CN109384840A - Protein modification with isolate and purify and the enzyme activity of thiol protease regulation method - Google Patents
Protein modification with isolate and purify and the enzyme activity of thiol protease regulation method Download PDFInfo
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Abstract
The invention discloses a kind of protein modification with isolate and purify and the enzyme activity of thiol protease regulation method, polymeric is connected on protein sulfhydryl, critical-temperature characteristic using polymer NIPAM at 32 DEG C, when temperature is more than 32 DEG C, albumen-polymer is precipitated from solution, albumen-polymer is centrifugation down again at this time, pure albumen-polymer is handled with DTT or other thio-alcohol reducing agents, breaks macromolecule to obtain former albumen, achievees the purpose that purify destination protein.The surface exposure sulfydryl of thiol protease and its enzymatic activity are closely related, its enzymatic activity can also be lost after surface sulfydryl is destroyed, and when the protein is for thiol protease, the regulation to enzyme activity is may be implemented in this method.
Description
Technical field
The invention discloses a kind of protein modification with isolate and purify and the enzyme activity of thiol protease regulation method.
Background technique
Currently, there are many kinds of the isolation and purification methods of protein: (1) it according to molecular size difference is isolated and purified, it can
It is divided into dialysis, ultrafiltration, centrifugation and gel filtration；(2) it is isolated and purified according to different solubility, common method has isoelectric point
Precipitating and pH value are adjusted, the salt of protein is molten and are saltoutd, organic solvent method, aqueous two-phase system, reverse micelle extraction method etc.；(3)
According to the charge of protein, that is, acid-base property difference method of separating protein, there is two class of electrophoresis and ion-exchange chromatography.It is above-mentioned
Separation method or cumbersome or separation are not thorough, some also need several separation methods to be used in combination.
Protein especially protease is in various environmental changes under the conditions of such as variation of temperature change, pH, ultraviolet irradiation
It is easy to lose vigor, so the protection of enzyme activity is a very important project.At present to the protection scheme of prolease activity
The mainly crucial group of sealase active centre.The enzyme activity of thiol protease is just controlled by its surface sulfydryl, some environment
Variation cause sulfydryl destroy, fracture, oxidation etc. will lead to thiol protease lose enzyme activity.Protection thiol protease at present
The method of enzyme activity mainly adds some reducing agents such as cysteine, three (2- carboxyethyl) phosphines etc. and its sulfydryl is protected not aoxidized
Damage.These methods need to add always reagent and maintain the sulfydryl of protease not by oxidative damage, and Expenses Cost is higher and operates
Trouble.
Summary of the invention
It is an object of the present invention to provide a kind of modification of protein with isolate and purify, connected on protein sulfhydryl
Polymeric, the critical-temperature characteristic using polymer NIPAM at 32 DEG C, albumen-polymer when temperature is more than 32 DEG C
It is precipitated from solution, is at this time again centrifugation down albumen-polymer, by pure albumen-polymer DTT or other sulphur
The processing of alcohols reducing agent breaks macromolecule to obtain former albumen, achievees the purpose that purify destination protein.
A kind of modification and isolation and purification method containing thiol protein, the albumen after modification and modification including protein
Matter isolates and purifies, and the modification of the protein is the coupling NIPAM polymer on the sulfydryl of protein, obtains albumen-NIPAM
Polymer；The isolation and purification method of the protein are as follows: by the mixed solution containing albumen-NIPAM polymer and other materials
32 DEG C or more are heated to, so that albumen-NIPAM polymer is precipitated from mixed solution, precipitating is collected by centrifugation, obtains pure egg
White-NIPAM polymer finally handles albumen-NIPAM polymer with thio-alcohol reducing agent, then dialyses, the albumen purified
Matter.
Specifically, the method for modifying of the protein the following steps are included:
1) protein and thio-alcohol reducing agent (such as TCEP) are dissolved separately in acetate buffer solution, are separately connected nitrogen deoxygenation
Device deoxygenation 7-10min, respectively obtains protein solution and thio-alcohol reducing agent solution；Thio-alcohol reducing agent solution is slowly added dropwise
Into protein solution, magnetic agitation reacts 7-10min under the conditions of nitrogen deoxygenation, mixed liquor is moved into bag filter after reaction, thoroughly
Analysis bag merging buffer in dialyses three times, for the first time dialysis use acetate buffer solution, after twice dialysis use phosphate buffer,
Entire dialysis procedure connects nitrogen deaerating plant, and liquid, that is, dialyzate A in bag filter is collected after dialysis；
2) pH value of dialyzate A is adjusted to being not less than 8.0, by initiator bis- [2- (2 '-bromo isobutyl acyloxy) ethyls] two sulphur
Compound (Bis [2- (2 '-bromoisobutyryloxy) ethyl] disulfide, hereinafter referred to as BiBOEDS) solution slowly by
It is added dropwise in dialyzate A, magnetic agitation reacts 30-35min, the mixed liquor after reaction is transferred in bag filter, using phosphoric acid
Buffer is dialysed three times, dialyses the dimethyl sulfoxide for being separately added into final concentration of 7.7%, 3.85%, 1% every time into phosphate buffer,
After dialysis, with the liquid in 0.22 μm of filter filtering bag filter, dialyzate B is obtained；
3) it takes 5 mL dialyzate B to move into round-bottomed flask A and connects nitrogen deaerating plant deoxygenation at least 23min；
Take 6 mg CuCl2, 10 mL pure water, 5 mL methanol, 7 μ L PMDETA and 600mg NIPAM monomers are added to circle
In the flask B of bottom, be added 7 mg ascorbic acid deoxygenation 20min again after nitrogen deoxygenation 25min, the above operation under the conditions of being protected from light into
Row；Reaction system nitrogen in round-bottomed flask B is pressed into round-bottomed flask A and nitrogen deoxygenation is kept to react 30min, reaction knot
Mixture is moved into bag filter after beam and is dialysed three times in phosphate buffer, after dialysis, with 0.22 μm of filter
Filter the liquid in bag filter, the protein after being modified, i.e. albumen-NIPAM polymer.
Specifically, protein after the modification isolate and purify the following steps are included:
1) by albumen-NIPAM polymer in the at a temperature of water-bath 3-4min not less than 32 DEG C, then 11000rpm centrifugation is laid equal stress on
It is outstanding, in triplicate more than, obtain pure albumen-NIPAM polymer；
2) thio-alcohol reducing agent (such as DTT) is added in above-mentioned albumen-NIPAM polymer, handles 10-15min, reaction solution
The protein purified three times with buffer dialysis.
It is another object of the present invention to provide a kind of enzyme activity of thiol protease to regulate and control method, in thiol protease
Polymeric is connected on sulfydryl, is closed sulfydryl with macromolecule, can be protected sulfydryl under violent environmental change not in this way
It is damaged, and enzyme activity temporary closure can be made in the case where specifically not needing enzyme activity.Under relatively mild environment
Or need to break polymer with dithiothreitol (DTT) reducing agent when enzyme activity, then it can restore the activity of thiol protease.
To achieve the goals above the invention adopts the following technical scheme:
A kind of enzyme activity of thiol protease regulates and controls method comprising the enzyme activity suppressing method of thiol protease and thiol protease
Enzyme activity restoration methods；The enzyme activity suppressing method of the thiol protease are as follows: NIPAM polymerization is coupled on the sulfydryl of thiol protease
Object, obtains thiol protease-NIPAM polymer, and the thiol protease-NIPAM polymer makes the sulfydryl of thiol protease
It is closed by NIPAM polymer, to inhibit enzyme activity；
The enzyme activity restoration methods of the thiol protease are as follows: will be heated containing the mixed solution of thiol protease-NIPAM polymer
To 32 DEG C or more, so that thiol protease-NIPAM polymer is precipitated from mixed solution, precipitating is collected by centrifugation, precipitating is dissolved
To remove impurity in PBS, thiol protease-NIPAM polymer solution is obtained, finally handles sulfydryl egg with thio-alcohol reducing agent
White enzyme-NIPAM polymer solution, then dialyses, the thiol protease for the enzyme activity that is restored.
The thiol protease is papain or bromelain.
Specifically, the enzyme activity suppressing method of the thiol protease the following steps are included:
1) thiol protease and thio-alcohol reducing agent (such as TCEP) are dissolved separately in acetate buffer solution, are separately connected nitrogen
Deaerating plant deoxygenation 7-10min, respectively obtains protein enzyme solution and thio-alcohol reducing agent solution；Thio-alcohol reducing agent solution is delayed
Slowly it is added drop-wise in protein enzyme solution, magnetic agitation reacts 7-10min under the conditions of nitrogen deoxygenation, moves into mixed liquor thoroughly after reaction
Analyse bag, bag filter merging buffer in dialyse three times, for the first time dialysis use acetate buffer solution, after twice dialysis use phosphate
Buffer, entire dialysis procedure connect nitrogen deaerating plant, and liquid, that is, dialyzate A in bag filter is collected after dialysis；
2) pH value of dialyzate A is adjusted to being not less than 8.0, and initiator solution BiBOEDS(is slowly added dropwise in dialyzate A,
Magnetic agitation reacts 30-35min, and the mixed liquor after reaction is transferred in bag filter, three times using phosphate buffer dialysis, often
The secondary dimethyl sulfoxide for being separately added into final concentration of 7.7%, 3.85%, 1% of dialysing is into phosphate buffer, after dialysis, with 0.22
μm filter filtering bag filter in liquid, obtain dialyzate B；
3) it takes 5 mL dialyzate B to move into round-bottomed flask A and connects nitrogen deaerating plant deoxygenation at least 23min；
Take 6 mg CuCl2, 10 mL pure water, 5 mL methanol, 7 μ L PMDETA and 600mg NIPAM monomers are added to circle
In the flask B of bottom, 7 mg ascorbic acid deoxygenation 20min again is added after nitrogen deoxygenation 25min；
Reaction system nitrogen in round-bottomed flask B is pressed into round-bottomed flask A and nitrogen deoxygenation is kept to react 30min, reaction
After mixture moved into bag filter and dialyse three times in phosphate buffer, after dialysis, with 0.22 μm of filtering
Device filters the liquid in bag filter, obtains the repressed thiol protease-NIPAM polymer of enzyme activity, the above operation is being protected from light item
It is carried out under part.
Specifically, the enzyme activity restoration methods of the thiol protease the following steps are included:
1) after thiol protease-NIPAM polymer being incubated for 3-4min in the water-bath not less than 32 DEG C, lotion is collected and 37
DEG C with 11000rpm be centrifuged 5min, then with pipette remove supernatant, sediment is dissolved in PBS at room temperature, is obtained pure
Thiol protease-NIPAM the polymer solution of change；
2) the DTT solution of final concentration 10mM is added in the thiol protease-NIPAM polymer solution of purifying, will be molten after reaction
Liquid is dialysed with phosphate buffer, the thiol protease for the enzyme activity that is restored.
The present invention relates to albumen-polymer prepare (protein modified), separation and purification of protein, enzyme activity regulation new technology.
This is the new technology for being grafted upper polymer to albumen using a kind of method for being referred to as " controllable atom polymerization ", by coupling polymerization
The protein of object modification can improve its property and function, improve protein solubility and stability etc..Due to NIPAM polymer
Solution generates muddiness at 32 DEG C or more, NIPAM polymer can be allowed to precipitate under high speed centrifugation, so polymerizeing in NIPAM
32 DEG C or more can be heated in object and the miscible solution of other materials, in this approach by NIPAM polymer and other objects
Matter separates, to achieve the purpose that isolate and purify in NIPAM polymer.The present invention is exactly this for utilizing NIPAM polymer
Characteristic carry out albumen isolate and purify and the enzyme activity of thiol protease regulation.
The thiol proteases such as papain all have exposed with bovine serum albumin (or other albumen containing sulfydryl) surface
Sulfydryl, albumen and initiator are connected into initiator by the Sulfhydryl Groups after certain mol proportion (such as 1:5) mixing in albumen；
It will connect and obtain albumen-after albumen and NIPAM monomer according to a certain mass ratio (such as 1:40) hybrid reaction after initiator and polymerize
Object product, then utilize NIPAM polymer peculiar property, be not less than 32 DEG C under conditions of by protein from other materials
It is separated in miscible solution, finally the key that albumen is connect with polymer is broken with reducing agent dithiothreitol (DTT), with sudden and violent
The sulfydryl for exposing protein surface makes albumen restore to the original state.
The surface exposure sulfydryl of the thiol proteases such as papain and its enzymatic activity are closely related, and surface sulfydryl is destroyed
Its enzymatic activity can also be lost afterwards.So being tie point to the sulfydryls albumen such as papain using the surface sulfydryl of thiol protease
After enzyme is modified, activity will be lost, and thus may be implemented in the case where different environment needs controllably to sulfydryl albumen
The activity of enzyme carries out switch-mode regulation；After thiol protease Sulfhydryl Groups are conjugated upper polymer, so that it may in environment abrupt change
When its sulfydryl is protected and preferably protects its enzymatic activity.And after breaking coupling polymer from tie point with reducing agent DTT
The activity of thiol protease is again to restore.
Detailed description of the invention
Fig. 1 is bovine serum albumin(BSA) (BSA), bovine serum albumin(BSA) and casein mixture (BSA- after SDS-PAGE electrophoresis
C), the polymerization modification of BSA is schemed with purifying in fetal calf serum (FBS)；
Fig. 2 is papain and bromelain enzymatic polymerization modification purifying figure；
Wherein, left figure Lane1:Mark；Lane2:Pap(papain, papain)；Lane3:Pap-PC(papain-
Polymer complex wooden pipe protease-polymer conjugates)；
Right figure Lane1:Mark；Lane2:Bro(bromelain, bromelain)；Lane3:Bro-R(bromelain-
Polymer complex bromelain-polymer conjugates)；
Fig. 3 is protein concentration canonical plotting；
Fig. 4 is the enzyme activity experimental result picture of papain and bromelain.
Specific embodiment
The modification of 1 protein of embodiment with isolate and purify
One, protein-polymer prepares (protein modified)
1 TCEP protein surface restores sulfydryl
1) TCEP of 80mg BSA and 11mg are dissolved into the acetate buffer solution of 10mL by 1:1 respectively in molar ratio, are separately connected nitrogen
Gas deaerating plant deoxygenation 7min；Respectively obtain TCEP solution and protein solution；
2) TCEP is slowly dropped in protein solution, magnetic agitation reacts 7min under the conditions of nitrogen deoxygenation, will mixing after reaction
Liquid move into bag filter, bag filter merging buffer in dialyse three times, for the first time use acetate buffer solution, after use phosphate-buffered twice
Liquid, entire dialysis procedure connect nitrogen deaerating plant, and liquid, that is, dialyzate A in bag filter is collected after dialysis.
2 protein surface sulfydryl modification initiators
1) it takes 10mL dialyzate A to be added in sizeable weighing bottle, adjusts solution ph not after 2mL Buffer 8.2 is added
Less than 8.0.
2) 1mL initiator solution BiBOEDS being slowly added dropwise in weighing bottle, magnetic stirrer reacts 30min,
Mixed liquor after abundant reaction is transferred in bag filter and is dialysed, three times with phosphate buffer dialysis, dialysis is separately added into every time
The dimethyl sulfoxide of final concentration 7.7%, 3.85%, 1% is into dialyzate, after the completion of dialysis, collects the disposable filter with 0.22 μm
Filtered sample obtains dialyzate B.
3 albumen substrate atoms transferring free-radical polymerizations
1) 5 mL dialyzate B move into the round-bottomed flask A of a 50mL, are connected to nitrogen deaerating plant deoxygenation at least 23min；
2) 6 mg CuCl are added in round-bottomed flask B2, 10 mL pure water, 5 mL methanol, 7 μ L PMDETA and 600mg
NIPAM monomer, 7 mg ascorbic acid are added after nitrogen deoxygenation 25min, and the black polybag of deoxygenation 20min(covers flask and is protected from light again)；
3) round-bottomed flask A and round-bottomed flask B is connected with hollow finer wire, will includes the polymerization reaction of monomer in round-bottomed flask B
It is mixed with albumen in system nitrogen indentation round-bottomed flask A and nitrogen deoxygenation is kept to react the black polybag of 30min(and cover flask and kept away
Light),.Mixture immigration bag filter is dialysed three times in phosphate buffer after reaction, after 0.22 μm of filter filtering
Collect the liquid in filter bag filter, the protein after being modified, i.e. BSA albumen-NIPAM polymer.
Two, protein isolates and purifies
1) above-mentioned BSA albumen-NIPAM polymer is centrifuged and is resuspended in 45 DEG C of water-bath 3min, 11000rpm, in triplicate to obtain
Obtain pure albumen-NIPAM polymer.
2) DTT is added, after handling 10min, reaction solution is dialysed three times with buffer, the BSA albumen purified.
Three, electrophoresis detection
Albumen after being restored with SDS-PAGE electrophoresis detection albumen-NIPAM polymer and with DTT, as a result as shown in Figure 1.
The present embodiment polymerize modification purifying cow's serum also in bovine serum albumin(BSA) and casein mixture, in fetal calf serum
Albumin, the further success of verifying polymerization modification and purifying, the same bovine serum albumin(BSA) of specific embodiment, as a result such as Fig. 1
It is shown.
Fig. 1 shows bovine serum albumin(BSA) (BSA), bovine serum albumin(BSA) and casein mixture after SDS-PAGE electrophoresis
(BSA-C) and the polymerization of BSA is modified in fetal calf serum (FBS) and purifying is schemed.
The enzyme activity of 2 thiol protease of embodiment regulates and controls
One, the enzyme activity of thiol protease inhibits
1 TCEP protein surface restores sulfydryl
1) in molar ratio 1:1 by the thiol protease albumen of 80mg (the present embodiment respectively with bromelain Bromelain and
For Papain papain) and the TCEP of 11mg be dissolved into the acetate buffer solution of 10mL respectively, be separately connected nitrogen deoxygenation
Device deoxygenation 7min；Respectively obtain protein enzyme solution and TCEP solution；
2) TCEP solution is slowly dropped in protein solution, magnetic agitation reacts 7min under the conditions of nitrogen deoxygenation, will after reaction
Mixed liquor move into bag filter, bag filter merging buffer in dialyse three times, for the first time use acetate buffer solution, after use phosphate twice
Buffer, entire dialysis procedure connect nitrogen deaerating plant, and liquid, that is, dialyzate A in bag filter is collected after dialysis.
2 protease surface sulfydryl modification initiators
1) it takes 10mL dialyzate A to be added in sizeable weighing bottle, adjusts solution ph not after 2mL Buffer 8.2 is added
Less than 8.0.
2) 1mL initiator solution BiBOEDS being slowly added dropwise in weighing bottle, magnetic stirrer reacts 30min,
Mixed liquor after abundant reaction is transferred in bag filter and is dialysed, three times with phosphate buffer dialysis, dialysis is separately added into every time
The dimethyl sulfoxide of final concentration 7.7%, 3.85%, 1% is into dialyzate, after the completion of dialysis, collects the disposable filter with 0.22 μm
Filtered sample obtains dialyzate B.
3 protease-based bottom atom transfer radical polymerization
1) 5 mL dialyzate B move into the round-bottomed flask A of a 50mL, are connected to nitrogen deaerating plant deoxygenation at least 23min；
2) 6 mg CuCl are added in round-bottomed flask B2, 10 mL pure water, 5 mL methanol, 7 μ L PMDETA and 600mg
NIPAM monomer, 7 mg ascorbic acid are added after nitrogen deoxygenation 25min, and the black polybag of deoxygenation 20min(covers flask and is protected from light again)；
3) round-bottomed flask A and round-bottomed flask B is connected with hollow finer wire, will includes the polymerization reaction of monomer in round-bottomed flask B
It is mixed with albumen in system nitrogen indentation round-bottomed flask A and nitrogen deoxygenation is kept to react the black polybag of 30min(and cover flask and kept away
Light),.Mixture immigration bag filter is dialysed three times in phosphate buffer after reaction, after 0.22 μm of filter filtering
The liquid in filter bag filter is collected, the repressed Papain-NIPAM polymer of enzyme activity and Bromelain-NIPAM are respectively obtained
Polymer.
Two, the enzyme activity of thiol protease is restored
1) after Papain-NIPAM polymer (or Bromelain-NIPAM polymer) is incubated for 3min in 40 DEG C of water-baths, solution
Become white and opaque shape, lotion is centrifuged 5min at 37 DEG C with 11000rpm, then removes supernatant, room temperature with pipette
It is lower that sediment is dissolved in PBS, repeat the program 3 times thoroughly to remove other soluble impurities, the Papain- purified
NIPAM polymer (or Bromelain-NIPAM polymer).
2) the DTT solution of final concentration 10mM is added to Papain-NIPAM polymer (or the Bromelain-NIPAM of purifying
Polymer) in disconnect disulfide bond, then solution phosphate buffer is dialysed to remove extra DTT and broken high score
Son, Papain or Bromelai after collecting reduction, enzyme activity is at this time to be restored.
Three, electrophoresis detection
It is restored with SDS-PAGE electrophoresis detection Papain-NIPAM polymer and Bromelain-NIPAM polymer and with DTT
Papain and Bromelain afterwards, as a result as shown in Figure 2.
Embodiment 3
Polymer is done to the Protection of thiol protease vigor, using 4 kinds of different disposals with papain and bromelain respectively
Method carries out enzyme activity comparison.
According to Fig. 3 protein concentration standard curve determination Papain, Papain-NIPAM polymer, Bromelain and
The concentration of Bromelain-NIPAM polymer, and concentration is all adjusted to 0.1mg/mL and surveys enzyme activity, because of concentration casein hydrolysis thus
Measurement light absorption value is in the measurable OK range of ultraviolet specrophotometer afterwards.
1) four kinds of processing modes are as follows:
Papain, Papain-NIPAM polymer are identical with the processing mode of bromelain, Bromelain-NIPAM polymer,
Illustrate by taking papain as an example below.
A) pH2.0 is handled
Comprehensively consider sample size, experimental error, facilitate the factors such as pH adjusting, respectively takes a certain amount of Papain and Papain-NIPAM
Polymer is diluted to 15mL to 50mL centrifuge tube, with PBS buffer solution, adjusts pH to 2.0 with hydrochloric acid solution, overnight rear (about 12h)
PH is adjusted back 7.4 again, is settled to 20mL, concentration 0.1mg/mL.
B) pH12.0 is handled
Processing mode is same a)
C) ± 80 DEG C of processing
Papain and Papain-NIPAM polymer is all adjusted to the concentration to 0.1 mg/mL, 80 DEG C of water-bath 30min wait temperature
Degree, which is cooled to room temperature, is placed on -80 DEG C of refrigerator freezing 30min.
D) Papain-NIPAM polymer treatment
The exposure surface Papain sulfydryl, surveys enzyme activity after restoring its activity after adding DTT to break polymer.
2) processing result is as shown in Figure 4.
A) figure and C) in figure, Pap and Bro are the enzyme activity of papain and bromelain protoenzyme；
Pap-i and Bro-i is that two kinds of enzymes connect the enzyme activity after initiator；
Pap-PC and Bro-PC is the enzyme activity on two kinds of enzymatic polymerizations after Nippam；
Pap-R and Bro-R is reply active enzyme activity of the polymer of two kinds of enzymes after DDT is broken.
B) figure and D) in figure, Pap and Pap-R are protoenzyme and the enzyme after DDT reduction treatment breaks polymer respectively；
Conrol group is that protoenzyme and polymer enzyme do not do condition processing, by the control group enzyme activity after the reduction of polymer enzyme enzymatic activity
Power；
2.0 groups of pH for protoenzyme and polymer enzyme, the pH after the processing of 2.0 acid condition of pH recalls to 7.0 enzyme activities measured, (DTT
Restore the enzyme activity of polymer enzyme)；
12.0 groups of pH for protoenzyme and polymer enzyme, the pH after the processing of 12.0 acid condition of pH recalls to 7.0 enzyme activities measured,
(enzyme activity of DTT recovery polymer enzyme)；
Temp group is that protoenzyme and polymer enzyme measure enzyme activity (DTT recovery polymer enzyme after ± 80 DEG C of processing under normal temperature condition
Enzyme activity).
Experimental result:
A) and C) enzyme activity of papain (Pap) and bromelain (Bro) and passes through all in relatively high level in figure
Albumen-high molecular polymer Pap-PC and Bro-PC enzymatic activity after reaction modification almost disappears.This is because polymerization reaction
Decorating site is in the sulfydryl of protein, and papain surface sulfydryl and its enzymatic activity are closely related, so the closing of sulfydryl
Directly result in the forfeiture of enzyme activity.It disconnects protein and connection initiator when with reducing agent dithiothreitol (DTT) in figure and gathers
The sulfydryl of albumen is exposed again after closing the high molecular disulfide bond of object, and the vigor of enzyme is restored at this time, as schemed A) and C) in
Pap-R and Bro-R.The enzyme activity recovery extent of papain and bromelain respectively reaches 88%(Pap-R/Pap) and
83%(Bro-R/Bro).
Still it can restore its most enzyme activity after papain and bromelain modified high molecular, illustrate egg
White matter loss of enzymatic activity during polymerization reaction is a series of be it is smaller, the activity of protein does not receive very big
Influence.Moreover, the phenomenon that macromolecule closed protein sulfydryl makes protease be temporarily lost with enzymatic activity is considered in turn,
This closing is also the protection to proteinase activity site sulfydryl.It can make protease sulphydryl activity position after macromolecule closing sulfydryl
Point will not thoroughly be destroyed in intense environment variation.
The present embodiment is measured respectively in pole acid condition (pH 2.0), pole alkaline condition (pH 12.0) and ± 80 DEG C of conditions
Protection of the lower macromolecule to protein sulfhydryl and enzyme activity.As a result such as figure B) and D), wherein Control group is not do environmental treatment
Control, Pap and Bro are the former protease of papain and bromelain by various treated enzyme activities, Pap-R and
Bro-R is that macromolecule modified papain and bromelain are adding DTT to break macromolecule exposure after various processing
Enzyme activity after sulfydryl.Under the acidic environment processing of pH 2.0, Pap and Bro enzyme activity retain 5% respectively [Pap(pH 2.0)/
Pap(Control)] and 1% [Bro(pH 2.0)/Bro(Control)], Pap-R and Bro-R enzyme activity retains 56% respectively
[Pap-R(pH 2.0)/Pap-R(Control)] and 31% [Bro-R(pH 2.0)/Bro-R(Control)]；In pH 12.0
Alkaline environment processing under, Pap and Bro enzyme activity retains 44% respectively [Pap(pH 12.0)/Pap(Control)] and 2% [Bro
(pH 12.0)/Bro(Control)], Pap-R and Bro-R enzyme activity retain 87% respectively [Pap-R(pH 12.0)/Pap-R
And 62% [Bro-R(pH 12.0)/Bro-R(Control)] (Control)]；Under ± 80 DEG C of environmental treatments, Pap and Bro enzyme
Vigor retains 64% respectively [Pap(± 80 DEG C)/Pap(Control)] and 4% [Bro(± 80 DEG C)/Bro(Control)], Pap-R
With Bro-R enzyme activity retain 68% respectively [Pap-R(± 80 DEG C)/Pap-R(Control)] and 58% [Bro-R(± 80 DEG C)/
Bro-R(Control)].Experimental result absolutely prove after albumen enzyme modification macromolecule to it enzyme activity in the environment of acute variation
Power has extraordinary protecting effect.
Claims (10)
1. a kind of modification and isolation and purification method containing thiol protein, the protein after modification and modification including protein
Isolate and purify, it is characterised in that: the modification of the protein be on the sulfydryl of protein coupling NIPAM polymer, obtain
Albumen-NIPAM polymer；
The isolation and purification method of the protein are as follows: will be heated containing the mixed solution of albumen-NIPAM polymer and other materials
To 32 DEG C or more, so that albumen-NIPAM polymer is precipitated from mixed solution, precipitating is collected by centrifugation, obtains pure albumen-
NIPAM polymer finally handles albumen-NIPAM polymer with thio-alcohol reducing agent, then dialyses, the protein purified.
2. a kind of modification and isolation and purification method containing thiol protein according to claim 1, it is characterised in that: described
The method of modifying of protein the following steps are included:
1) protein and thio-alcohol reducing agent are dissolved separately in acetate buffer solution, are separately connected nitrogen deaerating plant deoxygenation 7-
10min respectively obtains protein solution and thio-alcohol reducing agent solution；Thio-alcohol reducing agent solution is slowly dropped to protein solution
In, magnetic agitation reacts 7-10min under the conditions of nitrogen deoxygenation, and mixed liquor is moved into bag filter after reaction, and bag filter merging is slow
Dialyse in fliud flushing three times, for the first time dialysis use acetate buffer solution, after twice dialysis use phosphate buffer, entirely dialysed
Journey connects nitrogen deaerating plant, and liquid, that is, dialyzate A in bag filter is collected after dialysis；
2) pH value of dialyzate A is adjusted to being not less than 8.0, by initiator bis- [2- (2 '-bromo isobutyl acyloxy) ethyls] two sulphur
Compound solution is slowly added dropwise in dialyzate A, and magnetic agitation reacts 30-35min, and the mixed liquor after reaction is transferred to dialysis
In bag, three times using phosphate buffer dialysis, the dimethyl sulfoxide that dialysis is separately added into final concentration of 7.7%, 3.85%, 1% every time is arrived
In phosphate buffer, after dialysis, with the liquid in 0.22 μm of filter filtering bag filter, dialyzate B is obtained；
3) it takes 5 mL dialyzate B to move into round-bottomed flask A and connects nitrogen deaerating plant deoxygenation at least 23min；
Take 6 mg CuCl2, 10 mL pure water, 5 mL methanol, 7 μ L PMDETA and 600mg NIPAM monomers are added to round bottom
In flask B, 7 mg ascorbic acid deoxygenation 20min again is added after nitrogen deoxygenation 25min, the above operation carries out under the conditions of being protected from light；
Reaction system nitrogen in round-bottomed flask B is pressed into round-bottomed flask A and nitrogen deoxygenation is kept to react 30min, reaction terminates
Mixture is moved into bag filter afterwards and is dialysed three times in phosphate buffer, after dialysis, with 0.22 μm of filter mistake
Filter the liquid in bag filter, the protein after being modified, i.e. albumen-NIPAM polymer.
3. a kind of modification and isolation and purification method containing thiol protein according to claim 2, it is characterised in that: described
Thio-alcohol reducing agent is TCEP.
4. a kind of modification and isolation and purification method containing thiol protein according to claim 1, it is characterised in that: described
Protein after modification isolate and purify the following steps are included:
1) by albumen-NIPAM polymer in the at a temperature of water-bath 3-4min not less than 32 DEG C, then 11000rpm centrifugation is laid equal stress on
It is outstanding, in triplicate more than, obtain pure albumen-NIPAM polymer；
2) thio-alcohol reducing agent is added in above-mentioned albumen-NIPAM polymer, handles 10-15min, reaction solution buffer is saturating
Analyse the protein purified three times.
5. a kind of modification and isolation and purification method containing thiol protein according to claim 4, it is characterised in that: described
Thio-alcohol reducing agent is DTT.
6. a kind of enzyme activity of thiol protease regulates and controls method, it is characterised in that: it includes the enzyme activity suppressing method of thiol protease
With the enzyme activity restoration methods of thiol protease;
The enzyme activity suppressing method of the thiol protease are as follows: be coupled NIPAM polymer on the sulfydryl of thiol protease, obtain mercapto
Base protease-NIPAM polymer, the thiol protease-NIPAM polymer gather the sulfydryl of thiol protease by NIPAM
It closes object to be closed, to inhibit enzyme activity；
The enzyme activity restoration methods of the thiol protease are as follows: will be heated containing the mixed solution of thiol protease-NIPAM polymer
To 32 DEG C or more, so that thiol protease-NIPAM polymer is precipitated from mixed solution, precipitating is collected by centrifugation, precipitating is dissolved
To remove impurity in PBS, thiol protease-NIPAM polymer solution is obtained, finally handles sulfydryl egg with thio-alcohol reducing agent
White enzyme-NIPAM polymer solution, then dialyses, the thiol protease for the enzyme activity that is restored.
7. a kind of enzyme activity of thiol protease according to claim 6 regulates and controls method, it is characterised in that: the sulfydryl albumen
Enzyme is papain or bromelain.
8. a kind of enzyme activity of thiol protease according to claim 6 regulates and controls method, it is characterised in that: the sulfydryl albumen
The enzyme activity suppressing method of enzyme the following steps are included:
1) thiol protease and thio-alcohol reducing agent are dissolved separately in acetate buffer solution, are separately connected nitrogen deaerating plant and remove
Oxygen 7-10min respectively obtains protein enzyme solution and thio-alcohol reducing agent solution；Thio-alcohol reducing agent solution is slowly dropped to egg
In white enzyme solutions, magnetic agitation reacts 7-10min under the conditions of nitrogen deoxygenation, and mixed liquor is moved into bag filter after reaction, dialyses
Bag merging buffer in dialyse three times, for the first time dialysis use acetate buffer solution, after twice dialysis use phosphate buffer, it is whole
A dialysis procedure connects nitrogen deaerating plant, and liquid, that is, dialyzate A in bag filter is collected after dialysis；
2) pH value of dialyzate A is adjusted to being not less than 8.0, by initiator bis- [2- (2 '-bromo isobutyl acyloxy) ethyls] two sulphur
Compound solution is slowly added dropwise in dialyzate A, and magnetic agitation reacts 30-35min, and the mixed liquor after reaction is transferred to dialysis
In bag, three times using phosphate buffer dialysis, the dimethyl sulfoxide that dialysis is separately added into final concentration of 7.7%, 3.85%, 1% every time is arrived
In phosphate buffer, after dialysis, with the liquid in 0.22 μm of filter filtering bag filter, dialyzate B is obtained；
3) it takes 5 mL dialyzate B to move into round-bottomed flask A and connects nitrogen deaerating plant deoxygenation at least 23min；
Take 6 mg CuCl2, 10 mL pure water, 5 mL methanol, 7 μ L PMDETA and 600mg NIPAM monomers are added to round bottom
In flask B, 7 mg ascorbic acid deoxygenation 20min again is added after nitrogen deoxygenation 25min；
Reaction system nitrogen in round-bottomed flask B is pressed into round-bottomed flask A and nitrogen deoxygenation is kept to react 30min, reaction
After mixture moved into bag filter and dialyse three times in phosphate buffer, after dialysis, with 0.22 μm of filtering
Device filters the liquid in bag filter, obtains the repressed thiol protease-NIPAM polymer of enzyme activity, the above operation is being protected from light item
It is carried out under part.
9. a kind of enzyme activity of thiol protease according to claim 8 regulates and controls method, it is characterised in that: the thio-alcohol is also
Former agent is TCEP.
10. a kind of enzyme activity of thiol protease according to claim 6 regulates and controls method, it is characterised in that: the sulfydryl egg
The enzyme activity restoration methods of white enzyme the following steps are included:
1) after thiol protease-NIPAM polymer being incubated for 3-4min in the water-bath not less than 32 DEG C, lotion is collected and 37
DEG C with 11000rpm be centrifuged 5min, then with pipette remove supernatant, sediment is dissolved in PBS at room temperature, is obtained pure
Thiol protease-NIPAM the polymer solution of change；
2) the DTT solution of final concentration 10mM is added in the thiol protease-NIPAM polymer solution of purifying, will be molten after reaction
Liquid is dialysed with phosphate buffer, the thiol protease for the enzyme activity that is restored.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111848720A (en) * | 2020-08-10 | 2020-10-30 | 福州大学 | Method for performing macromolecule functional modification on glycosylated protein |
| CN114470865A (en) * | 2022-01-12 | 2022-05-13 | 武汉大学 | Method for simply, conveniently and quickly separating temperature-sensitive polymer and connector thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005039641A2 (en) * | 2003-10-15 | 2005-05-06 | The Regents Of The University Of California | Biomacromolecule polymer conjugates |
| CN1723232A (en) * | 2003-01-06 | 2006-01-18 | 尼克塔治疗亚拉巴马公司 | Thiol-selective water-soluble polmer derivatives |
| WO2014176279A1 (en) * | 2013-04-22 | 2014-10-30 | Carnegie Mellon Iniversity | Polymer-based protein engineering methods to rationally tune enzyme activity, ph-dependence and stability |
| CN104497097A (en) * | 2014-06-18 | 2015-04-08 | 深圳市第二人民医院 | Protein or polypeptide-polymer grafted copolymer with reduction responsiveness, synthesis method and protein or polypeptide drug thereof |
| CN104862288A (en) * | 2015-05-08 | 2015-08-26 | 苏州大学 | Synthesis of novel temperature-responsive inorganic pyrophosphatase conjugate and application of conjugate in enhanced polymerase chain reaction |
| CN105112397A (en) * | 2015-08-20 | 2015-12-02 | 天津科技大学 | Preparation method of polyquaternium ionic liquid enzyme reactor |
| WO2017062374A1 (en) * | 2015-10-05 | 2017-04-13 | The Regents Of The University Of California | Compositions and methods for generating oligodendrocyte precursors |
| US20170267716A1 (en) * | 2016-03-16 | 2017-09-21 | General Electric Company | Precipitation of polypeptides by aptamer-polymer conjugates |
| CN108210934A (en) * | 2011-11-04 | 2018-06-29 | 韩美科学株式会社 | It is used to prepare the method for physiological active polypeptide compound |
-
2018
- 2018-10-23 CN CN201811281530.2A patent/CN109384840A/en not_active Withdrawn
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1723232A (en) * | 2003-01-06 | 2006-01-18 | 尼克塔治疗亚拉巴马公司 | Thiol-selective water-soluble polmer derivatives |
| WO2005039641A2 (en) * | 2003-10-15 | 2005-05-06 | The Regents Of The University Of California | Biomacromolecule polymer conjugates |
| CN108210934A (en) * | 2011-11-04 | 2018-06-29 | 韩美科学株式会社 | It is used to prepare the method for physiological active polypeptide compound |
| WO2014176279A1 (en) * | 2013-04-22 | 2014-10-30 | Carnegie Mellon Iniversity | Polymer-based protein engineering methods to rationally tune enzyme activity, ph-dependence and stability |
| CN104497097A (en) * | 2014-06-18 | 2015-04-08 | 深圳市第二人民医院 | Protein or polypeptide-polymer grafted copolymer with reduction responsiveness, synthesis method and protein or polypeptide drug thereof |
| CN104862288A (en) * | 2015-05-08 | 2015-08-26 | 苏州大学 | Synthesis of novel temperature-responsive inorganic pyrophosphatase conjugate and application of conjugate in enhanced polymerase chain reaction |
| CN105112397A (en) * | 2015-08-20 | 2015-12-02 | 天津科技大学 | Preparation method of polyquaternium ionic liquid enzyme reactor |
| WO2017062374A1 (en) * | 2015-10-05 | 2017-04-13 | The Regents Of The University Of California | Compositions and methods for generating oligodendrocyte precursors |
| US20170267716A1 (en) * | 2016-03-16 | 2017-09-21 | General Electric Company | Precipitation of polypeptides by aptamer-polymer conjugates |
Non-Patent Citations (7)
| Title |
|---|
| NOR H.ABU SAMAH等: "Enhanced in vitro transdermal delivery of caffeine using a temperature-and pH-sensitive nanogel,poly(NIPAM-co-AAc)", 《INTERNATIONAL JOURNAL OF PHARMACEUTICS》 * |
| YUANZI WU等: "Cytocompatible Modification of Thermoresponsive Polymers on Living Cells for Membrane Proteomic Isolation and Analysis", 《ANALYTICAL CHEMISTRY》 * |
| YUANZI WU等: "Protein purification by chemo-selective precipitation using thermoresponsive polymers", 《BIOPOLYMERS》 * |
| 巫水根: "环境响应型高分子修饰蛋白质及其在蛋白质分离中的应用", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
| 焦云鹏等: "《酶制剂生产与应用》", 31 August 2015, 中国轻工业出版社 * |
| 蒋中华等: "《生物分子固定化技术及应用》", 31 July 1998, 化学工业出版社 * |
| 金佳钰: "改性聚N-异丙基丙烯酰胺协助蛋白质体外复性研究", 《中国优秀硕士学位论文工程科技Ⅰ辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111848720A (en) * | 2020-08-10 | 2020-10-30 | 福州大学 | Method for performing macromolecule functional modification on glycosylated protein |
| CN114470865A (en) * | 2022-01-12 | 2022-05-13 | 武汉大学 | Method for simply, conveniently and quickly separating temperature-sensitive polymer and connector thereof |
| CN114470865B (en) * | 2022-01-12 | 2023-03-10 | 武汉大学 | Method for simply, conveniently and quickly separating temperature-sensitive polymer and connector thereof |
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