CN100500841C - Expression of soluble TRAIL protein - Google Patents
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- CN100500841C CN100500841C CNB2005100303560A CN200510030356A CN100500841C CN 100500841 C CN100500841 C CN 100500841C CN B2005100303560 A CNB2005100303560 A CN B2005100303560A CN 200510030356 A CN200510030356 A CN 200510030356A CN 100500841 C CN100500841 C CN 100500841C
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Abstract
The present invention discloses a method for the expression of soluble TRAIL protein, which comprises the following steps: an extracellular region gene of the TRAIL is cloned to an expression vector cThe invention opened a method to express the soluble TRAIL protein which includes cloning the extracellular gene of the TRAIL into the expression carrier with the PL promotor of the lambda bacteriophaontaining a PL promoter of a lambda bacteriophage, a cI<+> host bacterium is converted, and a low temperature induction method is adopted to express the recombination TRAIL protein. On the basis of loge; Transforming the cI+ host bacteria; expressing the recombinative TRAIL protein by the low-temperature induction. Also it can add the Zn2+ or the synthetic inhibiter of the prokaryotic protein intow temperature induction, a bivalent metal ion, namely zinc or a synthesis inhibitor of prokaryotic cell protein can be further added to a culture medium in the stage of inducing expression. Most of th the medium in the inducing stage. The recombinative TRAIL protein is soluble, so it can simplify the isolation process. e recombination TRAIL protein expressed by the method of the present invention is soluble, which greatly simplifies the working procedures of separation and purification.
Description
Technical field
The present invention relates to the expression method of biological activity protein, relate in particular to the proteic expression method of tumor necrosin relative death inducing ligand (TRAIL).
Technical background
Tumor necrosin relative death inducing ligand (tumor necrosis factor-related apoptosis-inducingligand, be called for short TRAIL) be one of TNF family member who finds recently, claim again plain two parts of apoptosis (apoptotin 2ligand, Apo-2L).1996, Pitti at first separated and has identified TRAIL and inductive apoptosis thereof, and finder's trail dna total length 1769bp is positioned karyomit(e) 3q26.Trail protein is a kind of II type transmembrane protein, is made up of 281 amino acid, and molecular weight is 32.5kD, iso-electric point 7.63, and the 114-281 amino acids of its C-terminal constitutes extracellular region, and molecular weight is about 24kD, is the position of mainly bringing into play function.The biologic activity form of TRAIL is a homotrimer, exist on the monomer whose interface zine ion binding site (Sarah G etc., Biochem, 2000,39:633-640).
The bound energy of TRAIL extracellular region and its specific receptors is induced the apoptosis of kinds of tumor cells system rapidly and normal tissue cell is not had apoptosis-induced effect, causes people's extensive concern, is a kind of biological regulator that is used for oncotherapy with very big potentiality.People such as Walczak and Ashkenazi has obtained good therapeutic action at the preclinical study that mouse and monkey are done on one's body.No matter be that TRAIL treats separately or share with chemotherapeutics, all obtained the result consistent with cell strain, promptly cause the degeneration of tumour, it is slack-off to grow, even the tumour extinction tests occurs, does not have severe side effect.Along with going deep into of research, people find that also TRAIL plays the part of important role in processes such as antiviral immunity.
The development of recombinant DNA technology makes the scale operation of many rare somebodies source pharmaceutical protein become possibility.Many natural forms are nonglycosylated in these albumen, and the less general first-selected escherichia expression system of non-glycosylated protein production of molecular weight.Yet the recombinant protein of Escherichia coli system high expression level is easy to form does not have active inclusion body, changes into the biologically active recombinant protein in order to make inclusion body, must be with solubilization of inclusion bodies (separating folding) back renaturation.Because the lower and process complexity of the common renaturation yield of renaturing inclusion bodies, thereby renaturing inclusion bodies is the difficult problem of puzzlement scientific circles and industry member always, also limited the application of escherichia expression system to a certain extent.Therefore,, reduce the formation of inclusion body and the solubility expression of raising recombinant protein, will promote the development and the technical progress of engineered protein drug manufacture from the angle of recombinant protein industrialized development.
People such as The 2nd Army Medical College Wang Liang China utilize escherichia coli expression recombinant human TRAIL to achieve success, they are construction recombination plasmid pBV-TRAIL, it has the gene of ampicillin resistance gene and coding people's trail protein extracellular region (114-281 amino acids), be subjected to the control of λ PL promotor in the encoding gene upstream, the 42 ℃ of expression that can induce people's trail protein down.For solubility directly has activity, 70%～80% is inclusion body about expressed TRAIL20%, and the renaturing inclusion bodies rate is not high.
Therefore, this area presses for the expression technology of improving soluble TRAIL protein, solves trail protein as the technical barrier in antitumor drug research, the exploitation.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing the escherichia coli expression trail protein, to overcome inclusion body content height in the prior art, the problem that soluble TRAIL protein content is low.
The contriver thinks that for temperature-induced type expression system, the efficient of transcribing of thermal induction system is subjected to the derepression Temperature Influence, along with the rising of derepression temperature, induces intensity to strengthen, and the synthetic recombinant protein condenses the formation inclusion body easily.Otherwise, if,, help forming the solubility recombinant protein by reducing the synthesis rate of recombinant protein lower temperature-induced.Equally, add the microbiotic of the inhibition prokaryotic cell prokaryocyte protein synthesis of suitable concentration, comprise paraxin, tsiklomitsin etc., also can reduce the synthesis rate of recombinant protein, help the solubility Recombinant Protein Expression.
On the other hand, burying a divalent-metal ion (zine ion) binding site near the top based on trail protein tripolymer interface, the contriver imagines and adds divalent-metal ion zinc and help the correct folding of trail protein, forms to have activity the recombinant protein that exists with soluble form.
Design of the present invention is such: in the trail protein abduction delivering stage, by reducing means such as abduction delivering temperature, interpolation lower concentration microbiotic, control proteinic synthesis rate, perhaps in substratum, add the zine ion that promotes trail protein stability, reduce the formation of inclusion body, obtain a high proportion of soluble TRAIL protein.
The invention discloses a kind of method of expressing soluble TRAIL protein, TRAIL extracellular region (114-281 amino acids) gene clone in the expression vector that contains lambda particles phage PL promotor, is expressed suitable host bacterium, the abduction delivering temperature is 37-41 ℃.
The present invention is not limited to specific expression vector, as long as this expression vector contains the PL promotor; TRAIL extracellular region gene and expression vector are recombinated according to a conventional method, form recombinant expression vector, the invention is not restricted to specific recombination form, as long as with PL promotor and exercisable connection of TRAIL extracellular region gene; Recombinant expression vector can import suitable host cells according to a conventional method, and the present invention is not limited to any specific host cell, as long as it can express described recombinant expression vector.In a concrete example, expression vector is selected the pBV220 prokaryotic expression carrier for use, and the host bacterium is selected Escherichia coli C600 (supeE44 hsdR thi-1thr-1 leuB6 lacY1 tonA21) for use.
In a preferred scheme, disclose a kind of method of expressing soluble TRAIL protein, the gene clone of TRAIL extracellular region in the expression vector that contains lambda particles phage PL promotor, has been expressed suitable host bacterium, the abduction delivering temperature is 37-41 ℃, preferred 38 ℃; Add simultaneously divalent zinc ion in substratum, divalent zinc ion is selected a kind of in zinc sulfate, zinc chloride, zinc carbonate, the zinc acetate for use, and the concentration of the zine ion of interpolation is 0.1mmol/L to 5.0mmol/L, preferred 1mmol/L.
In another preferred scheme, disclose a kind of method of expressing soluble TRAIL protein, the gene clone of TRAIL extracellular region in the expression vector that contains lambda particles phage PL promotor, has been expressed suitable host bacterium, the abduction delivering temperature is 37-41 ℃, preferred 38 ℃; The microbiotic that add to suppress the prokaryotic cell prokaryocyte protein synthesis in substratum, microbiotic are selected from one or more the combination in paraxin, tsiklomitsin, kantlex, the gentamicin; Preferred paraxin is or/and tsiklomitsin, and wherein chloramphenicol concentration is 0.1-10 μ g/mL, more preferably 1 μ g/mL, and tsiklomitsin concentration is 1-20 μ g/mL, more preferably 8 μ g/mL.
Beneficial effect of the present invention is embodied in:
In pBV-TRAIL/Escherichia coli C600 expression system, at 42 ℃ of following energy abduction deliverings, its soluble fractions is lower than 20% of total expression amount routinely, and most trail protein exists with the inclusion body form of non-activity.
The present invention expresses 38 ℃ of low temperature inductions, compares with 42 common abduction deliverings, and the soluble TRAIL protein expression amount improves more than 50%, and the inclusion body protein amount is lower than 30% of total expression amount;
38 ℃ of low temperature inductions, add in the zine ion scheme, to compare with 42 common abduction deliverings, the soluble TRAIL protein expression amount improves more than 100%, and the inclusion body protein amount is lower than 20% of total expression amount;
38 ℃ of low temperature inductions, add in paraxin, the tsiklomitsin scheme, to compare with 42 common abduction deliverings, the soluble TRAIL protein expression amount improves more than 70%, and the inclusion body protein amount is lower than 10% of total expression amount.
As seen, expression method of the present invention has improved the expression of soluble TRAIL protein, greatly reduces the ratio of the target protein that exists with the inclusion body form.
Description of drawings
The total trail protein of expressing when Fig. 1, inducing temperature are 38 ℃ and 42 ℃ and the SDS-PAGE of soluble TRAIL protein be collection of illustrative plates relatively.
1,2,38 ℃ of supernatant liquors of 38 ℃ of full bacterium lysates
3,4,42 ℃ of supernatant liquors of 42 ℃ of full bacterium lysates
Fig. 2,38 ℃ of SDS-PAGE comparison collection of illustrative plates of inducing preceding total trail protein that adds to express when 1mmol/L zine ion and 42 is directly induced and soluble TRAIL protein.
1,38 ℃, the full bacterium lysate of 1mmol/L zine ion
2,38 ℃, 1mmol/L zine ion supernatant liquor
3,4,42 ℃ of supernatant liquors of 42 ℃ of full bacterium lysates
Before inducing, Fig. 3,38 adds the SDS-PAGE comparison collection of illustrative plates of 0.75mmol/L zine ion and 42 ℃ of total trail proteins of expressing when directly inducing and soluble TRAIL protein.
1,38 ℃, the full bacterium lysate of 0.75mmol/L zine ion
2,38 ℃, 0.75mmol/L zine ion supernatant liquor
3,4,42 ℃ of supernatant liquors of 42 ℃ of full bacterium lysates
Fig. 4,38 ℃ of SDS-PAGE comparison collection of illustrative plates of inducing preceding total trail protein that adds to express when 8 μ g/mL tsiklomitsins and 42 are directly induced and soluble TRAIL protein.
1,38 ℃, the full bacterium lysate of 8 μ g/mL tsiklomitsins
2,38 ℃, 8 μ g/mL tsiklomitsin supernatant liquors
3,4,42 ℃ of supernatant liquors of 42 ℃ of full bacterium lysates
Before inducing, Fig. 5,38 ℃ add 8 μ g/mL tsiklomitsins, 4 μ g/mL tsiklomitsins, 0.6 μ g/mL paraxin, 1 μ g/mL paraxin and 42 ℃ of total trail proteins of expressing when directly inducing and the comparison diagram of soluble TRAIL protein.
1,38 ℃, the total trail protein of 8 μ g/mL tsiklomitsins, soluble TRAIL protein;
2,38 ℃, the total trail protein of 4 μ g/mL tsiklomitsins, soluble TRAIL protein;
3,38 ℃, the total trail protein of 0.6 μ g/mL paraxin, soluble TRAIL protein;
4,38 ℃, the total trail protein of 1 μ g/mL paraxin, soluble TRAIL protein;
5, express when directly inducing for 42 ℃.
Embodiment
The present invention's said " trail protein " refers to the extracellular region of natural TRAIL, the part that the 114-281 amino acids is formed among the promptly natural TRAIL; The TRAIL extracellular region gene amino acid whose cDNA sequence of TRAIL (114-281 position) that refers to encode.The trail protein that exists with solubility, activity form that the present invention's said " soluble TRAIL protein " refers to produce in the thalline behind abduction delivering, corresponding with it with the inclusion body form exist, inactive trail protein.
The said temperature-induced type expression system of the present invention comprises the expression vector that contains lambda particles phage PL promotor, and the corresponding cI that contains lambda particles phage responsive to temperature type repressor gene (cIts857)
+Intestinal bacteria.When transformed bacteria 28-30 ℃ of whens growth, the aporepressor (being λ responsive to temperature type repressor) that temperature-sensitive gene cIts857 produces combines with operator gene, the transcribing of prevention PL promotor, so thalline produces in a large number.If temperature is raise (being generally 42 ℃), the aporepressor inactivation, RNA polymerase is attached on the λ PL promotor, starts the efficient transcript and expression of foreign gene.
The present invention's said " exercisable connection " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leading peptide) DNA is exactly the exercisable polypeptid DNA that is connected in so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can translate, it is operationally to be connected in encoding sequence so.
The structure of expression plasmid pBV220-TRAIL is seen Chinese patent CN 02110880.3 among the present invention, and expression vector pBV220-TRAIL is CN 02110880.3 disclosed pBV-Apo-2L I
100PBV220-TRAIL transforms CaCl routinely
2Conversion method is transformed among the host bacterium C600, has obtained reorganization bacterium C600-TRAIL.
Penbritin is purchased the company in Genebase, and paraxin, tsiklomitsin are purchased the company in Sigma, and peptone, yeast powder are purchased the company in OXOID, and other reagent are homemade analytical reagent.
Below in conjunction with embodiment, the present invention is described further, but the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
After recombinant escherichia coli strain C600-TRAIL activated in 30 ℃ of following 30mLLB substratum, the inoculum size by 1% was seeded to 30 cultivations in the 30mLLB substratum, and all need add penbritin to concentration before the inoculation is 100 μ g/mL.Be cultured to OD
600Be 0.58, put into 42 ℃ shaking table and induced 4 hours shaking bottle, vegetative period and inductive phase rotating speed be 200rpm.
Wherein LB substratum composition is 10g/L peptone, 5g/L yeast powder, 5g/L sodium-chlor, and pH transfers to 7.0.
Thalline after above-mentioned the inducing is abandoned supernatant after centrifugal, and (pH8.5) resuspended washing to be removing the impurity of carrying secretly in the thalline such as nutrient media components that do not utilized for TrisHCl 20mM/L, EDTA1Mm/L, and is centrifugal and abandon supernatant liquor, and repeated washing once again with the TE damping fluid.It is resuspended with thalline to add 20mL TE damping fluid, after mixing, utilizes ultrasonication thalline (power 300W, working hour 3S, off time 3S, totally 150 times), the reorganization trail protein is fully discharged from thalline, then by centrifugation, the supernatant that obtains.Supernatant liquor and full bacterium lysate carry out the SDS-PAGE electrophoresis, it is quantitative to carry out target product through the scanning electrophoretogram, soluble TRAIL protein accounts for 3.5% of supernatant total protein in the supernatant liquor as a result, be lower than 20% of total trail protein expression amount, see Fig. 1, among the figure, tip indication position is the trail protein band.
After recombinant escherichia coli strain C600-TRAIL activated in 30 ℃ of following 30mL LB substratum, the inoculum size by 1% was seeded to 30 ℃ of cultivations in the 30mL LB substratum, and all need add penbritin to concentration before the inoculation is 100 μ g/mL.Be cultured to OD
600Be 0.60, put into 38 ℃ shaking table and induced 8 hours shaking bottle, vegetative period and inductive phase rotating speed be 200rpm.
Wherein LB substratum composition is 10g/L peptone, 5g/L yeast powder, 5g/L sodium-chlor, and pH transfers to 7.0.
Press embodiment 1 identical method and handle thalline, supernatant liquor and full bacterium lysate are carried out the SDS-PAGE electrophoresis, it is quantitative that the scanning electrophoretogram carries out target product, sees Fig. 1.Compare with 42 ℃ of common abduction deliverings, with 38 ℃ as inducing temperature, the solubility expression amount improves more than 50%, the inclusion body protein amount is lower than 30% of total expression amount.
The solubility expression that the 1mmol/L zine ion improves the reorganization trail protein is induced, added to embodiment 3,38 degree
After activating in 30 ℃ of following 30mL LB substratum with this recombinant escherichia coli strain C600-TRAIL, the inoculum size by 1% is seeded in the 30mL LB substratum, cultivates down for 30 ℃, and all need add penbritin to concentration before the inoculation is 100 μ g/mL.Be cultured to OD
600Be 0.585, under aseptic condition, add divalent zinc ion, put into 38 ℃ shaking table and induce shaking bottle to concentration 1mmol/L, vegetative period and inductive phase rotating speed be 200rpm, induced 8 hours.
Wherein, LB substratum composition is 10g/L peptone, 5g/L yeast powder, 5g/L sodium-chlor, and pH transfers to 7.0.Adopt ZnSO
4The aqueous solution.
The solubility expression that the 0.75mmol/L zine ion improves the reorganization trail protein is induced, added to embodiment 4,38 degree
After activating in 30 ℃ of following 30mL LB substratum with this recombinant escherichia coli strain C600-TRAIL, the inoculum size by 1% is seeded in the 30mL LB substratum, cultivates down for 30 ℃, and all need add penbritin to concentration before the inoculation is 100 μ g/mL.Be cultured to OD
600Be 0.55, under aseptic condition, add divalent zinc ion, put into 38 ℃ shaking table and induce shaking bottle to concentration 0.75mmol/L, vegetative period and inductive phase rotating speed be 200rpm, induced 8 hours.
Wherein, LB substratum composition is 10g/L peptone, 5g/L yeast powder, 5g/L sodium-chlor, and pH transfers to 7.0.Adopt ZnSO
4The aqueous solution.
The solubility expression that arrestin synthetic tsiklomitsin improves the reorganization trail protein is induced, added to embodiment 5,38 degree
After activating in 30 ℃ of following 30mL LB substratum with this recombinant escherichia coli strain C600-TRAIL, the inoculum size by 1% is seeded in two parts of 30mL LB substratum, cultivates down for 30 ℃, and all need add penbritin to concentration before the inoculation is 100 μ g/mL.Two parts of cultures are cultured to OD respectively
600Be respectively 0.530,0.562, adding tsiklomitsin to concentration under aseptic condition is 4 μ g/mL, 8 μ g/mL.Put into 38 ℃ shaking table and induce shaking bottle, vegetative period and inductive phase rotating speed be 200rpm, induced 8 hours.
Wherein, LB substratum composition is 10g/L peptone, 5g/L yeast powder, 5g/L sodium-chlor, and pH transfers to 7.0.Microbiotic adopts aqueous solution form to add.
The solubility expression that arrestin synthetic paraxin improves the reorganization trail protein is induced, added to embodiment 6,38 degree
After activating in 30 ℃ of following 30mL LB substratum with this recombinant escherichia coli strain C600-TRAIL, the inoculum size by 1% is seeded in two parts of 30mL LB substratum, cultivates down for 30 ℃, and all need add penbritin to concentration before the inoculation is 100 μ g/mL.Two parts of cultures are cultured to OD respectively
600Be respectively 0.498,0.515, adding paraxin to concentration under aseptic condition is 0.6 μ g/mL, 1 μ g/mL.Put into 38 shaking table and induce shaking bottle, vegetative period and inductive phase rotating speed be 200rpm, induced 8 hours.
Wherein, LB substratum composition is 10g/L peptone, 5g/L yeast powder, 5g/L sodium-chlor, and pH transfers to 7.0.Microbiotic adopts aqueous solution form to add.
Compare with 42 ℃ of common abduction deliverings, the solubility expression amount improves more than 70%, and the inclusion body protein amount is lower than 10% of total expression amount, when particularly adding 1 μ g/mL paraxin, produces inclusion body albumen hardly.
Claims (2)
1, a kind of method of expressing soluble TRAIL protein comprises that gene clone with the 114-281 amino acids of coding trail protein C-terminal is in the expression vector that contains lambda particles phage PL promotor, at cI
+Express in the host bacterium, it is characterized in that, the abduction delivering temperature is 37-41 ℃;
CI wherein
+Finger is from the responsive to temperature type repressor gene cIts857 of lambda particles phage; CI
+The host bacterium refers to contain the coli strain that the cIts857 gene also can correctly be expressed;
Add divalent zinc ion during abduction delivering in the substratum or add the microbiotic that suppresses the prokaryotic cell prokaryocyte protein synthesis;
The microbiotic that suppresses the prokaryotic cell prokaryocyte protein synthesis is selected from a kind of in paraxin and the tsiklomitsin;
Said divalent zinc ion is selected from a kind of in zinc sulfate, zinc chloride, zinc carbonate, the zinc acetate;
Divalent zinc ion concentration is 0.1-5.0mmol/L in the substratum;
Chloramphenicol concentration is 0.6～1 μ g/mL in the substratum, and tsiklomitsin concentration is 4～8 μ g/mL.
2, the method for claim 1 is characterized in that expression vector is selected pBV220 for use, and the host bacterium is selected Escherichia coli C600 for use.
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| WO2009009919A1 (en) * | 2007-07-13 | 2009-01-22 | National Center Of Biomedical Analysis | An escherichia coli expressing trail protein and its construction method and applications |
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Non-Patent Citations (4)
| Title |
|---|
| Induction of apoptosis by a new member of the tumor necrosisfactor cyctokine family. Pitti RM et al.J Bio l Chem,Vol.271 No.22. 1996 |
| Induction of apoptosis by a new member of the tumor necrosisfactor cyctokine family. Pitti RM et al.J Bio l Chem,Vol.271 No.22. 1996 * |
| 可溶性TRAIL 蛋白的高密度培养及补料策略研究. 章越等.生物工程学报，第20卷第3期. 2004 |
| 可溶性TRAIL 蛋白的高密度培养及补料策略研究. 章越等.生物工程学报，第20卷第3期. 2004 * |
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