CN104862330A - Production and purification method of storage protein type alpha-amylase inhibitor with Cupin structure function domain - Google Patents
Production and purification method of storage protein type alpha-amylase inhibitor with Cupin structure function domain Download PDFInfo
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Abstract
The invention discloses a production and purification method of a storage protein type alpha-amylase inhibitor with a Cupin structure function domain, comprising the steps of: 1, construction of a rokaryotic expression system and bacterium solution cultivation inducible expression; 2, prokaryotic expression of soluble protein purification; and/or 3, prokaryotic expression of inclusion body protein purification and protein renaturation. At present, multiple repeated experiments test that, the protein obtained through producing the alpha-amylase inhibitor by using the technical process provided by the invention is high in yield and purity, the production process is simple and convenient, the production cycle is short, expensive devices are not needed, and thereby the production and purification method is an excellent bioprocess technology for producing and purifying the alpha-amylase inhibitor.
Description
Technical field
The invention belongs to bioprocess technology and food processing field, relate to a kind of production and the purification process with the storage protein type alpha-amylase inhibitor of Cupin Structure and function domain, be specifically related to production and the purification process of storage protein type alpha-amylase inhibitor in garbanzo seed.
Background technology
These three kinds of nutritive elements of carbohydrate, fat and protein for human body provide the overwhelming majority energy, the heat that wherein carbohydrate provides accounts for whole 60%-70%, hoard in body if carbohydrate intake too much will change into fat, too much absorption and residue will cause " rich man diseases " such as obesity, hyperglycemia, diabetes.
Garbanzo, its seed point, as olecranon, makes again peach beans, sheepshead beans, Nuo Hu carry, has the cultivation history of more than 2500 year in Xinjiang of China.Garbanzo is Uygur nationality's medicinal herbs most in use, there is very high pharmaceutical use, the traditional Chinese medical science is used for damping antidiarrheal, detoxicating, relieving inflammation, strong bone stomach invigorating etc., in addition, prove that garbanzo has obvious curative effect to more than 70 kinds of severe malnutritions by a large amount of clinical experience, therefore garbanzo becomes the important vegetable material of exploitation to human health's nutritive ingredient.Saccharan Starch Hydrolysis can be simple sugar glucose by α-amylase, and then as the energy derive that body absorbs, plays an important role in people's life meals.The α-amylase that alpha-amylase inhibitor can be secreted with human oral cavity, gi tract salivaryα-amylase and pancreas is effectively combined the mixture forming enzyme-inhibitor, alpha-amylase activity is lost or reduces the hydrolysis and the digestion that hinder carbohydrate in food.Based on the feature of alpha-amylase inhibitor self, alpha-amylase inhibitor has wide practical use in medical science and agriculture production, alpha-amylase inhibitor can as the medicine of the diseases such as control and treatment hyperglycemia, diabetes and hyperlipidemia on the one hand, also can be applied in daily life on the other hand and keep healthy, keep on a diet, reduce fat; Alpha-amylase inhibitor gene and product are prevented and treated agricultural insect and are reduced sterilant use, important using value of preserving the ecological environment simultaneously.
We obtain one to garbanzo seed alpha-amylase inhibitor crude extract and have the albumen suppressing alpha-amylase activity early stages after the separation and purification of ammonium sulfate precipitation, ion-exchange chromatography and reverse-phase chromatography, it belongs to storage protein type (it is numbered Q9SMJ4 in NCBI), by Q9SMJ4 Gene Handling, being different from known alpha-amylase inhibitor type completely, is a kind of new type.This albumen is made up of alpha subunit (or protein chain) and beta subunit (or protein chain) 2 subunits, is amyloid protein precursor, and it is sheared the most at last in vivo and generates alpha chain and beta chain.Respectively a Cupin structure (as Fig. 1) is contained in alpha peptide chain and beta peptide chain protein steric structural.We study further and find that the albumen of all Cupin of having structures (functional domain) generally has α-amylase inhibit activities, and Cupin structure is crucial functional domain.Because this storage protein type alpha-amylase inhibitor content in garbanzo seed is little, adopt natural garbanzo seed material to carry out extraction purification, process is complicated, and cost intensive, is unsuitable for extraction and isolation and the exploitation of this proteinoid.Adopt biotechnology, use rational bioprocess technology flow process to can be the production of alpha-amylase inhibitor in garbanzo and purifying provides new way.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a kind of production and the purification process with the storage protein type alpha-amylase inhibitor of Cupin Structure and function domain are provided.
Object of the present invention realizes by following technical scheme:
There is production and the purification process of the storage protein type alpha-amylase inhibitor of Cupin Structure and function domain, comprise the following steps (1) and be selected from or two in step (2) and step (3):
(1) prokaryotic expression system builds and bacterium liquid cultivation abduction delivering:
The ORF of target protein is cloned into prokaryotic expression carrier, and builds Q9SMJ4 albumen escherichia coli prokaryotic expression system; Cultivate Q9SMJ4 albumen escherichia coli prokaryotic expression system, and induce target protein to express; Wherein said prokaryotic expression carrier contains His label protein encoding gene;
(2) prokaryotic expression soluble proteins purifying:
A. every 50mL cultivates the resuspended thalline of thalline 10mL damping fluid 1 that bacterium liquid is collected, and add the TritonX-100 of 150 ~ 200 μ the L 35% and PMSF of 50 ~ 100 μ L 100mM;
B. resuspended bacterium liquid is placed on ice, ultrasonication thalline;
C. by centrifugal for the bacterium liquid of ultrasonication, collect supernatant, abandon precipitation;
D. the supernatant liquor containing His label protein is added in Ni post, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely;
E. Ni post or until A is rinsed with the damping fluid 2 of 6 ~ 10 times of column volumes
280value reaches stable;
F. wash-out His tag expression albumen adopts gradient elution mode, and each gradient elution volume is 4 ~ 5 column volumes, collects each gradient elution albumen;
G. by each gradient eluted protein purity in SDS-PAGE electrophoresis detection previous step, the elution fraction being applicable to collecting is determined;
H. the eluted protein liquid of collection being loaded molecular weight cut-off is in the dialysis tubing of 3600Da, and put into ultrapure water dialysis 20 ~ 24h desalination, period rocks dialysis tubing and changes ultrapure water;
I. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% (w/v) is concentrated into proper volume, is prokaryotic expression and the object soluble proteins solution of purified pool;
(3) prokaryotic expression inclusion body protein purifying and protein renaturation:
A. every 50mL cultivates the resuspended thalline of thalline 0.1M phosphoric acid buffer 10mL that bacterium liquid is collected;
B. resuspended bacterium liquid is placed on ice, ultrasonication thalline;
C. by centrifugal for the bacterium liquid of ultrasonication, collecting precipitation, abandons supernatant;
D. the washing precipitation of 0.1 ~ 0.3M phosphoric acid buffer is used, centrifugal, collecting precipitation;
E. use 10mL lysate dissolution precipitation, at room temperature hatch 1-2h;
F. centrifugal 30 ~ 40min, collects supernatant liquor, abandons precipitation insolubles;
G. balance Ni post with 8 ~ 10 times of column volumes of buffer 3, coutroi velocity is 0.5-1.0mL/min;
H. the supernatant liquor containing His label protein collected in step f is added in Ni post, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely, loading volume: column volume ratio is 15:1 to the maximum; I. again Ni post is balanced, coutroi velocity 0.5-1.0mL/min with 4 ~ 6 times of column volumes of buffer 3;
J. Ni post or until A is rinsed with the damping fluid 4 of 2 ~ 3 times of column volumes
280value reaches stable, and flow rate control is 1.0mL/min;
K. be 1.0mL/min by the damping fluid 5 wash-out inclusion body protein flow rate control of 5 ~ 7 times of column volumes;
L.SDS-PAGE electrophoresis detection previous step eluted protein purity and purification effect;
M. the inclusion body protein nature renaturation collected by wash-out under 4 DEG C of conditions, slowly adds ultrapure water, makes urea concentration in solution carry out gradient dilution in solution;
N. it is in the dialysis tubing of 3600Da that protein solution renaturation terminated loads molecular weight cut-off, and put into ultrapure water dialysis 20 ~ 25h desalination, period rocks dialysis tubing and changes ultrapure water;
O. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% (w/v) is concentrated into proper volume, is Prokaryotic expression, purification and collects and the object inclusion body protein solution of renaturation.
Cupin structure refers to the barrel-like structure formed by least 6 beta-pleated sheets, cupin structural domain is made up of two conservative elementary zones, individual containing two β-pleated sheet structure secondary structures in each region, be connected by one section of transformable ring texture between two basic conservative regions; The basic comprising of a conservative region is G (X)
5hXH (X)
3,4e (X)
6g, another conservative region basic comprising is G (X)
5pXG (X)
2h (X)
3n, but the amino acid in conservative region shows non-conservation in some cupin family proteins.
Described cupin protein structure domain derives from high green plants, genus bacillus, ascomycetes, comprises seed 11S and 7S storage protein containing cupin domain protein family, sprouts element and several functions albumen.
One as the inventive method is preferred, the described storage protein type alpha-amylase inhibitor with Cupin Structure and function domain is storage protein type alpha-amylase inhibitor in garbanzo seed, this inhibitor is numbered Q9SMJ4 in NCBI, called after Q9SMJ4 albumen.Described garbanzo storage protein Q9SMJ4 albumen contains 2 cupin structural domains, lays respectively on itself α and β subunit chain.
One as the inventive method is preferred, and described (1) prokaryotic expression system builds and bacterium liquid cultivation abduction delivering preferably includes further:
A. grow the cDNA of seed for template with garbanzo, obtained the ORF sequence of coding storage protein type alpha-amylase inhibitor by PCR method;
B. utilize round pcr to add restriction enzyme site sequence EcoR I and Not I respectively at 5 ' and 3 ' end of coding Q9SMJ4 albumen ORF sequence, obtain goal gene fragment; Wherein Q9SMJ4 albumen ORF sequence removes proteins encoded signal peptide sequence;
C. utilize the goal gene fragment that obtains in restriction enzyme EcoR I and Not I couple of step b and pET-28a (+) prokaryotic expression carrier plasmid to carry out enzyme respectively to cut, the goal gene fragment that glue recycled and plasmid DNA fragment;
D. by previous step obtain with the DNA fragmentation T that restriction enzyme ferment treatment is crossed
4ligase enzyme connects under 16 DEG C of conditions, obtains recombinant prokaryotic expression vector;
E. by the recombinant prokaryotic expression vector Plastid transformation that obtains in escherichia coli prokaryotic expression bacterial strain BL21 (DE3), obtain Q9SMJ4 albumen escherichia coli prokaryotic expression system bacterium liquid;
F. by the expression system bacterium liquid set up in step e in proportion (v/v) 1:100 to be inoculated in LB liquid nutrient medium and to load two volumes and cultivate in liquid nutrient medium Erlenmeyer flask;
G. the cultivation bacterium liquid in step f is carried out the protein induced expression of Q9SMJ4;
H.4800 ~ 5000rpm, 4 DEG C of centrifugal 5 ~ 10min collect the thalline of inducing and terminating in bacterium liquid, abandon supernatant.
Wherein, in step f, culture condition is preferably at 37 DEG C, carries out cultivation 6h in the incubator of rotating speed 200rpm, treats that bacterium liquid OD value reaches 0.6 ~ 0.8.
In step g, concrete abduction delivering condition optimization is: inducing temperature is 37 DEG C, and microbiotic Kan concentration is 25 μ g/mL, and the abduction delivering time is 6h, and inductor IPTG concentration is 0.5mM, and shaking speed is 200rpm.
One as the inventive method is preferred, and step (2) prokaryotic expression soluble proteins purifying comprises further:
A. every 50mL cultivates the resuspended thalline of thalline damping fluid 10mL that bacterium liquid is collected, and adds the TritonX-100 of 170 μ the L 35% and PMSF of 100 μ L 100mM;
B. be placed on ice by resuspended bacterium liquid, ultrasonication is until bacterium liquid becomes clear, and ultrasound condition is ultrasonication 3s, cooling 5s, and power is 400 watts;
C. by the bacterium liquid 12000rpm of ultrasonication, 4 DEG C of centrifugal 15min, collect supernatant, abandon precipitation;
D. add in Ni post by the supernatant liquor containing His label protein, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely, applied sample amount/column volume (v/v) ratio is 15:1 to the maximum;
E. Ni post or until A is rinsed with the damping fluid 2 of 8 times of column volumes
280value reaches stable, and flow rate control is 1.0mL/min;
F. wash-out His tag expression albumen adopts gradient elution mode, and each gradient elution volume is 4 column volumes, collects each gradient elution albumen;
G. by each gradient eluted protein purity in SDS-PAGE electrophoresis detection previous step, determine that collection composition is single, impurity is less and the albumen elution fraction that molecular weight of albumen is consistent with target protein;
H. the eluted protein liquid of collection being loaded molecular weight cut-off is in the dialysis tubing of 3600Da, and put into ultrapure water and to dialyse 24h desalination, period rocks dialysis tubing and changes ultrapure water;
I. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% (w/v) is concentrated into proper volume, is prokaryotic expression and the Q9SMJ4 soluble proteins solution of purified pool.
Wherein, described damping fluid 1 is 50mM NaH
2pO
4, 300mM NaCl, pH=8.0; Described damping fluid 2 is 50mM NaH
2pO
4, 300mM NaCl, 10mM imidazoles, pH=8.0; The elutriant that step f uses is 50mM NaH
2pO
4, 300mM NaCl, pH=8.0, imidazole concentration is respectively 50,100,150,200,250mM.
One as the inventive method is preferred, and step (3) prokaryotic expression inclusion body protein purifying and protein renaturation preferably include further:
A. every 50mL cultivates the resuspended thalline of thalline pH=8.0,0.1M phosphoric acid buffer 10mL that bacterium liquid is collected;
B. be placed on ice by resuspended bacterium liquid, ultrasonication is until bacterium liquid becomes clarification, and ultrasound condition is ultrasonication 3s, cooling 5s, and power is 400 watts;
C. by the bacterium liquid 12000rpm of ultrasonication, 4 DEG C of centrifugal 10min, collecting precipitation, abandons supernatant;
D. the washing precipitation of pH=8.0,0.1M phosphoric acid buffer is used once, 12000rpm, 4 DEG C of centrifugal 10min, collecting precipitation;
E. use 10mL lysate dissolution precipitation, at room temperature hatch 1-2h, period constantly rocks solution, makes resolution of precipitate complete;
F.12000rpm, centrifugal 30min under 4 DEG C of conditions, collects supernatant liquor, abandons precipitation insolubles;
G. balance Ni post with 8 times of column volumes of buffer 3, coutroi velocity is 0.5-1.0mL/min;
H. the supernatant liquor containing His label protein collected in step f is added in Ni post, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely, loading volume: column volume ratio is 15:1 to the maximum;
I. again Ni post is balanced, coutroi velocity 0.5-1.0mL/min with 4 times of column volumes of buffer 3;
J. Ni post or until A is rinsed with the damping fluid 4 of 2 times of column volumes
280value reaches stable, and flow rate control is 1.0mL/min;
K. be 1.0mL/min by the damping fluid 5 wash-out inclusion body protein flow rate control of 6 times of column volumes;
Eluted protein purity and purification effect in l.SDS-PAGE electrophoresis detection previous step;
M. the inclusion body protein nature renaturation collected by wash-out under 4 DEG C of conditions, slowly adds ultrapure water, makes urea concentration in solution carry out gradient dilution in solution;
N. by reviving the protein solution terminated, to load molecular weight cut-off be in the dialysis tubing of 3600Da, and put into ultrapure water and to dialyse 24 desalinations, period rocks dialysis tubing and changes ultrapure water;
O. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% (w/v) is concentrated into proper volume, is Prokaryotic expression, purification and collects and the Q9SMJ4 inclusion body protein solution of renaturation.
Wherein, described lysate is 100mM NaH
2pO
4, 10mM Tris-HCl, 100mM DTT, 8M urea, pH=8.0; Described damping fluid 3 is 100mM NaH
2pO
4, 10mM Tris-HCl, 8M urea, pH=8.0; Described damping fluid 4 is 100mM NaH
2pO
4, 10mM Tris-HCl, 8M urea, 10mM imidazoles, pH=8.0; Described damping fluid 5 is 100mMNaH
2pO
4, 10mM Tris-HCl, 8M urea, 200mM imidazoles, pH=8.0.
The gradient that in step m, urea concentration carries out diluting is preferably 8M-6M-4M-2M-1M-0.1M, each gradient duration
For 1h.
The solution preparation of the whole process of the inventive method all needs to use pure water.
Beneficial effect
The present invention is directed to a kind of storage protein type alpha-amylase inhibitor be present in garbanzo seed and be difficult to separation and purification from natural materials, removing other foreign proteins becomes α-amylase to be for this reason applied to an actual challenge difficult problem, sets up a set of production and the purifying process flow process that are suitable for this storage protein type alpha-amylase inhibitor first.
This cover bioprocess technology flow process has carried out biological preparation to this storage protein type alpha-amylase inhibitor in garbanzo seed first and follow-up protein purification reclaims, through SDS-PAGE electrophoresis detection be separated obtain to prepare protein-active high, preparation amount is many, easy and simple to handle, cost is lower, reproducible, the biological production of this alpha-amylase inhibitor can be directly applied to.
The whole technological process of production is applicable to production and the purifying of Q9SMJ4 albumen alpha chain (adopting the ORF of coding alpha chain) and beta chain (adopting the ORF of coding beta chain) equally respectively, also can be applicable to production and the purifying of storage protein type alpha-amylase inhibitor that different biological (animal, plant, microorganism) originates, that have Cupin structure (functional domain).
Accompanying drawing explanation
Fig. 1 Q9SMJ4 protein sequence and alpha and beta peptide sequence information pattern
Storage protein type alpha-amylase inhibitor (Q9SMJ4) prokaryotic expression soluble proteins Expression and purification result figure in Fig. 2 garbanzo seed
Expressing protein band is depicted as in red block, swimming lane is followed successively by from right to left: 1. albumen maker, 2. express the broken supernatant liquor of bacterium, 3. express bacterium broken throw out, 4. sample penetration peak, 5. elution peak (10mM imidazoles), 6. elution peak (50mM imidazoles), 7. elution peak (100mM imidazoles), 8. elution peak (150mM imidazoles), 9. elution peak (200mM imidazoles), 10. elution peak (250mM imidazoles)
Storage protein type alpha-amylase inhibitor (Q9SMJ4) prokaryotic expression inclusion body protein Expression and purification result figure in Fig. 3 garbanzo seed
Red arrow is depicted as storage protein type alpha-amylase inhibitor (Q9SMJ4) prokaryotic expression inclusion body protein
Embodiment
Embodiment 1
(1) prokaryotic expression system builds and bacterium liquid cultivation abduction delivering:
A. in growing with Desi type garbanzo, the cDNA of seed is for template, and obtained the ORF sequence of coding for alpha-amylase inhibitor protein (it is numbered Q9SMJ4 in NCBI) by PCR method, primer sequence is
F1:5'-TGTCATCATGGCTAAGCTTCTTG-3'(SEQ ID NO.1) and
R1:5'-TTGTTTCCGCTAAAAGGTACATG-3'(SEQ ID NO.2)；
B. utilize round pcr to add restriction enzyme site sequence EcoR I and Not I respectively at 5 ' and 3 ' end of coding Q9SMJ4 albumen ORF sequence (removing proteins encoded signal peptide sequence), obtain goal gene fragment, primer sequence is
F2:5'-CG
gAATTCtTGAGAGATCAACCT-3'(SEQ ID NO.3) and
R2:5'-TTTTCCTTTT
GCGGCCGCCAGCTGCTGCTTTGTT-3'(SEQ ID NO.4)；
C. utilize the goal gene fragment obtained in restriction enzyme EcoR I and Not I couple of step b under 37 DEG C of conditions, carry out 4 hours enzymes and cut process, the DNA fragmentation that glue recycled;
D. under utilizing restriction enzyme EcoR I and Not I pair of pET-28a (+) prokaryotic expression carrier plasmid to carry out 37 DEG C of conditions, enzyme cuts 4 hours, the plasmid DNA fragment that glue recycled;
E. by obtain in step c and steps d with the DNA fragmentation T that restriction enzyme ferment treatment is crossed
4ligase enzyme connects under 16 DEG C of conditions, obtains recombinant prokaryotic expression vector;
F. by the recombinant prokaryotic expression vector Plastid transformation that obtains in escherichia coli prokaryotic expression bacterial strain BL21 (DE3), obtain Q9SMJ4 albumen escherichia coli prokaryotic expression system bacterium liquid;
G. by the expression system bacterium liquid set up in step f in proportion (v/v) 1:100 to be inoculated in LB liquid nutrient medium and to load two volumes in liquid nutrient medium Erlenmeyer flask, at 37 DEG C, carry out cultivation 6h in the incubator of rotating speed 200rpm, treat that bacterium liquid OD value reaches about 0.7;
H. the cultivation bacterium liquid in step g is carried out the protein induced expression of Q9SMJ4, concrete abduction delivering condition is as follows: inducing temperature is 37 DEG C, and microbiotic Kan concentration is 25 μ g/mL, and the abduction delivering time is 6h, and inductor IPTG concentration is 0.5mM, and rotating speed is 200rpm;
I.5000rpm, 4 DEG C of centrifugal 5min collect the thalline of inducing and terminating in bacterium liquid, abandon supernatant.
(2) prokaryotic expression soluble proteins purification process:
A. every 50mL cultivates thalline damping fluid 1 (the 50mM NaH that bacterium liquid is collected
2pO
4, 300mM NaCl, pH=8.0) the resuspended thalline of 10mL, and add the TritonX-100 of 170 μ the L 35% and PMSF of 100 μ L 100mM;
B. be placed on ice by resuspended bacterium liquid, ultrasonication is until bacterium liquid becomes clear, and ultrasound condition is ultrasonication 3s, cooling 5s, and power is 400 watts;
C. by the bacterium liquid 12000rpm of ultrasonication, 4 DEG C of centrifugal 15min, collect supernatant, abandon precipitation;
D. the supernatant liquor containing His label protein is added in Ni post, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely, applied sample amount: column volume ratio is 15:1 to the maximum;
E. damping fluid 2 (the 50mM NaH of 8 times of column volumes is used
2pO
4, 300mM NaCl, 10mM imidazoles, pH=8.0) rinse Ni post or until A
280value reaches stable, and flow rate control is 1.0mL/min;
F. wash-out His tag expression albumen adopts gradient elution mode, and elutriant is 50mM NaH
2pO
4, 300mM NaCl, pH=8.0, imidazole concentration is respectively 50,100,150,200,250mM, each gradient elution volume is 4 column volumes, collects each gradient elution albumen;
G. by each gradient eluted protein purity in SDS-PAGE electrophoresis detection step f, the elution fraction being applicable to collecting is determined;
H. the eluted protein liquid of collection being loaded molecular weight cut-off is in the dialysis tubing of 3600Da, and put into ultrapure water and to dialyse 24h desalination, period rocks dialysis tubing and changes ultrapure water;
I. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% (w/v) is concentrated into proper volume, is prokaryotic expression and the solubility Q9SMJ4 protein solution of purified pool.
J. detected the solubility Q9SMJ4 albumen (Fig. 2) collected by SDS-PAGE glue, shown prokaryotic expression bacterium expression soluble proteins is higher, and purification effect is good, has higher activity, and it is higher that purifying reclaims protein content.
Embodiment 2
A.5000rpm, 4 DEG C of centrifugal 5min collect embodiment 1 step (1) and induce the thalline terminated in bacterium liquid, abandon supernatant;
B. every 50mL cultivates the resuspended thalline of thalline 0.1M phosphoric acid buffer (pH=8.0) 10mL that bacterium liquid is collected;
C. be placed on ice by resuspended bacterium liquid, ultrasonication is until bacterium liquid becomes clarification, and ultrasound condition is ultrasonication 3s, cooling 5s, and power is 400 watts;
D. by the bacterium liquid 12000rpm of ultrasonication, 4 DEG C of centrifugal 10min, collecting precipitation, abandons supernatant;
E. 0.1M phosphoric acid buffer (pH=8.0) washing precipitation is used once, 12000rpm, 4 DEG C of centrifugal 10min, collecting precipitation;
F. 10mL lysate (100mM NaH is used
2pO
4, 10mM Tris-HCl, 100mM DTT, 8M urea, pH=8.0) dissolution precipitation, at room temperature hatch 1-2h, period constantly rocks solution, makes resolution of precipitate complete;
G.12000rpm, centrifugal 30min under 4 DEG C of conditions, collects supernatant liquor, abandons precipitation insolubles;
H. 8 times of column volumes of buffer 3 (100mM NaH is used
2pO
4, 10mM Tris-HCl, 8M urea, pH=8.0) balance Ni post, coutroi velocity is 0.5-1.0mL/min;
I. the supernatant liquor containing His label protein collected in step g is added in Ni post, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely, loading volume: column volume (v/v) ratio is 15:1 to the maximum;
J. 4 times of column volumes of buffer 3 (100mM NaH is used
2pO
4, 10mM Tris-HCl, 8M urea, pH=8.0) again balance Ni post, coutroi velocity 0.5-1.0mL/min;
K. damping fluid 4 (the 100mM NaH of 2 times of column volumes is used
2pO
4, 10mM Tris-HCl, 8M urea, 10mM imidazoles, pH=8.0) rinse Ni post or until A280 value reaches stable, flow rate control is 1.0mL/min;
L. damping fluid 5 (the 100mM NaH of 6 times of column volumes is used
2pO
4, 10mM Tris-HCl, 8M urea, 200mM imidazoles, pH=8.0) wash-out inclusion body protein flow rate control is 1.0mL/min;
Eluted protein purity and purification effect in m.SDS-PAGE electrophoresis detection previous step;
N. the inclusion body protein nature renaturation under 4 DEG C of conditions, wash-out collected, in solution, slowly add ultrapure water, make urea concentration in solution carry out gradient dilution (8M-6M-4M-2M-1M-0.1M), each gradient duration is 1h;
O. it is in the dialysis tubing of 3600Da that the protein solution that renaturation terminated loads molecular weight cut-off, and put into ultrapure water and to dialyse 24h desalination, period rocks dialysis tubing and changes ultrapure water;
P. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% is concentrated into proper volume, is Prokaryotic expression, purification and collects and the Q9SMJ4 inclusion body protein solution of renaturation.
Y. the Q9SMJ4 inclusion bodies of protein (Fig. 3) collected is detected through SDS-PAGE glue, shown effect is that prokaryotic expression bacterium expression target protein amount is very high, abduction delivering condition is good, purifying recovering effect is good, there is higher activity, it is higher that purifying reclaims protein content, and foreign protein content is few.
Claims (10)
1. there is production and the purification process of the storage protein type alpha-amylase inhibitor of Cupin Structure and function domain, it is characterized in that one or two that comprises the following steps (1) and be selected from step (2) and step (3):
(1) prokaryotic expression system builds and bacterium liquid cultivation abduction delivering:
The ORF of target protein is cloned into prokaryotic expression carrier, and builds Q9SMJ4 albumen escherichia coli prokaryotic expression system; Cultivate Q9SMJ4 albumen escherichia coli prokaryotic expression system, and induce target protein to express; Wherein said prokaryotic expression carrier contains His label protein encoding gene;
(2) prokaryotic expression soluble proteins purifying:
A. every 50mL cultivates the resuspended thalline of thalline 10mL damping fluid 1 that bacterium liquid is collected, and add the TritonX-100 of 150 ~ 200 μ the L 35% and PMSF of 50 ~ 100 μ L 100mM;
B. resuspended bacterium liquid is placed on ice, ultrasonication thalline;
C. by centrifugal for the bacterium liquid of ultrasonication, collect supernatant, abandon precipitation;
D. the supernatant liquor containing His label protein is added in Ni post, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely;
E. Ni post or until A is rinsed with the damping fluid 2 of 6 ~ 10 times of column volumes
280value reaches stable;
F. wash-out His tag expression albumen adopts gradient elution mode, and each gradient elution volume is 4 ~ 5 column volumes, collects each gradient elution albumen;
G. by each gradient eluted protein purity in SDS-PAGE electrophoresis detection previous step, the elution fraction being applicable to collecting is determined;
H. the eluted protein liquid of collection being loaded molecular weight cut-off is in the dialysis tubing of 3600Da, and put into ultrapure water dialysis 20 ~ 24h desalination, period rocks dialysis tubing and changes ultrapure water;
I. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% (w/v) is concentrated into proper volume, is prokaryotic expression and the object soluble proteins solution of purified pool;
(3) prokaryotic expression inclusion body protein purifying and protein renaturation:
A. every 50mL cultivates the resuspended thalline of thalline 0.1M phosphoric acid buffer 10mL that bacterium liquid is collected;
B. resuspended bacterium liquid is placed on ice, ultrasonication thalline;
C. by centrifugal for the bacterium liquid of ultrasonication, collecting precipitation, abandons supernatant;
D. the washing precipitation of 0.1 ~ 0.3M phosphoric acid buffer is used, centrifugal, collecting precipitation;
E. use 10mL lysate dissolution precipitation, at room temperature hatch 1-2h;
F. centrifugal 30 ~ 40min, collects supernatant liquor, abandons precipitation insolubles;
G. balance Ni post with 8 ~ 10 times of column volumes of buffer 3, coutroi velocity is 0.5-1.0mL/min;
H. the supernatant liquor containing His label protein collected in step f is added in Ni post, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely, loading volume: column volume ratio is 15:1 to the maximum;
I. again Ni post is balanced, coutroi velocity 0.5-1.0mL/min with 4 ~ 6 times of column volumes of buffer 3;
J. Ni post or until A is rinsed with the damping fluid 4 of 2 ~ 3 times of column volumes
280value reaches stable, and flow rate control is 1.0mL/min;
K. be 1.0mL/min by the damping fluid 5 wash-out inclusion body protein flow rate control of 5 ~ 7 times of column volumes;
L.SDS-PAGE electrophoresis detection previous step eluted protein purity and purification effect;
M. the inclusion body protein nature renaturation collected by wash-out under 4 DEG C of conditions, slowly adds ultrapure water, makes urea concentration in solution carry out gradient dilution in solution;
N. the protein solution of reviving end being loaded molecular weight is in the dialysis tubing of 3600Da, and put into ultrapure water dialysis 20 ~ 25h desalination, period rocks dialysis tubing and changes ultrapure water;
O. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% (w/v) is concentrated into proper volume, is Prokaryotic expression, purification and collects and the object inclusion body protein solution of renaturation.
2. method according to claim 1, it is characterized in that the described storage protein type alpha-amylase inhibitor with Cupin Structure and function domain is storage protein type alpha-amylase inhibitor in garbanzo seed, this inhibitor is numbered Q9SMJ4 in NCBI, called after Q9SMJ4 albumen.
3. method according to claim 2, is characterized in that described (1) prokaryotic expression system builds and bacterium liquid cultivation abduction delivering comprises:
A. grow the cDNA of seed for template with garbanzo, obtained the ORF sequence of coding storage protein type alpha-amylase inhibitor by PCR method;
B. utilize round pcr to add restriction enzyme site sequence EcoR I and Not I respectively at 5 ' and 3 ' end of coding Q9SMJ4 albumen ORF sequence, obtain goal gene fragment; Wherein Q9SMJ4 albumen ORF sequence removes proteins encoded signal peptide sequence;
C. utilize the goal gene fragment that obtains in restriction enzyme EcoR I and Not I couple of step b and pET-28a (+) prokaryotic expression carrier plasmid to carry out enzyme respectively to cut, the goal gene fragment that glue recycled and plasmid DNA fragment;
What d. previous step obtained connects under 16 DEG C of conditions with the DNA fragmentation T4 ligase enzyme that restriction enzyme ferment treatment is crossed, obtains recombinant prokaryotic expression vector;
E. by the recombinant prokaryotic expression vector Plastid transformation that obtains in escherichia coli prokaryotic expression bacterial strain BL21 (DE3), obtain Q9SMJ4 albumen escherichia coli prokaryotic expression system bacterium liquid;
F. by the expression system bacterium liquid set up in step e in proportion (v/v) 1:100 to be inoculated in LB liquid nutrient medium and to load two volumes and cultivate in liquid nutrient medium Erlenmeyer flask;
G. the cultivation bacterium liquid in step f is carried out the protein induced expression of Q9SMJ4;
H.4800 ~ 5000rpm, 4 DEG C of centrifugal 5 ~ 10min collect the thalline of inducing and terminating in bacterium liquid, abandon supernatant.
4. method according to claim 3, is characterized in that in step f, culture condition is at 37 DEG C, carries out cultivation 6h, treats that bacterium liquid OD value reaches 0.6 ~ 0.8 in the incubator of rotating speed 200rpm.
5. method according to claim 3, is characterized in that in step g, concrete abduction delivering condition is: inducing temperature is 37 DEG C, and microbiotic Kan concentration is 25 μ g/mL, and the abduction delivering time is 6h, and inductor IPTG concentration is 0.5mM, and shaking speed is 200rpm.
6. method according to claim 2, is characterized in that step (2) prokaryotic expression soluble proteins purifying comprises:
A. every 50mL cultivates the resuspended thalline of thalline damping fluid 10mL that bacterium liquid is collected, and adds the TritonX-100 of 170 μ the L 35% and PMSF of 100 μ L 100mM;
B. be placed on ice by resuspended bacterium liquid, ultrasonication is until bacterium liquid becomes clear, and ultrasound condition is ultrasonication 3s, cooling 5s, and power is 400 watts;
C. by the bacterium liquid 12000rpm of ultrasonication, 4 DEG C of centrifugal 15min, collect supernatant, abandon precipitation;
D. the supernatant liquor containing His label protein is added in Ni post, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely, applied sample amount: column volume (v/v) ratio is 15:1 to the maximum;
E. Ni post or until A is rinsed with the damping fluid 2 of 8 times of column volumes
280value reaches stable, and flow rate control is 1.0mL/min;
F. wash-out His tag expression albumen adopts gradient elution mode, and each gradient elution volume is 4 column volumes, collects each gradient elution albumen;
G. by each gradient eluted protein purity in SDS-PAGE electrophoresis detection previous step, determine that collection composition is single, impurity is less and the albumen elution fraction that molecular weight of albumen is consistent with target protein;
H. the eluted protein liquid of collection being loaded molecular weight cut-off is in the dialysis tubing of 3600Da, and put into ultrapure water and to dialyse 24h desalination, period rocks dialysis tubing and changes ultrapure water;
I. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% (w/v) is concentrated into proper volume, is prokaryotic expression and the Q9SMJ4 soluble proteins solution of purified pool.
7. method according to claim 6, is characterized in that described damping fluid 1 is 50mM NaH
2pO
4, 300mM NaCl, pH=8.0; Described damping fluid 2 is 50mM NaH
2pO
4, 300mM NaCl, 10mM imidazoles, pH=8.0; The elutriant that step f uses is 50mM NaH
2pO
4, 300mM NaCl, pH=8.0, imidazole concentration is respectively 50,100,150,200,250mM.
8. method according to claim 2, is characterized in that step (3) prokaryotic expression inclusion body protein purifying and protein renaturation comprise:
A. every 50mL cultivates the resuspended thalline of thalline pH=8.0,0.1M phosphoric acid buffer 10mL that bacterium liquid is collected;
B. be placed on ice by resuspended bacterium liquid, ultrasonication is until bacterium liquid becomes clarification, and ultrasound condition is ultrasonication 3s, cooling 5s, and power is 400 watts;
C. by the bacterium liquid 12000rpm of ultrasonication, 4 DEG C of centrifugal 10min, collecting precipitation, abandons supernatant;
D. the washing precipitation of pH=8.0,0.1M phosphoric acid buffer is used once, 12000rpm, 4 DEG C of centrifugal 10min, collecting precipitation;
E. use 10mL lysate dissolution precipitation, at room temperature hatch 1-2h, period constantly rocks solution, makes resolution of precipitate complete;
F.12000rpm, centrifugal 30min under 4 DEG C of conditions, collects supernatant liquor, abandons precipitation insolubles;
G. balance Ni post with 8 times of column volumes of buffer 3, coutroi velocity is 0.5-1.0mL/min;
H. the supernatant liquor containing His label protein collected in step f is added in Ni post, coutroi velocity 0.5-1.0mL/min, until supernatant liquor flows through Ni post completely, loading volume: column volume ratio is 15:1 to the maximum;
I. again Ni post is balanced, coutroi velocity 0.5-1.0mL/min with 4 times of column volumes of buffer 3;
J. Ni post or until A is rinsed with the damping fluid 4 of 2 times of column volumes
280value reaches stable, and flow rate control is 1.0mL/min;
K. be 1.0mL/min by the damping fluid 5 wash-out inclusion body protein flow rate control of 6 times of column volumes;
Eluted protein purity and purification effect in l.SDS-PAGE electrophoresis detection previous step;
M. the inclusion body protein nature renaturation collected by wash-out under 4 DEG C of conditions, slowly adds ultrapure water, makes urea concentration in solution carry out gradient dilution in solution;
N. the protein solution of reviving end being loaded molecular weight cut-off is in the dialysis tubing of 3600Da, and put into ultrapure water dialysis 24 hours desalinations, period rocks dialysis tubing and changes ultrapure water;
O. PEG 6000 solution dialysis tubing that dialysis terminates being put into 40% (w/v) is concentrated into proper volume, is Prokaryotic expression, purification and collects and the Q9SMJ4 inclusion body protein solution of renaturation.
9. method according to claim 8, is characterized in that described lysate is 100mM NaH
2pO
4, 10mM Tris-HCl, 100mM DTT, 8M urea, pH=8.0; Described damping fluid 3 is 100mM NaH
2pO
4, 10mM Tris-HCl, 8M urea, pH=8.0; Described damping fluid 4 is 100mM NaH
2pO
4, 10mM Tris-HCl, 8M urea, 10mM imidazoles, pH=8.0; Described damping fluid 5 is 100mM NaH
2pO
4, 10mM Tris-HCl, 8M urea, 200mM imidazoles, pH=8.0.
10. method according to claim 8, it is characterized in that the gradient that in step m, urea concentration carries out diluting is 8M-6M-4M-2M-1M-0.1M, each gradient duration is 1h.
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| CN111551716B (en) * | 2020-06-10 | 2023-02-28 | 安徽大学 | Method for judging combination of starch binding domain of alpha-amylase and non-starch substrate |
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