CN107760744A - The preparation method of high yield and high stability recombinant lamprey Lj RGD3 albumen - Google Patents
The preparation method of high yield and high stability recombinant lamprey Lj RGD3 albumen Download PDFInfo
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Abstract
The present invention discloses a kind of preparation method of high yield and high stability recombinant lamprey Lj RGD3 albumen, using different culture mediums to positive recombinant high density fermentation and IPTG induced expressions, broken thalline is filtered with hollow fiber filter membrane, operated instead of existing high speed centrifugation and dead-end filtration etc., the reasonability and operability of technique after amplifying is effectively ensured, through nickel ion affinity chromatograph, anion-exchange chromatography and sieve chromatography, the rLj RGD3 stostes of hectogram level can be obtained, its purity of protein >=99%, suitable for large-scale industrial production.RLj RGD3 stostes and different component are coordinated, prepare rLj RGD3 albumen by being freeze-dried, stability is strong.
Description
Technical field
The present invention relates to a kind of protein preparation method, especially a kind of high yield simple to operate, purifying rate is high and height are steady
The preparation method of qualitative recombinant lamprey Lj-RGD3 albumen.
Background technology
Lj-RGD3 is the RGD toxin proteins from Japanese lamprey oral gland secretion, by 117 amino acid residues
Composition, Lj-RGD3 primary structure are removed special with three RGD die bodys and a pair of cysteine this typical RGD toxin proteins
Sign is outer, also containing 17 histidines, 17 arginine, be a kind of HRG basic protein, it has been disclosed that in Patent No.
200510083437.7th, the entitled " gene cloning and table of the Japanese lamprey oral gland RGD die body albumen of tool antitumor action
Up to " Chinese invention patent document in, that is, be named as Lampetrin3 albumen, there is anti-angiogenic rebirth, antitumor propagation, anti-
Thrombus and other effects.
China Patent No. discloses one kind for ZL201310600092.2 patent of invention and " removes affinitive layer purification label
The preparation method of recombinant lamprey Lj-RGD3 albumen ", is comprised the following steps that：Draw in the end of Lj-RGD3 gene orders 3 '
Enter terminator codon, be connected with carrier with Nde I and Hind III digestions site, positive transformants are carried out after being transformed into expression bacterium
The Screening and Identification of son；IPTG induced expressions are carried out to positive recombinant；The recombinant protein of expression is extracted；To the egg of extraction
Bai Yici is purified as follows：It is pure that albumen is carried out by metal chelate ions affinity column HiTrap IMAC HP
Change, cation-exchange chromatography, dialysis and freeze-drying are carried out using SP posts to collected albumen.But but there is as follows
Problem：
1. being not directed to large scale fermentation, products obtained therefrom low yield, the small-scale mg levels protein production in laboratory is suitable only for；
, it is necessary to remove cell fragment by centrifuge when 2. pair bacterial cell disruption liquid is clarified.More high-speed and continuous are wandered about as a refugee scheming
High equipment investment and rotor regular maintenance cost will be caused, unit is cumbersome, and labor intensity is big, parameter easily by it is artificial because
Element interference and it is uncontrollable, be not suitable for mass producing；
3. purge process can not effectively remove host DNA and heat source substance, it is too low to obtain protein concentration；
4. it is poor to freeze obtained rLj-RGD3 protein stabilities.
The content of the invention
The present invention is to solve the above-mentioned technical problem present in prior art, there is provided a kind of simple to operate, purifying rate
High high yield and the preparation method of high stability recombinant lamprey Lj-RGD3 albumen.
The present invention technical solution be：A kind of high yield and high stability recombinant lamprey Lj-RGD3 albumen
Preparation method, it is characterised in that carry out in accordance with the following steps successively：
A. terminator codon is introduced in the end of Lj-RGD3 gene orders 3 ', with carrier with Nde I and Hind III digestions site
It is connected, is transformed into the Screening and Identification of progress positive transformant after expression bacterium, obtains positive recombinant；
B. positive recombinant is expanded and cultivates and carry out IPTG induced expressions：
B.1 monoclonal is taken, is inoculated into LB culture mediums, 30 DEG C are cultivated 16 ~ 18 hours, reach 2 ~ 3 to OD600；
B.2 by volume 1:In high-concentration culturing base, 30 DEG C are cultivated 2 ~ 3 hours 10 two-stage inoculations, reach 1 ~ 2 to OD600；
B.3 it is inoculated into 30 DEG C of intermediate transit fermentation tank to continue to cultivate, when zymotic fluid OD600 reaches 2 ~ 3 or so, into culture medium
Add the trace element of fermentation volume 1/20, the glucose of fermentation volume 2/5, the Mg of fermentation volume 1/1002SO4·2H2O, send out
The CaCl of ferment volume 0.2/1002·2H2O and final concentration of 1 mM IPTG, 30 DEG C of cultures to OD are at least 5；
The component of the LB culture mediums is as follows：Tryptone 10g/L, dusty yeast 5g/L, NaCl 5g/L；
The component of the high-concentration culturing base is as follows：Tryptone 12g/L, the g/L of dusty yeast 24, the ml/L of 100% glycerine 4, lemon
Lemon acid 7.5 g/L, NaCl 0.5g/L, KH2PO42.31 g/L, K2HPO4·3H2The g/L of O 12.5,（NH4）2SO47g/L,
H3PO41.5 ml/L, the ml/L of defoamer 0.6；
C. thalline is collected, supernatant is filtered to obtain through homogeneous crusher machine, hollow fiber filter membrane；
D. gained supernatant obtains rLj-RGD3 stostes through nickel ion affinity chromatograph, anion-exchange chromatography and sieve chromatography.
The step c is as follows：
C.1 thalline is collected, bacterial cell disruption liquid is obtained through homogeneous crusher machine, bacterial cell disruption liquid is diluted 2 times, carried out using 1 μm of filter core
Filtering；
C.2 500 ~ 750 further ultrafiltration of K hollow fiber filter membranes are used, obtain clear filtrate；
C.3 3K hollow fiber filter membrane concentrating clarifying filtrates are used.
The Binding buffer compositions of the Step d nickel ion affinity chromatograph are 20mM Tris, 0.8M NaCl and 5mM
PH 7.9 imidazoles, Elution buffer compositions are 20mM Tris, 0.8M NaCl, 1M pH 7.9 imidazoles；Described the moon
The filler of ion-exchange chromatography is QFF, and buffer solution is Buffer A：20 mM PBS, pH7.4；Buffer B：100 mM PBS,
1M NaCl, pH7.4；The sieve chromatography filler is Superdex 75, and buffer solution is 10 ~ 30 mM PBS, pH7.4.
After the Step d into 250 μ g rLj-RGD3 stostes add mass percent be 1% ~ 5% mannitol or 50 ~
250 mM Gly, final concentration of 50 ~ 250 mM Arg and 10 ~ 30 mM pH7.4 PBS buffer, it is freeze-dried and
Into.
The present invention uses different culture mediums to positive recombinant high density fermentation and IPTG induced expressions, with doughnut
Membrane filtration crushes thalline, instead of existing high speed centrifugation and dead-end filtration etc. operate, be effectively ensured amplification after technique it is reasonable
Property and operability, through nickel ion affinity chromatograph, anion-exchange chromatography and sieve chromatography, the rLj- of hectogram level can be obtained
RGD3 stostes, its purity of protein >=99%, suitable for large-scale industrial production.RLj-RGD3 stostes and different component are coordinated,
RLj-RGD3 albumen is prepared by being freeze-dried, stability is strong.
Brief description of the drawings
Fig. 1 is the SDS-PAGE of rLj-RGD3 of embodiment of the present invention 300L fermentation expression results.
Fig. 2 is the SDS-PAGE that 750K of embodiment of the present invention hollow fiber filter membranes pass through end target protein.
Fig. 3 is nickel ion affinity chromatograph product result figure of the embodiment of the present invention.
Fig. 4 is anion-exchange chromatography product result figure of the embodiment of the present invention.
Fig. 5 is the polishing purification result figure of sieve chromatography of the embodiment of the present invention.
Fig. 6 is the HPLC testing results of the polishing purification albumen of sieve chromatography of the embodiment of the present invention.
Embodiment
The present invention high yield and high stability recombinant lamprey Lj-RGD3 albumen preparation method, successively according to
Following steps are carried out：
A. terminator codon is introduced in the end of Lj-RGD3 gene orders 3 ', with carrier with Nde I and Hind III digestions site
It is connected, is transformed into the Screening and Identification of progress positive transformant after expression bacterium, obtains positive recombinant；This step is according to Chinese special
Profit number discloses one kind for ZL201310600092.2 patent of invention and " goes the recombinant lamprey of affinitive layer purification label
The preparation method of Lj-RGD3 albumen " is carried out.
B. positive recombinant is expanded and cultivates and carry out IPTG induced expressions：
B.1 monoclonal is taken, is inoculated into 50 ml LB culture mediums, 30 DEG C are cultivated 16 ~ 18 hours, reach 2 ~ 3 to OD600；
B.2 by volume 1:In 1000 ml high-concentration culturing bases, 30 DEG C are cultivated 2 ~ 3 hours 10 two-stage inoculations, are reached to OD600
To 1 ~ 2；
B.3 it is inoculated into 30 DEG C of intermediate transit fermentation tank to continue to cultivate, samples once simultaneously record data every 1hr, treat zymotic fluid
When OD600 reaches 2 ~ 3 or so, transferred species in the production fermentation tank high-concentration culturing base to 100 more than L, i.e., added into culture medium
The trace element of fermentation volume 1/20（Formula：FeSO4.7H2O 3.36 g/L, MnSO4.H2O 0.51 g/L, ZnSO4.7H2O
0.84 g/L, NaMoD4.7H2O 0.25 g/L, CuSO4.5H2O 0.12 g/L, H3BO3 0.36 g/L, H3PO4 48
ml/L）, fermentation volume 2/5 glucose, the Mg of fermentation volume 1/1002SO4·2H2O, the CaCl of fermentation volume 2/10002·
2H2O and final concentration of 1 mM IPTG, 30 DEG C of cultures to OD are at least 5；
The component of the LB culture mediums is as follows：Tryptone 10g/L, dusty yeast 5g/L, NaCl 5g/L；
The component of the high-concentration culturing base is as follows：Tryptone 12g/L, the g/L of dusty yeast 24, the ml/L of 100% glycerine 4, lemon
Lemon acid 7.5 g/L, NaCl 0.5g/L, KH2PO42.31 g/L, K2HPO4·3H2The g/L of O 12.5,（NH4）2SO47g/L,
H3PO41.5 ml/L, the ml/L of defoamer 0.6；
Different time sample carries out SDS-PAGE detections（And scan）As a result it is as shown in Figure 1.
Lane 1 in Fig. 1：Shake-flask seed thalline； Lane 2：Canister seed thalline；Lane 3：Ferment 5 h sample points
Thalline；Lane 4：Ferment the thalline of 9 h sample points；Lane 5：Ferment the thalline of 13 h sample points；Lane 6：15 h that ferment take
The thalline of sampling point；Lane 7：Ferment the thalline of 17 h sample points；Lane 8：Ferment the thalline of 19 h sample points：Lane 9：Hair
The thalline of the h sample points of ferment 21；Lane 10：Ferment the thalline of 23 h sample points；Lane 11：Ferment 23 h nutrient solution supernatants；
Lane 12：STD HG； Lane 13： Marker.
Determine protein content：Last every liter of zymotic fluid total protein output capacity is 14 g, i.e. 50 L can obtain total egg with top fermentation
White 700 more than g.
C. thalline is collected, supernatant is filtered to obtain through homogeneous crusher machine, hollow fiber filter membrane：
C.1 thalline is collected, bacterial cell disruption liquid is obtained through homogeneous crusher machine, bacterial cell disruption liquid is diluted 2 times, carried out using 1 μm of filter core
Filtering；Ultrafiltration buffer：0.5 M NaCl, 5 mM imidazoles, 20 mM TrisHCl；Ultrafiltration apparatus：10 t/h centrifugal pumps or
Like device；Membranous type number：K06-E500-10-N or like product；Pump speed：26-30 t/h.
C.2 500 ~ 750 further ultrafiltration of K hollow fiber filter membranes are used, obtain clear filtrate；Ultrafiltration buffer：0.5 M
NaCl, 5 mM imidazoles, 20 mM TrisHCl；Ultrafiltration apparatus：10 t/h centrifugal pumps or similar devices；Membranous type number：UFP-3-
C-85 or like product；Pump speed：28 t/h.
C.3 3K hollow fiber filter membranes are used（Or like product）Concentrating clarifying filtrate, concentrate 2 times of volumes.
750K hollow fiber filter membranes are as shown in Figure 2 through the SDS-PAGE of end target protein.
SDS PAGE in Fig. 2（Sampled through end, electrophoresis loading 20ug）Display is filtered by 500 K Hollow fiber systems
Bacterium solution, through the collection situation of end target protein：Lane 1:Sample is passed through before first time filter wash; Lane 2:Sample before ultrafiltration
Product; Lane 3:Single-point 1 is passed through after ultrafiltration; Lane 4:Sample is passed through before first time filter wash; Lane 5: Marker;
Lane 6:Ultrafiltration passes through single-point 2; Lane 7:Ultrafiltration passes through single-point 3; Lane 8:Ultrafiltration passes through single-point 4; Lane
9:Ultrafiltration passes through single-point 5; Lane 10:Ultrafiltration passes through single-point 5.
D. gained supernatant obtains rLj-RGD3 stostes through nickel ion affinity chromatograph, anion-exchange chromatography and sieve chromatography：
The present invention carries out rLj-RGD3 first step purifying using the potent FF resins of Ni 6 of force trapping：
Previous step supernatant liquid is taken to balance 5CV, balance and loading, wash baselines are put down, the CV of gradient elution 1.5, and elution is linear.It is pure
Change equipment：AKTA PROCESS or same type purifier apparatus；Resin model：Ni 6FF；Pillar model：Model is greater than or equal to
BPG 200/500 pillar or similar products, more than the L of column volume 7.Binding buffer compositions are 20mM Tris, 0.8M
NaCl and 5mM pH 7.9 imidazoles, Elution buffer compositions are 20mM Tris, 0.8M NaCl, 1M pH 7.9 miaow
Azoles；According to the peak value detected, the sample for eluting each stage is collected.
Nickel ion affinity chromatograph result is as shown in Figure 3.
The SDS-PAGE, Lane 1 of Fig. 3 nickel ion affinity chromatograph albumen：Marker；Lane 2：Peak point sample；Lane
3：1B1 samples；Lane 4：1B2 samples；Lane 5：1B2 samples；Lane 6：1B2 samples.
Disposably purifying can capture more than the g of thick leach protein 200 as can be seen here to this step, and the FF resins of Ni 6 are to rLj-RGD3
Capture ability it is stronger.
By sample loading obtained by previous step ion affinity chromatography, purifier apparatus：AKTA PROCESS or same type purifying are set
It is standby；Resin model：Anion exchange resin Q FF；Pillar model：Model is greater than or equal to BPG300/500 pillar or similar
Product, more than the L of column volume 30.BufferA -20 mM PBS, PH 7.4；BufferB：100 mM PBS, 1M NaCl, PH
7.4.Balance 10 CV, balance and loading, gradient elution.According to the peak value detected, the sample for eluting each stage is collected, every batch
It is secondary to obtain more than the g of purifying protein 150.The host DNA and heat source substance in albumen stoste can effectively be removed.
Anion-exchange chromatography product result is as shown in Figure 4.
The SDS-PAGE of Fig. 4 Q anion-exchange chromatography protein purification samples.Lane 1：Marker；Lane 2：Q2 samples
The μ g of product 20；Lane 3：The μ g of Q2 samples 10；Lane 4：Q1 samples；Lane 5：Sample before Q.
Previous step Q anion exchanges sample is used for sieve chromatography.Purifier apparatus：AKTA PROCESS or same type are pure
Change equipment；Resin model：Anion exchange resin Q FF.Pillar model：Model greater than or equal to BPG300/950 pillar or
Similar products, more than the L of column volume 50.Filler：Superdex molecular sieve fillings or similar products；Buffer：20 mM PBS
（10 ~ 30 mM PBS）, pH 7.4；Flow velocity is 8.5 more than ml/min.According to the peak value detected, collect and elute each rank
The sample of section.More than the g of purification of samples 100 can be obtained per batch, can effectively remove the polymer in sample and can be to protein solution
Buffer system displacement 20 mM PBS into the PBS of pharmaceutical formulation, albumen stoste purity is up to more than 99%.
Sieve chromatography result is as shown in Figure 5：
Fig. 5 sieve chromatography protein 15 non-reduced SDS-PAGE of % gum concentrations, show each collecting pipe sample in peak 1.
Sieve chromatography polishing purification albumen is detected through HPLC, HPLC operating conditions are as follows：Chromatographic column TSKgel used
G2000-3000 swXL thin layer chromatography posts；Mobile phase is by 3.2 mM disodium hydrogen phosphates, 1.5 mM potassium dihydrogen phosphates, 400 mM
NaCl, PH7.3 are formed.The nm of Detection wavelength 214.
As a result it is as shown in Figure 6：As a result show that gained purity of protein reaches more than 99.94%（Abscissa minutes, indulge and sit
Mark AU）.
In order to improve rLj-RGD3 stability, take rLj-RGD3 stostes obtained by the 250 μ g embodiment of the present invention, to it in
Add mannitol or 50 ~ 250 mM Gly, final concentration of 50 ~ 250 mM Arg that final concentration mass percent is 1% ~ 5% and
10 ~ 30 mM pH7.4 PBS buffer, it is freeze-dried to form.
As a result show that the protective agents such as the mannitol of the embodiment of the present invention can play protein protection, lyophilized figuration and regulation simultaneously
Osmotic pressure function, effectively increase rLj-RGD3 stability.
Claims (4)
1. the preparation method of a kind of high yield and high stability recombinant lamprey Lj-RGD3 albumen, it is characterised in that successively
Carry out in accordance with the following steps：
A. terminator codon is introduced in the end of Lj-RGD3 gene orders 3 ', with carrier with Nde I and Hind III digestions site
It is connected, is transformed into the Screening and Identification of progress positive transformant after expression bacterium, obtains positive recombinant；
B. positive recombinant is expanded and cultivates and carry out IPTG induced expressions：
B.1 monoclonal is taken, is inoculated into LB culture mediums, 30 DEG C are cultivated 16 ~ 18 hours, reach 2 ~ 3 to OD600；
B.2 by volume 1:In high-concentration culturing base, 30 DEG C are cultivated 2 ~ 3 hours 10 two-stage inoculations, reach 1 ~ 2 to OD600；
B.3 it is inoculated into 30 DEG C of intermediate transit fermentation tank to continue to cultivate, when zymotic fluid OD600 reaches 2 ~ 3 or so, into culture medium
Add the trace element of fermentation volume 1/20, the glucose of fermentation volume 2/5, the Mg of fermentation volume 1/1002SO4·2H2O, send out
The CaCl of ferment volume 0.2/1002·2H2O and final concentration of 1 mM IPTG, 30 DEG C of cultures to OD are at least 5；
The component of the LB culture mediums is as follows：Tryptone 10g/L, dusty yeast 5g/L, NaCl 5g/L；
The component of the high-concentration culturing base is as follows：Tryptone 12g/L, the g/L of dusty yeast 24, the ml/L of 100% glycerine 4, lemon
Lemon acid 7.5 g/L, NaCl 0.5g/L, KH2PO42.31 g/L, K2HPO4·3H2The g/L of O 12.5,（NH4）2SO47g/L,
H3PO41.5 ml/L, the ml/L of defoamer 0.6；
C. thalline is collected, supernatant is filtered to obtain through homogeneous crusher machine, hollow fiber filter membrane；
D. gained supernatant obtains rLj-RGD3 stostes through nickel ion affinity chromatograph, anion-exchange chromatography and sieve chromatography.
2. high yield and the preparation method of high stability recombinant lamprey Lj-RGD3 albumen according to claim 1,
It is characterized in that the step c is as follows：
C.1 thalline is collected, bacterial cell disruption liquid is obtained through homogeneous crusher machine, bacterial cell disruption liquid is diluted 2 times, carried out using 1 μm of filter core
Filtering；
C.2 500 ~ 750 further ultrafiltration of K hollow fiber filter membranes are used, obtain clear filtrate；
C.3 3K hollow fiber filter membrane concentrating clarifying filtrates are used.
3. the preparation of high yield according to claim 1 or 2 and high stability recombinant lamprey Lj-RGD3 albumen
Method, it is characterised in that：The Binding buffer compositions of the Step d nickel ion affinity chromatograph are 20mM Tris, 0.8M
NaCl and 5mM pH 7.9 imidazoles, Elution buffer compositions are 20mM Tris, 0.8M NaCl, 1M pH 7.9 miaow
Azoles；The filler of the anion-exchange chromatography is QFF, and buffer solution is Buffer A：20 mM PBS, pH7.4；Buffer B：
100 mM PBS, 1M NaCl, pH7.4；The sieve chromatography filler is Superdex 75, and buffer solution is 10 ~ 30 mM
PBS, pH7.4.
4. high yield according to claim 3 and high stability recombinant lamprey Lj-RGD3 albumen
Preparation method, it is characterised in that：It is 1% to add mass percent into 250 μ g rLj-RGD3 stostes after the Step d
~ 5% mannitol or 50 ~ 250 mM Gly, final concentration of 50 ~ 250 mM Arg and 10 ~ 30 mM pH7.4 PBS
Buffer, it is freeze-dried to form.
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| CN114045299A (en) * | 2021-10-09 | 2022-02-15 | 安徽师范大学 | Protein extraction method for in vitro experiment |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694330A (en) * | 2013-11-25 | 2014-04-02 | 辽宁师范大学 | Preparation method of affinity chromatography purification tag-free genetic recombinant lamprey Lj-RGD3 protein |
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| CN103694330A (en) * | 2013-11-25 | 2014-04-02 | 辽宁师范大学 | Preparation method of affinity chromatography purification tag-free genetic recombinant lamprey Lj-RGD3 protein |
Non-Patent Citations (2)
| Title |
|---|
| JIANG Q, ET AL.: "rLj-RGD3 induces apoptosis via the mitochondrial-dependent pathway and inhibits adhesion, migration and invasion of human HeyA8 cells via FAK pathway", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| 罗红霞等: "重组大肠杆菌 BL21(DE3)/ rbLF- N高密度培养条件优化", 《中国乳品工业》 * |
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| CN114045299A (en) * | 2021-10-09 | 2022-02-15 | 安徽师范大学 | Protein extraction method for in vitro experiment |
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