CN109022389A - A kind of production method of Bacillus coli expression people Iduronate-2-sulfatase - Google Patents
A kind of production method of Bacillus coli expression people Iduronate-2-sulfatase Download PDFInfo
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Abstract
The invention discloses a kind of production methods of Bacillus coli expression people Iduronate-2-sulfatase, this method passes through the 5L fermentor High Density Cultivation and inducing expression of Escherichia coli recombinant strain, to the progress of inducing expression post-fermentation liquid, thalline were collected by centrifugation, high-pressure homogenization is crushed recombinant bacterium and obtains recombinant protein inclusion body, Ni column affinitive layer purification is hung after recombinant protein solubilization of inclusion bodies, obtains recombined human Iduronate-2-sulfatase through desalination and freeze-drying.The method of the present invention simplifies the cumbersome washing step of inclusion body, insoluble inclusion body protein is become into soluble protein by groping suitable ultrasound condition, avoid the denaturation renaturation manipulation using the strong denaturants such as 8M urea or 6M guanidine hydrochloride to inclusion body, the purity of protein as obtained by Ni column affinity chromatography is 95% or more, recovery efficiency has achieved the effect that simple and effective fast purifying 70% or more.
Description
Technical field
The present invention relates to a kind of production methods of Bacillus coli expression people Iduronate-2-sulfatase.
Background technique
Mucopolysaccharidosis II type is also known as Hunter syndrome, is x gene linkage stealth hereditary disease, which is cell
The gene mutation of Iduronate-2-sulfatase in lysosome, intracorporal glycosaminoglycan, which can not be metabolized, leads to dermatan
It causes a disease with Heparan sulfate bulk deposition in the various internal organs of whole body and tissue lysosome.With the product of glycosaminoglycan
Tired, being gradually accumulate in intracorporal mucopolysaccharide will lead to the diseases such as cell hyperemia, organ enlargement, disorganization and system dysfunction
Disease.The iduronic sulfatase injection of FDA on July 24 approval ShireHumanGenetic Therpapies company in 2006
Listing, trade name Elaprase, for the first drug for treating rare hereditary disease MPSII.Foreign countries have thin by mammal CHO
Cellular expression produces people's iduronate-2-sulfatase, document " Low-scale expression and purification of
an Active putative induronate 2-sulfate sulfatase-Like enzyme from
It expressed in Escherichia coli K12 " similar to people's Iduronate-2-sulfatase, but its gene order is not complete
Long sequence, and gene order passes through artificial reconstructed modification, and the country is found not yet at present utilizes Bacillus coli expression people Ai Du
The pertinent literature of uronic acid -2- sulfatase full-length gene.And people's N-acetylgalactosamine-6-sulfatase (GALNS) exists
E.coli (BL21) can be expressed in a manner of active (Poutou, 2006；et al.,2010)。
Summary of the invention
The object of the present invention is to provide a kind of production methods of Bacillus coli expression people Iduronate-2-sulfatase.
For above-mentioned purpose, the technical solution adopted in the present invention is made of following step:
1, picking E. coli clones are inoculated in the culture of the liquid LB containing 100 μ g/mL ampicillins in an aseptic environment
In base, 32~37 DEG C of 12~16h of constant-temperature table culture.
2, by the first order seed flame inoculation after step 1 culture in fermentation medium, inoculum concentration is 5%~10%, 32
~37 DEG C of 8~12h of constant temperature incubation, are cooled to 30~32 DEG C, then isopropylthiogalactoside is added in 1~2h of constant temperature incubation
Into fermentation liquid, its concentration is 0.6~1.0mmol/mL, and constant speed supplements supplemented medium into fermentation liquid, per hour magnitude of recruitment
It is the 5%~9% of fermentating liquid volume, carries out Escherichia coli inducing expression people's Iduronate-2-sulfatase, induces 6~10h
Stop fermentation afterwards, thallus is collected in room temperature centrifuge separation.
3, by thallus that step 2 is collected with the PBS buffer solution for being cooled to 4~8 DEG C in advance be resuspended to cell density be 100~
150g/L, 50~60min of high-pressure homogenization under 60~80MPa pressure, it is cooling with ice-water bath by the buffer of high pressure homogenizer
Ensure to export efflux temperature and be no more than 37 DEG C, room temperature is centrifugated after homogenate, collects precipitating, it is thick to obtain recombinant protein inclusion body
Product.
4, recombinant protein inclusion body crude product is resuspended with cleaning solution to its concentration is 6~10g/L, in 60~80MPa pressure
30~40min of lower high-pressure homogenization ensures that outlet efflux temperature does not surpass by the way that the cleaning solution ice-water bath of high pressure homogenizer is cooling
37 DEG C are crossed, room temperature is centrifugated after homogenate, is collected precipitating, is obtained recombinant protein inclusion body.
5, the recombinant protein inclusion body that step 4 obtains is resuspended with lysate to its concentration is 6~10g/L, uses ultrasonic wave
For broken instrument after 400~600W power, open cycle 5s, closed loop 5s, ice-water bath ultrasound 1h, supernatant is collected in room temperature centrifuge separation
Liquid.
6, the supernatant that step 5 is collected is diluted to the final concentration of 0.8~1.5mg/ of recombinant protein inclusion body with balance A liquid
ML, with 0.1M NaCl tune conductance to 2.0~2.5ms, dilute HCl tune pH 9.0~9.3；With ultrapure water with the punching of 10mL/min flow velocity
It is smooth to baseline to wash Ni Sepharose FF column, Ni Sepharose FF is then rinsed with 10mL/min flow velocity with balance A liquid
10 column volumes, then loading is overloaded with 4~8mL/min flow velocity, it is first rinsed after completion of the sample with balance A liquid with 10mL/min flow velocity
10 column volumes of Ni Sepharose FF, then the mixed liquor for being 3:7 with elution B liquid and balance A liquid volume ratio is with 10mL/min
Flow velocity elutes 10 column volumes of miscellaneous band, finally obtains target protein solution with elution B liquid with the elution of 10mL/min flow velocity.
7, the hyperfiltration membrane assembly that target protein solution is 10kD with molecular cut off is subjected to ultrafiltration desalination and be concentrated, vacuum
Freeze-drying, obtains recombined human Iduronate-2-sulfatase.
In above-mentioned steps 2, the composition of the fermentation medium are as follows: yeast powder 5g/L, peptone 5g/L, glycerol 5mL/L,
Na2HPO4·12H2O 25g/L、K2HPO4 4g/L、NH4Cl 1g/L、NH4SO42g/L, glycine 0.5g/L, MgCl2
0.25g/L, defoaming agent 0.5mL/L, nutrient 1mL/L；The composition of the supplemented medium are as follows: yeast powder 5g/L, peptone 5g/
L, glycerol 5mL/L, glycine 0.5g/L, MgCl20.25g/L, nutrient 1mL/L；The wherein composition of the nutrient are as follows:
CuSO4·5H2O 6g/L、KI 0.088g/L、MnSO4·H2O 3g/L、Na2MoO4·2H2O 0.2g/L、H3BO3 0.02g/L、
CoCl6·H2O 0.5g/L、ZnCl2 20g/L、FeSO4·7H2O 65g/L, Biotin 0.2g/L, dense H2SO4 5mL/L。
In above-mentioned steps 4, the composition of the cleaning solution are as follows: 15% sucrose, 0.25% NaTDC, 3% n-butanol,
0.8%Triton 100,20mM Tris-HCl, pH 9.3.
In above-mentioned steps 5, the composition of the lysate are as follows: 0.5% beta -mercaptoethanol, 10mM Na2PO4·12H2O、20mM
Tris-HCl, pH 9.3.
In above-mentioned steps 6, the balance A liquid is: 9.3,0.22 μm of 30mM imidazoles, 20mM Tris-HCl, pH filter membrane
Filtering；Elution B liquid is: 9.3,0.22 μm of 120mM imidazoles, 20mM Tris-HCl, pH membrane filtration.
The present invention expresses people's Iduronate-2-sulfatase using Escherichia coli system high density low temperature induction, passes through
High-pressure homogenization is crushed Bacillus coli cells and discharges people's Iduronate-2-sulfatase inclusion body protein, lysate dissolution packet
Contain body protein, simplify cumbersome inclusion body washing step, dissolved recombinant protein can directly upper Ni Sepharose FF
Affinity column avoids and carries out denaturation renaturation manipulation, gained to inclusion body using the strong denaturants such as 8M urea or 6M guanidine hydrochloride
Purity of protein can reach 95% or more, and 70% or more recovery efficiency is easy to operate, high-efficient.
The present invention has filled up domestic weight using Escherichia coli system expression people's Iduronate-2-sulfatase full length sequence
The blank of group expression people's Iduronate-2-sulfatase, since Escherichia coli are the tables of a kind of cheap and simple and mature safety
Up to system, metabolic pathway and genetic background understand, have cultivation cycle short, and fermentation density is high, and target protein expression amount is high, hair
The advantages that ferment is at low cost to be easy to industrialize amplification, and downstream purification technical maturity is stablized and the ideal place for becoming expression recombinant protein
It is main.
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis in embodiment 1 before and after recombinant protein N i Sepharose FF column affinitive layer purification
Figure, 1 is the broken 20 μ L of inclusion body crude product loading of recombinant protein high-pressure homogenization, and 2 is broken for recombinant protein high-pressure homogenization
30 μ L, 3 and 4 of inclusion body crude product loading is that the 20 μ L, 5 and 6 of recombinant protein crude product loading after cleaning solution washing is Ni
Recombinant protein sterling loading 20 μ L, M after Sepharose FF column affinitive layer purification are albumen Marker26610.
Fig. 2 is the chromatogram of the recombinant protein sterling after Ni Sepharose FF column affinitive layer purification.
Specific embodiment
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to
These embodiments.
Embodiment 1
1, picking E. coli clones are inoculated in liquid LB training of the 300mL containing 100 μ g/mL ampicillins under gnotobasis
Support in base, the composition of LB liquid medium are as follows: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, with dilute HCl or
Person NaOH adjusts 7.3,37 DEG C of constant-temperature table 250rpm of pH and cultivates 12h, obtains first order seed.
2, after 5L fermentor moist heat sterilization being cooled to 37 DEG C, flame inoculation first order seed 300mL is in 2L fermentation medium
In, the composition of the fermentation medium are as follows: yeast powder 5g/L, peptone 5g/L, glycerol 5mL/L, Na2HPO4·12H2O 25g/
L、K2HPO4 4g/L、NH4Cl 1g/L、NH4SO42g/L, glycine 0.5g/L, MgCl20.25g/L, defoaming agent 0.5mL/L,
Nutrient 1mL/L, the wherein composition of nutrient are as follows: CuSO4·5H2O 6g/L、KI 0.088g/L、MnSO4·H2O 3g/L、
Na2MoO4·2H2O 0.2g/L、H3BO3 0.02g/L、CoCl6·H2O 0.5g/L、ZnCl2 20g/L、FeSO4·7H2O
65g/L, Biotin 0.2g/L, dense H2SO4After 5mL/L, 300rpm37 DEG C of culture 10h, 32 DEG C are reduced the temperature to, 250rpm training
1h is supported, then addition inducer isopropylthio thiogalactoside to 0.8mmol/mL, starts the inducing expression of recombinant protein, and
Into fermentation liquid, constant speed supplements supplemented medium, and magnitude of recruitment is 165mL per hour, carries out Escherichia coli inducing expression people's idose
Aldehydic acid -2- sulfatase, the composition of the supplemented medium are as follows: yeast powder 5g/L, peptone 5g/L, glycerol 5mL/L, glycine
0.5g/L、MgCl20.25g/L, nutrient 1mL/L (its composition is identical with step 2)；Induce 8h after stop fermentation, tubular type from
Scheming 7000rpm room temperature is centrifuged 20min, collects thallus.
3, the thallus that step 2 is collected is resuspended with the PBS buffer solution for being cooled to 6 DEG C in advance to cell density is 100g/L, in
High-pressure homogenization 60min under 80MPa pressure ensures to export efflux temperature by the way that the buffer ice-water bath of high pressure homogenizer is cooling
No more than 37 DEG C, it is centrifuged 30min in tube centrifuge 10000rpm room temperature after homogenate, collects precipitating, precipitating is recombinant protein
Inclusion body crude product.
4, recombinant protein inclusion body crude product is resuspended with cleaning solution to its concentration is 10g/L, the composition of the cleaning solution are as follows:
15% sucrose, 0.25% NaTDC, 3% n-butanol, 0.8%Triton100,20mM Tris-HCl, pH 9.3,
High-pressure homogenization 30min under 80MPa pressure, being cooled with an ice bath by the cleaning solution of high pressure homogenizer ensures to export efflux temperature not
More than 37 DEG C.It is centrifuged 30min in tube centrifuge 10000rpm room temperature after homogenate, precipitating is collected, obtains recombinant protein inclusion body.
5, the obtained recombinant protein inclusion body of step 4 is resuspended with lysate to its concentration is 6g/L, the lysate
Composition are as follows: 0.5% beta -mercaptoethanol, 10mM Na2PO4·12H2O, 20mM Tris-HCl, pH 9.3.Use ultrasonic disruption
Instrument is in 400W power, open cycle 5s, and after closed loop 5s, ice-water bath ultrasound 1h, room temperature 10000rpm is centrifuged 30min, collects supernatant
Liquid.
6, the supernatant that step 5 is collected is diluted to the final concentration of 1.0mg/mL of recombinant protein inclusion body with balance A liquid, used
0.1M NaCl tune conductance is to 2.5ms, dilute HCl tune pH 9.3；Ni Sepharose is rinsed with 10mL/min flow velocity with ultrapure water
FF column is smooth to baseline, then with balance A liquid with 10mL/min flow velocity flushing 10 column volumes of Ni Sepharose FF, then with
5mL/min flow velocity overloads loading, first rinses Ni Sepharose FF 10 with balance A liquid after completion of the sample with 10mL/min flow velocity
A column volume, then with elution B liquid and the mixed liquor that A liquid volume ratio is 3:7 is balanced with 10mL/min flow velocity elution 10 columns of miscellaneous band
Volume finally obtains target protein solution with elution B liquid with the elution of 10mL/min flow velocity.Wherein balance A liquid is: 30mM imidazoles
9.3,0.22 μm of membrane filtration of 20mM Tris-HCl pH；Elution B liquid is: 120mM imidazoles 20mM Tris-HCl pH 9.3,
0.22 μm of membrane filtration.Ni Sepharose FF column first uses 20 column volumes of ultrapure water after use, then uses volume fraction
5 column volumes are rinsed for 20% ethanol water, save pillar.
7, the hyperfiltration membrane assembly that target protein solution is 10kD with molecular cut off is subjected to ultrafiltration desalination and be concentrated, vacuum
Freeze-drying, obtains recombined human Iduronate-2-sulfatase.
Fig. 1 is SDS-PAGE electrophoresis, and wherein swimming lane 1 and 2 is the broken recombinant protein inclusion body of recombinant bacterium；Swimming lane 3
With 4 be washing after recombinant protein inclusion body, swimming lane 5 and 6 be affinitive layer purification after recombinant protein sterling, swimming lane 7 be egg
White Marker (26610).As seen from the figure, inclusion body purity after washing is 80% or so, the rate of recovery 90%, by Ni
Recombinant protein purity after Sepharose F column affinitive layer purification is 95% or more, and the rate of recovery is 70% or more.
Fig. 2 is the recombinant protein chromatographic peak profile figure after Ni Sepharose F column affinitive layer purification, X-axis 450 in figure
Target peak is the eluting peak of recombinant protein.As seen from the figure, peak shape is single, and target peak absorption value is high, and forward and postpeak distance are remote,
Illustrate good separation.
Claims (6)
1. a kind of production method of Bacillus coli expression people Iduronate-2-sulfatase, it is characterised in that this method is under
State step composition:
(1) picking E. coli clones are inoculated in the LB liquid medium containing 100 μ g/mL ampicillins in an aseptic environment
In, 32~37 DEG C of 12~16h of constant-temperature table culture；
(2) by step (1) culture after first order seed flame inoculation in fermentation medium, inoculum concentration be 5%~10%, 32~
37 DEG C of 8~12h of constant temperature incubation are cooled to 30~32 DEG C, 1~2h of constant temperature incubation, and isopropylthiogalactoside then is added extremely
Its concentration is 0.6~1.0mmol/mL in fermentation liquid, and constant speed supplements supplemented medium into fermentation liquid, and magnitude of recruitment is per hour
The 5%~9% of fermentating liquid volume carries out Escherichia coli inducing expression people's Iduronate-2-sulfatase, after inducing 6~10h
Stop fermentation, thallus is collected in room temperature centrifuge separation；
(3) thallus that step (2) are collected is resuspended with the PBS buffer solution for being cooled to 4~8 DEG C in advance to cell density is 100~150g/
L, 50~60min of high-pressure homogenization under 60~80MPa pressure ensure by the buffer ice-water bath cooling of high pressure homogenizer
Mouth efflux temperature is no more than 37 DEG C, and room temperature is centrifugated after homogenate, collects precipitating, obtains recombinant protein inclusion body crude product；
(4) recombinant protein inclusion body crude product is resuspended with cleaning solution to its concentration is 6~10g/L, high under 60~80MPa pressure
Pressure 30~40min of homogenate ensures to export efflux temperature no more than 37 by the way that the cleaning solution ice-water bath of high pressure homogenizer is cooling
DEG C, room temperature is centrifugated after homogenate, is collected precipitating, is obtained recombinant protein inclusion body；
(5) the recombinant protein inclusion body that step (4) obtains is resuspended with lysate to its concentration is 6~10g/L, broken with ultrasonic wave
For broken instrument after 400~600W power, open cycle 5s, closed loop 5s, ice-water bath ultrasound 1h, supernatant is collected in room temperature centrifuge separation；
(6) supernatant that step (5) are collected is diluted to the final concentration of 0.8~1.5mg/ of recombinant protein inclusion body with balance A liquid
ML, with 0.1M NaCl tune conductance to 2.0~2.5ms, dilute HCl tune pH 9.0~9.3；With ultrapure water with the punching of 10mL/min flow velocity
It is smooth to baseline to wash Ni Sepharose FF column, Ni Sepharose FF is then rinsed with 10mL/min flow velocity with balance A liquid
10 column volumes, then loading is overloaded with 4~8mL/min flow velocity, it is first rinsed after completion of the sample with balance A liquid with 10mL/min flow velocity
10 column volumes of Ni Sepharose FF, then the mixed liquor for being 3:7 with elution B liquid and balance A liquid volume ratio is with 10mL/min
Flow velocity elutes 10 column volumes of miscellaneous band, finally obtains target protein solution with elution B liquid with the elution of 10mL/min flow velocity；
Above-mentioned balance A liquid is: 9.3,0.22 μm of 30mM imidazoles, 20mM Tris-HCl, pH membrane filtration；Elute B liquid:
9.3,0.22 μm of 120mM imidazoles, 20mM Tris-HCl, pH membrane filtration；
(7) hyperfiltration membrane assembly that target protein solution is 10kD with molecular cut off is subjected to ultrafiltration desalination and be concentrated, vacuum is cold
It is lyophilized dry, obtains recombined human Iduronate-2-sulfatase.
2. the production method of Bacillus coli expression people Iduronate-2-sulfatase according to claim 1, special
Sign is: in step (2), the composition of the fermentation medium are as follows: yeast powder 5g/L, peptone 5g/L, glycerol 5mL/L,
Na2HPO4·12H2O 25g/L、K2HPO4 4g/L、NH4Cl 1g/L、NH4SO42g/L, glycine 0.5g/L, MgCl2
0.25g/L, defoaming agent 0.5mL/L, nutrient 1mL/L.
3. the production method of Bacillus coli expression people Iduronate-2-sulfatase according to claim 1, special
Sign is: in step (2), the composition of the supplemented medium are as follows: yeast powder 5g/L, peptone 5g/L, glycerol 5mL/L, sweet ammonia
Sour 0.5g/L, MgCl20.25g/L, nutrient 1mL/L.
4. the production method of Bacillus coli expression people Iduronate-2-sulfatase according to claims 2 or 3,
It is characterized by: the composition of the nutrient are as follows: CuSO4·5H2O 6g/L、KI 0.088g/L、MnSO4·H2O 3g/L、
Na2MoO4·2H2O 0.2g/L、H3BO3 0.02g/L、CoCl6·H2O 0.5g/L、ZnCl2 20g/L、FeSO4·7H2O
65g/L, Biotin 0.2g/L, dense H2SO4 5mL/L。
5. the production method of Bacillus coli expression people Iduronate-2-sulfatase according to claim 1, special
Sign is: in step (4), the composition of the cleaning solution are as follows: 15% sucrose, 0.25% NaTDC, 3% n-butanol, 0.8%
Triton 100,20mM Tris-HCl, pH 9.3.
6. the production method of Bacillus coli expression people Iduronate-2-sulfatase according to claim 1, special
Sign is: in step (5), the composition of the lysate are as follows: 0.5% beta -mercaptoethanol, 10mM Na2PO4·12H2O、20mM
Tris-HCl, pH 9.3.
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