CN104862330B - A kind of production of storage protein type alpha-amylase inhibitor with Cupin Structure and function domains and purification process - Google Patents
A kind of production of storage protein type alpha-amylase inhibitor with Cupin Structure and function domains and purification process Download PDFInfo
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Abstract
Production and purification process the invention discloses a kind of storage protein type alpha amylase inhibitor with Cupin Structure and function domains；Including (1) prokaryotic expression system structure and bacterium solution culture induced expression；(2) prokaryotic expression soluble protein purifies；And/or the purifying of (3) prokaryotic expression inclusion body protein and protein renaturation.At present, by repeatedly experiment proves repeatedly, using present invention process flow, technique productions are carried out to such a alpha-amylase inhibitor, obtained protein yield height, purity height, simple production process is convenient, with short production cycle, it is a kind of excellent bioprocess technology method for being suitable for the production purifying of this alpha-amylase inhibitor without expensive equipment.
Description
Technical field
The invention belongs to bioprocess technology and food processing field, is related to a kind of storage egg with Cupin Structure and function domains
The production of white type alpha-amylase inhibitor and purification process, and in particular to storage protein type alpha-amylase in chick-pea seed
The production of inhibitor and purification process.
Background technology
These three nutrients of carbohydrate, fat and protein provide the energy of the overwhelming majority, wherein carbon for human body
The heat that hydrate provides accounts for whole 60%-70%, if carbohydrate intake will excessively change into fat
Hoard in body, excessive intake and residue will trigger obesity, hyperglycaemia, diabetes etc. " rich people's disease ".
Chick-pea, its seed point such as olecranon, is called peach beans, sheepshead beans, Nuo Hu and carries, more than 2500 years existing in Xinjiang of China
Cultivation history.Chick-pea is Uygur nationality's medicinal herbs most in use, has very high medical value, and the traditional Chinese medical science is used for damping antidiarrheal, removing toxic substances
Anti-inflammatory, strong bone stomach invigorating etc., in addition, proving that chick-pea has obvious treatment to more than 70 kinds of severe malnutritions by a large amount of clinical experiences
Effect, therefore chick-pea becomes important vegetable material of the exploitation to human health's nutritional ingredient.Alpha-amylase can be by polysaccharide
Starch Hydrolysis is simple sugar glucose, and then the energy source absorbed as body, is played in people's life meals important
Effect.The alphalise starch that alpha-amylase inhibitor can effectively be secreted with human oral cavity, intestines and stomach salivaryα-amylase and pancreas
Enzyme combines to form the compound of enzyme-inhibitor, alpha-amylase activity is lost or is reduced to hinder carbohydrate in food
Hydrolysis and digestion.The characteristics of based on alpha-amylase inhibitor itself, alpha-amylase inhibitor have extensively in medicine and agricultural production
General application prospect, one side alpha-amylase inhibitor, which can be used as, prevents and treats the diseases such as hyperglycaemia, diabetes and hyperlipidemia
The medicine of disease, on the other hand can also be applied to health care in daily life, keep on a diet, and reduce fat；Alpha-amylase suppression at the same time
Preparation gene and product agricultural insect is prevented and is reduced insecticide using, preserving the ecological environment has important application value.
Our to chick-pea seed alpha-amylase inhibitor crude extract through ammonium sulfate precipitation, ion-exchange chromatography early periods
There is the albumen that suppresses alpha-amylase activity with obtaining one after the isolating and purifying of reverse-phase chromatography, it belongs to storage protein type
(it is Q9SMJ4 that it is numbered in NCBI), is controlled by Q9SMJ4 genes, is totally different from known alpha-amylase inhibitor type,
It is a kind of new type.The albumen is made of 2 subunits of alpha subunits (or protein chain) and beta subunits (or protein chain), is
Amyloid protein precursor, most shearing generates alpha chains and beta chains at last in vivo for it.Alpha peptide chains and beta peptides catenin are empty
Between respectively contain a Cupin structure (such as Fig. 1) in structure.We are further study show that all have Cupin structures (functional domain)
Albumen generally there is alpha-amylase inhibitory activity, Cupin structures are crucial functional domain.Due to the storage in chick-pea seed
Protein types alpha-amylase inhibitor content is seldom, and extraction purification is carried out using natural chick-pea seed material, and process is complicated, into
This costliness, is unsuitable for the extraction separation and utilization of this albuminoid.Using biotechnology, with rational bioprocess technology flow
New way can be provided for the production and purifying of alpha-amylase inhibitor in chick-pea.
The content of the invention
The purpose of the present invention is the above-mentioned deficiency for the prior art, there is provided a kind of storage with Cupin Structure and function domains
Hide production and the purification process of protein types alpha-amylase inhibitor.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of production of storage protein type alpha-amylase inhibitor with Cupin Structure and function domains and purification process,
Comprise the following steps (1) and one in step (2) and step (3) or two：
(1) prokaryotic expression system structure and bacterium solution culture induced expression：
The ORF of destination protein is cloned into prokaryotic expression carrier, and builds Q9SMJ4 albumen escherichia coli prokaryotic expression bodies
System；Q9SMJ4 albumen escherichia coli prokaryotic expression systems are cultivated, and induce destination protein to express；The wherein described prokaryotic expression carries
Body contains His label protein encoding genes；
(2) prokaryotic expression soluble protein purifies：
A. thalline is resuspended with 10mL buffer solutions 1 in the thalline that culture bacterium solution is collected per 50mL, and add 150~200 μ L
The PMSF of 35% TritonX-100 and 50~100 μ L 100mM；
B. resuspended bacterium solution is placed on ice, ultrasonication thalline；
C. the bacterium solution of ultrasonication is centrifuged, collects supernatant, abandon precipitation；
D. will be added containing the supernatant of His label proteins in Ni columns, coutroi velocity 0.5-1.0mL/min, until supernatant
Liquid flows completely through Ni columns；
E. Ni columns or until A are rinsed with the buffer solution 2 of 6~10 times of column volumes280Value reaches stable；
F. elution His tag expressions albumen uses gradient elution mode, and each gradient elution volume is 4~5 column volumes,
Collect each gradient elution albumen；
G. purity of protein is eluted by each gradient in SDS-PAGE electrophoresis detection previous steps, determines to be adapted to that collects to wash
De- component；
H. the elution protein liquid of collection is fitted into the bag filter that molecular cut off is 3600Da, is put into ultra-pure water and dialyses
20~24h desalinations, during which rock bag filter and replace ultra-pure water；
I. the bag filter for end of dialysing is put into 6000 solution of PEG of 40% (w/v) and is concentrated into proper volume, be
The purpose soluble protein solution of prokaryotic expression and purified pool；
(3) purifying of prokaryotic expression inclusion body protein and protein renaturation：
A. thalline is resuspended with 0.1M phosphate buffers 10mL in the thalline that culture bacterium solution is collected per 50mL；
B. resuspended bacterium solution is placed on ice, ultrasonication thalline；
C. the bacterium solution of ultrasonication is centrifuged, collects precipitation, abandon supernatant；
D. precipitation is washed with 0.1~0.3M phosphate buffers, centrifuged, collect precipitation；
E. with 10mL lysates dissolving precipitation, it is incubated at room temperature 1-2h；
F. 30~40min is centrifuged, collects supernatant, abandons precipitation insoluble matter；
G. Ni columns, coutroi velocity 0.5-1.0mL/min are balanced with 8~10 times of column volumes of buffer 3；
H. the supernatant containing His label proteins being collected into step f is added in Ni columns, coutroi velocity 0.5-
1.0mL/min, until supernatant flows completely through Ni columns, loading volume:Column volume ratio is up to 15:1；I. with 4~6 times of cylinders
Product buffer solution 3 balances Ni columns, coutroi velocity 0.5-1.0mL/min again；
J. Ni columns or until A are rinsed with the buffer solution 4 of 2~3 times of column volumes280Value reaches stable, flow control 1.0mL/
min；
K. it is 1.0mL/min to elute inclusion body protein flow control with the buffer solution 5 of 5~7 times of column volumes；
L.SDS-PAGE electrophoresis detections previous step elutes purity of protein and purification effect；
M. the inclusion body protein nature renaturation being collected into will be eluted under the conditions of 4 DEG C, ultra-pure water is slowly added into solution,
Urea concentration in solution is set to carry out gradient dilution；
N. protein solution renaturation terminated is fitted into the bag filter that molecular cut off is 3600Da, is put into ultra-pure water thoroughly
20~25h desalinations are analysed, bag filter is during which rocked and replaces ultra-pure water；
O. the bag filter for end of dialysing is put into 6000 solution of PEG of 40% (w/v) and is concentrated into proper volume, be
Prokaryotic expression, purification is collected and the purpose inclusion body protein solution of renaturation.
Cupin structures refer to the barrel-like structure formed by least six beta-pleated sheet, and cupin domains are conservative by two
Fundamental region forms, a in each region to contain two β-pleated sheet secondary structures, can by one section between two basic conservative regions
The cyclic structure of change is connected；One conservative region is configured to G (X) substantially5HXH(X)3,4E(X)6G, another conserved region
Domain is configured to G (X) substantially5PXG(X)2H(X)3N, but the amino acid in conservative region is shown in some cupin family proteins
Non-conservation.
The cupin protein structure domains are tied in high green plants, bacillus, sac fungus containing cupin
Structure domain protein family includes seed 11S and 7S storage protein, sprouts element and several functions albumen.
The storage protein type α-shallow lake with Cupin Structure and function domain preferred, described as one kind of the method for the present invention
Powder enzyme inhibitor is storage protein type alpha-amylase inhibitor in chick-pea seed, which numbers in NCBI is
Q9SMJ4, is named as Q9SMJ4 albumen.The chick-pea storage protein Q9SMJ4 albumen contains 2 cupin domains, respectively
On itself α and β subunit chain.
Preferably, described (1) prokaryotic expression system is built and bacterium solution culture induced expression for one kind as the method for the present invention
Further preferably include：
A. using the cDNA of chick-pea development seed as template, coding storage protein type alphalise starch is obtained by PCR method
The ORF sequences of enzyme inhibitor；
B. restriction enzyme site is added respectively at 5 ' and 3 ' ends of coding Q9SMJ4 albumen ORF sequences using round pcr
Sequence EcoR I and Not I, obtains target gene fragment；Wherein Q9SMJ4 albumen ORF sequences remove encoding proteins signal peptide sequence
Row；
C. using restriction enzyme EcoR I and Not I to the target gene fragment and pET-28a that are obtained in step b
(+) prokaryotic expression carrier plasmid carries out digestion, the processed target gene fragment of glue reclaim and Plasmid DNA fragment respectively；
D. by previous step acquisition with the processed DNA fragmentation T of restriction enzyme4Ligase is under the conditions of 16 DEG C
Connection, obtains recombinant prokaryotic expression vector；
E. the recombinant prokaryotic expression vector plasmid of acquisition is converted into escherichia coli prokaryotic expression bacterial strain BL21 (DE3),
Obtain Q9SMJ4 albumen escherichia coli prokaryotic expression system bacterium solutions；
F. by the expression system bacterium solution set up in step e (v/v) 1 in proportion:100 are inoculated into LB fluid nutrient mediums and fill
Enter two volumes to be cultivated in fluid nutrient medium conical flask；
G. the culture bacterium solution in step f is subjected to the protein induced expression of Q9SMJ4；
H.4800~5000rpm, 4 DEG C of 5~10min of centrifugation collect the thalline in induction end bacterium solution, abandon supernatant.
Wherein, condition of culture preferably carries out culture 6h in 37 DEG C, the incubator of rotating speed 200rpm in step f, treats bacterium solution
OD values reach 0.6~0.8.
Specific induced expression condition is preferably in step g：Inducing temperature is 37 DEG C, and antibiotic Kan concentration is 25 μ g/mL,
The induced expression time is 6h, and derivant IPTG concentration is 0.5mM, shaking speed 200rpm.
Preferably, the purifying of step (2) prokaryotic expression soluble protein further comprises one kind as the method for the present invention：
A. thalline is resuspended with buffer solution 10mL in the thalline that culture bacterium solution is collected per 50mL, and adds 170 μ L's 35%
The PMSF of TritonX-100 and 100 μ L 100mM；
B. resuspended bacterium solution is placed on ice, until bacterium solution becomes clear, ultrasound condition is ultrasonication for ultrasonication
3s, cools down 5s, and power is 400 watts；
C. by the bacterium solution 12000rpm of ultrasonication, 4 DEG C of centrifugation 15min, collect supernatant, abandon precipitation；
D. will be added containing the supernatant of His label proteins in Ni columns, coutroi velocity 0.5-1.0mL/min, until supernatant
Liquid flows completely through Ni columns, and applied sample amount/column volume (v/v) ratio is up to 15:1；
E. Ni columns or until A are rinsed with the buffer solution 2 of 8 times of column volumes280Value reaches stable, flow control 1.0mL/
min；
F. elution His tag expressions albumen uses gradient elution mode, and each gradient elution volume is 4 column volumes, receives
Collect each gradient elution albumen；
G. purity of protein is eluted by each gradient in SDS-PAGE electrophoresis detection previous steps, determines to collect composition list
First, the albumen wash-out component that impurity is less and molecular weight of albumen is consistent with destination protein；
H. the elution protein liquid of collection is fitted into the bag filter that molecular cut off is 3600Da, is put into ultra-pure water and dialyses
24h desalinations, during which rock bag filter and replace ultra-pure water；
I. the bag filter for end of dialysing is put into 6000 solution of PEG of 40% (w/v) and is concentrated into proper volume, be
The Q9SMJ4 soluble protein solution of prokaryotic expression and purified pool.
Wherein, the buffer solution 1 is 50mM NaH2PO4, 300mM NaCl, pH=8.0；The buffer solution 2 is
50mM NaH2PO4, 300mM NaCl, 10mM imidazoles, pH=8.0；The eluent that step f is used is 50mM NaH2PO4、300mM
NaCl, pH=8.0, imidazole concentration is respectively 50,100,150,200,250mM.
Preferably, the purifying of step (3) prokaryotic expression inclusion body protein and protein renaturation are into one for one kind as the method for the present invention
Step preferably includes：
A. thalline is resuspended with pH=8.0,0.1M phosphate buffer 10mL in the thalline that culture bacterium solution is collected per 50mL；
B. resuspended bacterium solution is placed on ice, until bacterium solution becomes to clarify, ultrasound condition is ultrasonication 3s, cold for ultrasonication
But 5s, power are 400 watts；
C. by the bacterium solution 12000rpm of ultrasonication, 4 DEG C of centrifugation 10min, collect precipitation, abandon supernatant；
D. precipitation is washed once with pH=8.0,0.1M phosphate buffer, 12000rpm, 4 DEG C of centrifugation 10min, it is heavy to collect
Form sediment；
E. with 10mL lysates dissolving precipitation, 1-2h is incubated at room temperature, during which constantly rocks solution, has dissolved precipitation
Entirely；
F.12000rpm, 30min is centrifuged under the conditions of 4 DEG C, collect supernatant, abandon precipitation insoluble matter；
G. Ni columns, coutroi velocity 0.5-1.0mL/min are balanced with 8 times of column volumes of buffer 3；
H. the supernatant containing His label proteins being collected into step f is added in Ni columns, coutroi velocity 0.5-
1.0mL/min, until supernatant flows completely through Ni columns, loading volume:Column volume ratio is up to 15:1；
I. Ni columns, coutroi velocity 0.5-1.0mL/min are balanced again with 4 times of column volumes of buffer 3；
J. Ni columns or until A are rinsed with the buffer solution 4 of 2 times of column volumes280Value reaches stable, flow control 1.0mL/
min；
K. it is 1.0mL/min to elute inclusion body protein flow control with the buffer solution 5 of 6 times of column volumes；
Purity of protein and purification effect are eluted in l.SDS-PAGE electrophoresis detection previous steps；
M. the inclusion body protein nature renaturation being collected into will be eluted under the conditions of 4 DEG C, ultra-pure water is slowly added into solution,
Urea concentration in solution is set to carry out gradient dilution；
N. the protein solution for reviving end is fitted into the bag filter that molecular cut off is 3600Da, be put into ultra-pure water thoroughly
24 desalinations are analysed, bag filter is during which rocked and replaces ultra-pure water；
O. the bag filter for end of dialysing is put into 6000 solution of PEG of 40% (w/v) and is concentrated into proper volume, be
Prokaryotic expression, purification is collected and the Q9SMJ4 inclusion body protein solution of renaturation.
Wherein, the lysate is 100mM NaH2PO4, 10mM Tris-HCl, 100mM DTT, 8M ureas, pH=8.0；
The buffer solution 3 is 100mM NaH2PO4, 10mM Tris-HCl, 8M ureas, pH=8.0；The buffer solution 4 is 100mM
NaH2PO4, 10mM Tris-HCl, 8M ureas, 10mM imidazoles, pH=8.0；The buffer solution 5 is 100mM NaH2PO4、10mM
Tris-HCl, 8M urea, 200mM imidazoles, pH=8.0.
The gradient that urea concentration is diluted in step m is preferably 8M-6M-4M-2M-1M-0.1M, each gradient duration
For 1h.
The solution of the method for the present invention whole process is prepared and is both needed to use pure water.
Beneficial effect
The present invention is directed to a kind of storage protein type alpha-amylase inhibitor being present in chick-pea seed from natural material
It is difficult to isolate and purify in material, removes other foreign proteins and be applied to actual challenge problem as this alpha-amylase, establish one first
Set is suitable for production and the purifying process flow of this storage protein type alpha-amylase inhibitor.
This set bioprocess technology flow first carries out this storage protein type alpha-amylase inhibitor in chick-pea seed
Biology prepare and follow-up protein purification recycling, through SDS-PAGE electrophoresis detections it is isolated prepare protein active height, prepare
Amount is more, and easy to operate, cost is relatively low, reproducible, may be directly applied to the biological production of this alpha-amylase inhibitor.
The whole technological process of production is equally respectively suitable for Q9SMJ4 albumen alpha chains (using the ORF of coding alpha chains)
With the production and purifying of beta chains (using the ORF of coding beta chains), different biological (animal, plant, micro- lifes are can also be applied to
Thing) source, have Cupin structures (functional domain) storage protein type alpha-amylase inhibitor production and purifying.
Brief description of the drawings
Fig. 1 Q9SMJ4 protein sequences and alpha and beta peptide sequence hum patterns
Storage protein type alpha-amylase inhibitor (Q9SMJ4) prokaryotic expression soluble protein table in Fig. 2 chick-pea seeds
Reach and purification result figure
Expressing protein band is shown in red block, swimming lane is followed successively by from right to left：1. albumen maker, 2. expression bacterium are broken
Broken supernatant, 3. expression bacterium crush sediment, 4. sample penetration peaks, 5. eluting peaks (10mM imidazoles), 6. eluting peaks (50mM miaows
Azoles), 7. eluting peaks (100mM imidazoles), 8. eluting peaks (150mM imidazoles), 9. eluting peaks (200mM imidazoles), 10. eluting peaks
(250mM imidazoles)
Storage protein type alpha-amylase inhibitor (Q9SMJ4) prokaryotic expression inclusion body protein table in Fig. 3 chick-pea seeds
Reach and purification result figure
Red arrow show storage protein type alpha-amylase inhibitor (Q9SMJ4) prokaryotic expression inclusion body protein
Embodiment
Embodiment 1
(1) prokaryotic expression system structure and bacterium solution culture induced expression：
A. the cDNA of seed obtains coding alpha-amylase by PCR method and presses down as template in being developed using Desi types chick-pea
The ORF sequences of preparation albumen (it is Q9SMJ4 that it is numbered in NCBI), primer sequence are
F1:5'-TGTCATCATGGCTAAGCTTCTTG-3'(SEQ ID NO.1) and
R1:5'-TTGTTTCCGCTAAAAGGTACATG-3'(SEQ ID NO.2)；
B. using round pcr the 5 ' and 3 ' of coding Q9SMJ4 albumen ORF sequences (removing encoding proteins signal peptide sequence)
Restriction enzyme site sequence EcoR I and Not I is added at end respectively, obtains target gene fragment, and primer sequence is
F2:5'-CGGAATTCTTGAGAGATCAACCT-3'(SEQ ID NO.3) and
R2:5'-TTTTCCTTTTGCGGCCGCCAGCTGCTGCTTTGTT-3'(SEQ ID NO.4)；
C. utilize restriction enzyme EcoR I and Not I to the target gene fragment obtained in step b in 37 DEG C of conditions
It is lower to carry out 4 hour digestion processing, the processed DNA fragmentation of glue reclaim；
D. 37 DEG C of bars are carried out to pET-28a (+) prokaryotic expression carrier plasmid using restriction enzyme EcoR I and Not I
When digestion 4 is small under part, the processed Plasmid DNA fragment of glue reclaim；
E. will be obtained in step c and step d with the processed DNA fragmentation T of restriction enzyme4Ligase is 16
Connected under the conditions of DEG C, obtain recombinant prokaryotic expression vector；
F. the recombinant prokaryotic expression vector plasmid of acquisition is converted into escherichia coli prokaryotic expression bacterial strain BL21 (DE3),
Obtain Q9SMJ4 albumen escherichia coli prokaryotic expression system bacterium solutions；
G. by the expression system bacterium solution set up in step f (v/v) 1 in proportion:100 are inoculated into LB fluid nutrient mediums and fill
Enter two volumes in fluid nutrient medium conical flask, culture 6h is carried out in 37 DEG C, the incubator of rotating speed 200rpm, treat bacterium solution OD
Value reaches 0.7 or so；
H. the culture bacterium solution in step g is subjected to the protein induced expression of Q9SMJ4, specific induced expression condition is as follows：Induction
Temperature is 37 DEG C, and antibiotic Kan concentration is 25 μ g/mL, and the induced expression time is 6h, and derivant IPTG concentration is 0.5mM, rotating speed
For 200rpm；
I.5000rpm, 4 DEG C of centrifugation 5min collect the thalline in induction end bacterium solution, abandon supernatant.
(2) prokaryotic expression soluble protein purification process：
A. thalline (the 50mM NaH of buffer solution 1 that culture bacterium solution is collected per 50mL2PO4, 300mM NaCl, pH=8.0)
Thalline is resuspended in 10mL, and adds the PMSF of the TritonX-100 and 100 μ L 100mM of 170 μ L 35%；
B. resuspended bacterium solution is placed on ice, until bacterium solution becomes clear, ultrasound condition is ultrasonication for ultrasonication
3s, cools down 5s, and power is 400 watts；
C. by the bacterium solution 12000rpm of ultrasonication, 4 DEG C of centrifugation 15min, collect supernatant, abandon precipitation；
D. will be added containing the supernatant of His label proteins in Ni columns, coutroi velocity 0.5-1.0mL/min, until supernatant
Liquid flows completely through Ni columns, applied sample amount:Column volume ratio is up to 15:1；
E. with (the 50mM NaH of buffer solution 2 of 8 times of column volumes2PO4, 300mM NaCl, 10mM imidazoles, pH=8.0) rinse Ni
Column or until A280Value reaches stable, flow control 1.0mL/min；
F. elution His tag expressions albumen uses gradient elution mode, and eluent is 50mM NaH2PO4, 300mM NaCl,
PH=8.0, imidazole concentration is respectively 50,100,150,200,250mM, each gradient elution volume is 4 column volumes, is collected each
A gradient elution albumen；
G. purity of protein is eluted by each gradient in SDS-PAGE electrophoresis detection steps f, determines to be adapted to that collects to wash
De- component；
H. the elution protein liquid of collection is fitted into the bag filter that molecular cut off is 3600Da, is put into ultra-pure water and dialyses
24h desalinations, during which rock bag filter and replace ultra-pure water；
I. the bag filter for end of dialysing is put into 6000 solution of PEG of 40% (w/v) and is concentrated into proper volume, be
The soluble Q9SMJ4 protein solutions of prokaryotic expression and purified pool.
J. the soluble Q9SMJ4 albumen (Fig. 2) being collected into by the detection of SDS-PAGE glue, shown prokaryotic expression bacterium expression
Soluble protein is higher, and purification effect is good, has higher activity, and purifying recycling protein content is higher.
Embodiment 2
A.5000rpm, 4 DEG C of centrifugation 5min collect the thalline in 1 step of embodiment (1) induction end bacterium solution, abandon supernatant；
B. thalline is resuspended with 0.1M phosphate buffers (pH=8.0) 10mL in the thalline that culture bacterium solution is collected per 50mL；
C. resuspended bacterium solution is placed on ice, until bacterium solution becomes to clarify, ultrasound condition is ultrasonication 3s, cold for ultrasonication
But 5s, power are 400 watts；
D. by the bacterium solution 12000rpm of ultrasonication, 4 DEG C of centrifugation 10min, collect precipitation, abandon supernatant；
E. precipitation is washed once with 0.1M phosphate buffers (pH=8.0), 12000rpm, 4 DEG C of centrifugation 10min, it is heavy to collect
Form sediment；
F. 10mL lysates (100mM NaH are used2PO4, 10mM Tris-HCl, 100mM DTT, 8M ureas, pH=8.0) dissolving
Precipitation, is incubated at room temperature 1-2h, during which constantly rocks solution, makes precipitation dissolving complete；
G.12000rpm, 30min is centrifuged under the conditions of 4 DEG C, collect supernatant, abandon precipitation insoluble matter；
H. with 8 times of (100mM NaH of column volumes of buffer 32PO4, 10mM Tris-HCl, 8M ureas, pH=8.0) balance Ni
Column, coutroi velocity 0.5-1.0mL/min；
I. the supernatant containing His label proteins being collected into step g is added in Ni columns, coutroi velocity 0.5-
1.0mL/min, until supernatant flows completely through Ni columns, loading volume:Column volume (v/v) ratio is up to 15:1；
J. with 4 times of (100mM NaH of column volumes of buffer 32PO4, 10mM Tris-HCl, 8M ureas, pH=8.0) balance again
Ni columns, coutroi velocity 0.5-1.0mL/min；
K. with (the 100mM NaH of buffer solution 4 of 2 times of column volumes2PO4, 10mM Tris-HCl, 8M ureas, 10mM imidazoles, pH=
8.0) rinse Ni columns or until A280 values reach stable, flow control 1.0mL/min；
L. with (the 100mM NaH of buffer solution 5 of 6 times of column volumes2PO4, 10mM Tris-HCl, 8M ureas, 200mM imidazoles, pH=
8.0) it is 1.0mL/min to elute inclusion body protein flow control；
Purity of protein and purification effect are eluted in m.SDS-PAGE electrophoresis detection previous steps；
N. the inclusion body protein nature renaturation being collected into will be eluted under the conditions of 4 DEG C, ultra-pure water is slowly added into solution,
Urea concentration in solution is set to carry out gradient dilution (8M-6M-4M-2M-1M-0.1M), each gradient duration is 1h；
O. protein solution renaturation terminated is fitted into the bag filter that molecular cut off is 3600Da, is put into ultra-pure water thoroughly
24h desalinations are analysed, bag filter is during which rocked and replaces ultra-pure water；
P. the bag filter for end of dialysing is put into 40% 6000 solution of PEG and is concentrated into proper volume, be protokaryon table
Up to purified pool and the Q9SMJ4 inclusion body protein solution of renaturation.
Y. the Q9SMJ4 inclusion bodies of protein (Fig. 3) collected by the detection of SDS-PAGE glue, shown effect is prokaryotic expression bacterium
Express express target protein amount is very high, and induced expression condition is good, and purifying recovering effect is good, has higher activity, purifying recycling
Protein content is higher, and foreign protein content is few.
Claims (4)
1. production and the purification process of a kind of storage protein type alpha-amylase inhibitor with Cupin Structure and function domains, its
It is characterized in that comprising the following steps (1) and one in step (2) and step (3) or two：
(1) prokaryotic expression system structure and bacterium solution culture induced expression：
The ORF of Q9SMJ4 albumen is cloned into prokaryotic expression carrier, and builds Q9SMJ4 albumen escherichia coli prokaryotic expression systems；
Q9SMJ4 albumen escherichia coli prokaryotic expression systems are cultivated, and induce destination protein to express；The wherein described prokaryotic expression carrier
Contain His label protein encoding genes；
(2) prokaryotic expression soluble protein purifies：
A. thalline is resuspended with 1 10mL of buffer solution in the thalline that culture bacterium solution is collected per 50mL, and adds 170 μ L's 35%
The PMSF of TritonX-100 and 100 μ L 100mM；
B. resuspended bacterium solution is placed on ice, until bacterium solution becomes clear, ultrasound condition is ultrasonication 3s, cold for ultrasonication
But 5s, power are 400 watts；
C. by the bacterium solution 12000rpm of ultrasonication, 4 DEG C of centrifugation 15min, collect supernatant, abandon precipitation；
D. will be added containing the supernatant of His label proteins in Ni columns, coutroi velocity 0.5-1.0mL/min, until supernatant is complete
Flow through Ni columns, applied sample amount entirely:Column volume (v/v) ratio is up to 15:1；
E. Ni columns or until A are rinsed with the buffer solution 2 of 8 times of column volumes280Value reaches stable, flow control 1.0mL/min；
F. elution His tag expressions albumen uses gradient elution mode, and each gradient elution volume is 4 column volumes, collects each
A gradient elution albumen；
G. purity of protein is eluted by each gradient in SDS-PAGE electrophoresis detection previous steps, determines that collection composition is single, miscellaneous
The albumen wash-out component that matter is less and molecular weight of albumen is consistent with destination protein；
H. the elution protein liquid of collection is fitted into the bag filter that molecular cut off is 3600Da, is put into ultra-pure water the 24h that dialyses
Desalination, during which rocks bag filter and replaces ultra-pure water；
I. the bag filter for end of dialysing is put into 6000 solution of PEG of 40% (w/v) and is concentrated into proper volume, be protokaryon
Express the Q9SMJ4 soluble protein solution of simultaneously purified pool；
Wherein, the buffer solution 1 is 50mM NaH2PO4, 300mM NaCl, pH=8.0；The buffer solution 2 is 50mM
NaH2PO4, 300mM NaCl, 10mM imidazoles, pH=8.0；The eluent that step f is used is 50mM NaH2PO4、300mM
NaCl, pH=8.0, imidazole concentration is respectively 50,100,150,200,250mM；
(3) purifying of prokaryotic expression inclusion body protein and protein renaturation：
A. thalline is resuspended with pH=8.0,0.1M phosphate buffer 10mL in the thalline that culture bacterium solution is collected per 50mL；
B. resuspended bacterium solution is placed on ice, until bacterium solution becomes to clarify, ultrasound condition is ultrasonication 3s for ultrasonication, cooling
5s, power are 400 watts；
C. by the bacterium solution 12000rpm of ultrasonication, 4 DEG C of centrifugation 10min, collect precipitation, abandon supernatant；
D. precipitation is washed once with pH=8.0,0.1M phosphate buffer, 12000rpm, 4 DEG C of centrifugation 10min, collect precipitation；
E. with 10mL lysates dissolving precipitation, 1-2h is incubated at room temperature, during which constantly rocks solution, make precipitation dissolving complete；
F.12000rpm, 30min is centrifuged under the conditions of 4 DEG C, collect supernatant, abandon precipitation insoluble matter；
G. Ni columns, coutroi velocity 0.5-1.0mL/min are balanced with 8 times of column volumes of buffer 3；
H. the supernatant containing His label proteins being collected into step f is added in Ni columns, coutroi velocity 0.5-1.0mL/
Min, until supernatant flows completely through Ni columns, loading volume:Column volume ratio is up to 15:1；
I. Ni columns, coutroi velocity 0.5-1.0mL/min are balanced again with 4 times of column volumes of buffer 3；
J. Ni columns or until A are rinsed with the buffer solution 4 of 2 times of column volumes280Value reaches stable, flow control 1.0mL/min；
K. it is 1.0mL/min to elute inclusion body protein flow control with the buffer solution 5 of 6 times of column volumes；
Purity of protein and purification effect are eluted in l.SDS-PAGE electrophoresis detection previous steps；
M. the inclusion body protein nature renaturation being collected into will be eluted under the conditions of 4 DEG C, ultra-pure water is slowly added into solution, is made molten
Urea concentration carries out gradient dilution in liquid；
N. protein solution renaturation terminated is fitted into the bag filter that molecular cut off is 3600Da, is put into ultra-pure water and dialyses 24
Hour desalination, during which rocks bag filter and replaces ultra-pure water；
The bag filter for end of dialysing is put into 6000 solution of PEG of 40% (w/v) and is concentrated into proper volume, is protokaryon table
Up to purified pool and the Q9SMJ4 inclusion body protein solution of renaturation；
Wherein, the lysate is 100mM NaH2PO4, 10mM Tris-HCl, 100mM DTT, 8M ureas, pH=8.0；It is described
Buffer solution 3 be 100mM NaH2PO4, 10mM Tris-HCl, 8M ureas, pH=8.0；The buffer solution 4 is 100mM
NaH2PO4, 10mM Tris-HCl, 8M ureas, 10mM imidazoles, pH=8.0；The buffer solution 5 is 100mM NaH2PO4、10mM
Tris-HCl, 8M urea, 200mM imidazoles, pH=8.0；The gradient that urea concentration is diluted in step m is 8M-6 M-4 M-2 M-1
M-0.1M, each gradient duration are 1h.
2. according to the method described in claim 1, it is characterized in that (1) prokaryotic expression system structure and bacterium solution culture lure
Leading expression includes：
A. using the cDNA of chick-pea development seed as template, coding storage protein type alpha-amylase is obtained by PCR method and is pressed down
The ORF sequences of preparation；
B. restriction enzyme site sequence is added respectively at 5 ' and 3 ' ends of coding Q9SMJ4 albumen ORF sequences using round pcr
EcoR I and Not I, obtains target gene fragment；Wherein Q9SMJ4 albumen ORF sequences remove encoding proteins signal peptide sequence；
C. it is former to the target gene fragment and pET-28a (+) obtained in step b using restriction enzyme EcoR I and Not I
Nuclear expression vector plasmid carries out digestion, the processed target gene fragment of glue reclaim and Plasmid DNA fragment respectively；
D. connected what previous step obtained with restriction enzyme processed DNA fragmentation T4 ligases under the conditions of 16 DEG C
Connect, obtain recombinant prokaryotic expression vector；
E. the recombinant prokaryotic expression vector plasmid of acquisition is converted into escherichia coli prokaryotic expression bacterial strain BL21 (DE3), obtained
Q9SMJ4 albumen escherichia coli prokaryotic expression system bacterium solutions；
F. by the expression system bacterium solution set up in step e (v/v) 1 in proportion:100 are inoculated into LB fluid nutrient mediums and load two
Times volume is cultivated in fluid nutrient medium conical flask；
G. the culture bacterium solution in step f is subjected to the protein induced expression of Q9SMJ4；
H.4800~5000rpm, 4 DEG C of 5~10min of centrifugation collect the thalline in induction end bacterium solution, abandon supernatant.
3. according to the method described in claim 2, it is characterized in that in step f condition of culture be at 37 DEG C, rotating speed 200rpm's
Culture 6h is carried out in incubator, treats that bacterium solution OD values reach 0.6~0.8.
4. according to the method described in claim 2, it is characterized in that specific induced expression condition is in step g：Inducing temperature is
37 DEG C, antibiotic Kan concentration is 25 μ g/mL, and the induced expression time is 6h, and derivant IPTG concentration is 0.5mM, and shaking speed is
200rpm。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102559640A (en) * | 2012-02-15 | 2012-07-11 | 上海禧月食品有限公司 | Separation technology of chickpea alpha-amylase and application thereof |
| CN102924592A (en) * | 2012-11-29 | 2013-02-13 | 南京农业大学 | Method for extracting and separating protein type alpha-amylase inhibitors in chickpea seeds |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102559640A (en) * | 2012-02-15 | 2012-07-11 | 上海禧月食品有限公司 | Separation technology of chickpea alpha-amylase and application thereof |
| CN102924592A (en) * | 2012-11-29 | 2013-02-13 | 南京农业大学 | Method for extracting and separating protein type alpha-amylase inhibitors in chickpea seeds |
Non-Patent Citations (6)
| Title |
|---|
| Characterization of a Novel Legumin α-Amylase Inhibitor from Chickpea (Cicer arietinum L.) Seeds;Xiaoyan HAO等;《Biosci. Biotechnol. Biochem.》;20090507;第73卷(第5期);1200-1202 * |
| Fuqiang Yin等.Analysis of common bean expressed sequence tags identifies sulfur metabolic pathways active in seed and sulfur-rich proteins highly expressed in the absence of phaseolin and major lectins.《BMC Genomics》.2011,第12卷(第268期), * |
| Mandaokar,A.D.等.Accession Number:Q9SMJ4.《UniProt》.2014, * |
| 刘晓洁等.鹰嘴豆α-淀粉酶抑制剂的提取及性质研究.《时珍国医国药》.2011,第22卷(第7期), * |
| 贾彦凤等.鹰嘴豆α-淀粉酶抑制剂基因CL-AI的原核表达、纯化及生物学特性.《核农学报》.2017,第31卷(第5期), * |
| 郝小燕.鹰嘴豆蛋白样a-淀粉酶抑制剂的分离纯化、候选基因.《中国博士学位论文全文数据库》.2016,(第08期), * |
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