240mg/mL (for 480 times of lysozyme concentration), and solution is that 30 ℃, rotating speed are renaturation 20h in the 140rpm shaking table in temperature, and the result as shown in Figure 2.The antalzyme activity rate of recovery reaches 58.5% after the renaturation, and the activity recovery of dilution refolding only is 34.8% under the same terms.The sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), and pH 8.5.Renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.
Embodiment 7
1) get the NaOH solution that 50mL concentration is 0.3mol/L, add the excessive propene aqueous acid, behind the stirring reaction 6h, solvent removed by evaporation at reduced pressure and remaining vinylformic acid can be collected and obtain the sodium acrylate solid;
2) the tensio-active agent tween-80 is dissolved in to make its concentration in the 100mL organic phase whiteruss be 1% (g/mL), adds reactor, feed nitrogen, stir 25min, tensio-active agent is fully disperseed.The aqueous solution of raw material that contains various reactive components that with volume is organic phase volume 1/3 then slowly is added dropwise in the reactor with liquid-transfering gun; aqueous solution of raw material consists of main monomer concentration 14% (g/g); degree of crosslinking is 10% (g/g); monomer N-N-isopropylacrylamide is 15.7:1 with the mol ratio that self-control is total to the monomer sodium acrylate; the concentration of oxygenant ammonium persulphate is 0.5% (g/mL); under nitrogen protection; after stirring 40min; slowly add initiator N, N, N '; N '-Tetramethyl Ethylene Diamine 2 μ L; 20 ℃ of polymerization temperatures, stirring velocity 280rpm, reaction 3h discharging.Product dehydrated alcohol/water washing by soaking is repeatedly dried in loft drier.Granular P (NIPA-co-SA) the gel copolymer maximum swelling multiplying power that obtains is 9, its lower critical solution temperature (LCST) is 39 ℃, increase significantly than granular PNIPA gel under the corresponding conditions (maximum swelling multiplying power 6.3,33 ℃ of lower critical solution temperatures), the result as shown in Figure 1.The median size of gel is 0.3mm, is distributed in the narrower zone of 0.2～0.5mm, good dispersion property, and the gel particles surface arrangement some amount diameter is arranged is the hole of 1～2 μ m.This granulated gel has temperature sensitivity preferably, reaches swelling and the shrink balance only needs about 15min in the aqueous solution.
3) N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, be mixed with the solution that concentration is 10mg/mL, place 37 ℃, 100rpm shaking table to handle 120min solution, obtain the unfolding attitude lysozyme soln of non-activity, get the unfolding attitude lysozyme soln of 0.1mL non-activity, slowly join in the renaturation buffer, mixing, the final volume of renaturation system is 10mL, wherein, the add-on of granular P (NIPA-co-SA) gel copolymer is 48mg/mL (for 480 times of lysozyme concentration), and solution is that 30 ℃, rotating speed are renaturation 20h in the 140rpm shaking table in temperature, and the result as shown in Figure 2.The antalzyme activity rate of recovery reaches 81.7% after the renaturation, and dilution refolding only can obtain 74.3% activity recovery under the same terms.The sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), and pH 8.5.Renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.
Embodiment 8
Renaturation process finishes the back only to be needed solution is placed 45 ℃ thermostatic bath 30min, and gel is fully shunk, and realizes separating of target protein after gel and the renaturation.Place the water-bath of 4 ℃ and 50 ℃ to make its swelling, shrink repeatedly respectively granular P (NIPA-co-SA) gel copolymer after separating, repeat 10 times after, can realize the recovery of gel easily and quickly.After P (NIPA-co-SA) gel copolymer that cleans up displaced wherein water with dehydrated alcohol, be put in the baking oven and dry, can be used for proteinic renaturation experiment once more.The gel that reclaims by this method can be used for the lysozyme renaturation experiment repeatedly, and the result as shown in Figure 3.When the N,O-Diacetylmuramidase starting point concentration was 250 μ g/mL, gel still can reach 58.1% the antalzyme activity rate of recovery after reusing 6 times, improved about 13% than dilution refolding.
Claims (5)
1. the method for granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer assisting lysozyme in vitro refolding by means is characterized in that the step of method is as follows:
1) get the NaOH solution that 40～60mL concentration is 0.2～0.4mol/L, add excessive acrylic acid aqueous solution, behind stirring reaction 4～6h, solvent removed by evaporation at reduced pressure and remaining vinylformic acid can be collected and obtain the sodium acrylate solid;
2) the tensio-active agent tween-80 is dissolved in 75～100mL organic phase whiteruss, making its concentration is 1～1.5% (g/mL), add reactor, feed nitrogen, stir 20～30min, tensio-active agent is fully disperseed, the aqueous solution of raw material that contains various reactive components that with volume is organic phase volume 1/3 then slowly is added dropwise in the reactor with liquid-transfering gun, aqueous solution of raw material consists of: mass percent concentration is the main monomer of 14% (g/g), degree of crosslinking is 10% (g/g), the monomer N-N-isopropylacrylamide mol ratio of monomer sodium acrylate together is 19:1～9:1, concentration is the oxygenant ammonium persulphate of 0.5～0.8% (g/mL), under nitrogen protection, after stirring 30～40min, slowly add initiator N, N, N ', N '-Tetramethyl Ethylene Diamine 2～3 μ L, polymerization temperature is controlled at 20～25 ℃, stirring velocity is controlled at 250～280rpm, reaction 3～4h discharging, product dehydrated alcohol/water washing by soaking repeatedly, in vacuum drying oven, dry, can obtain granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer; Wherein, main monomer comprises monomer N-N-isopropylacrylamide and linking agent N, N '-methylene-bisacrylamide, and degree of crosslinking is meant linking agent N, N '-methylene-bisacrylamide shared mass percent in main monomer;
3) N,O-Diacetylmuramidase is dissolved in the sex change damping fluid, be mixed with the solution that concentration is 10mg/mL, place 37 ℃, 100～120rpm shaking table to handle 90～120min solution, obtain the unfolding attitude lysozyme soln of non-activity, get the unfolding attitude lysozyme soln of 0.1～0.5mL non-activity, slowly add renaturation buffer, mixing obtains 10mL renaturation system solution;
4) in the renaturation system solution, add granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer, gel quality affects concentration is 160～560 times of N,O-Diacetylmuramidase mass concentration in the renaturation system solution, and solution is that 20～40 ℃, rotating speed are renaturation 16～24h in the shaking table of 100～140rpm in temperature.
2. the method for a kind of granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer assisting lysozyme in vitro refolding by means according to claim 1 is characterized in that described N-N-isopropylacrylamide adopts the normal hexane recrystallization.
3. the method for a kind of granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer assisting lysozyme in vitro refolding by means according to claim 1, it is characterized in that described sex change damping fluid is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 8mol/L urea, 1mmol/L sodium ethylene diamine tetracetate and 30mmol/L dithiothreitol (DTT), pH 8.5.
4. the method for a kind of granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer assisting lysozyme in vitro refolding by means according to claim 1, it is characterized in that described renaturation buffer is 0.1mol/L three (methylol) aminomethane-hydrochloric acid soln that contains 1mmol/L sodium ethylene diamine tetracetate, 3mol/L urea, 0.15mol/L sodium-chlor, 3mmol/L reduced glutathion and 0.375mmol/L Sleep-promoting factor B, pH8.5.
5. the method for a kind of granular poly-(N-N-isopropylacrylamide-sodium acrylate) gel copolymer assisting lysozyme in vitro refolding by means according to claim 1, it is characterized in that described gel can be by elevated temperature to the gel transformation temperature after lysozyme renaturation is finished, thereby make the protein solution in the gel shrink discharge gel network, carrying out centrifuging again can reuse gel Separation and Recovery from renaturation solution.
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| CN102675505A (en) * | 2012-05-16 | 2012-09-19 | 温州医学院 | Synthesis method of structure-controllable crylic acid copolymer and application of crylic acid copolymer in promoting basic protein in vitro renaturation |
| CN109692069A (en) * | 2018-12-25 | 2019-04-30 | 徐红花 | A kind of intelligent warming patch with overheating protection function |
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| CN102675505A (en) * | 2012-05-16 | 2012-09-19 | 温州医学院 | Synthesis method of structure-controllable crylic acid copolymer and application of crylic acid copolymer in promoting basic protein in vitro renaturation |
| CN109692069A (en) * | 2018-12-25 | 2019-04-30 | 徐红花 | A kind of intelligent warming patch with overheating protection function |
| CN109692069B (en) * | 2018-12-25 | 2021-07-06 | 东阳市善水环境工程有限公司 | Intelligent warm keeping patch with overheat protection function |
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