CN105801671A - Biosynthesis method for recombinant RonaCare cyclic pentapeptide cosmetics - Google Patents
Biosynthesis method for recombinant RonaCare cyclic pentapeptide cosmetics Download PDFInfo
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
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Abstract
The invention relates to a biosynthesis method for recombinant RonaCare cyclic pentapeptide cosmetics. The method comprises the following steps that 1, RonaCare cyclic pentapeptide analogues are subjected to molecular docking; 2, gene design is performed on linear RonaCare cyclic pentapeptide analogues; 3, recombinant pTwin-RC5-DE3 is fermented and RonaCare cyclic peptides are obtained; 4, the RonaCare cyclic peptides are detected. The advantages of chemical synthesis and chemical enzymatic synthesis are integrated, reaction conditions are easy to obtain, the reaction period is short, and the product purity and yield are high.
Description
Technical field
The invention belongs to the technical field of RonaCare ring pentapeptide Cosmetic Manufacture method, be specifically related to one and utilize base
The method producing RonaCare ring pentapeptide because of engineering vitro recombination.
Background technology
Along with social development, constantly lifting, the high speed development of biotechnology of people's quality of life.People are to cosmetics
Demand not only shows that basic cleaning, maintenance and appearance beautifies the aspects such as modification, but pursues more many effects,
Such as: prevent and wrinkle reduction, anti-and slow down aging, whitening and speckle dispelling, moisturizing, anti-acne and the aspect such as sun-proof.Therefore, both effectiveness
The study hotspot of the research of composition always cosmetics.Unfortunately, good in current market cosmetic industry composition
Being chemosynthesis material, often because there is the problem such as safety and self stability, and making its use be obstructed.Therefore, one is found
Planting environmental protection, high-efficiency and economic, safely and effectively synthesis mode, exploitation has the natural active matter of healthy skin, it has also become
The research tendency in functional cosmetics field.
Research shows, there is preferable affinity, therefore, active polypeptide is applied to cosmetics between polypeptide and Skin Cell
In, its biological activated energy is fully played.Many peptides skin care item, in cosmetic field history the most for many years, have every year
Declare many peptides new product release in a large number.But many peptides raw material on the market is linear polypeptide, and RonaCare ring pentapeptide
Cosmetic (the fine luxurious foster light sensation essence of face) is first pure cyclic peptide skin-protection product of Merck (Merck) whole world, linear many compared to institute
For peptide, ring pentapeptide pastes because of stable structure and higher molecular rigidity and cell surface and property is fine, so opening " lock " transmission carefully
The ability of intracellular external signal is higher, is the set of all polypeptide series products skin-care effects.Through skin deep layer is repaiied more rapidly
Multiple damaged cell, and suppress the activity of lipase, reduce hoarding of superabundant fats, reinvent skin support force, contend with centrifugal force.
But compared with optimistic market prospect, RonaCare ring pentapeptide cosmetic chemistry synthesis price is sufficiently expensive, exist the most again anti-
Should the deficiency such as cycle length, low, the poor stability of efficiency.This will make its facing lots of obstruction in the popularization of the marketization, and then is difficult to favour
And it is popular.
Cyclic peptide synthesizes at present mainly three kinds of methods: chemical method, enzyme process, recombinant DNA technology.Every kind of method has it excellent scarce
Point.
The first is chemical method, and such as Kong Yi et al. is with dichloro resin as solid phase carrier, and HBTU, PyBOP are condensing agent, Gu
It is combined to liquid phase synthesis cyclic peptide after linear peptides, uses preparing high-efficient liquid chromatography of oppisite phase to be purified.Product purity is
98.3%, total recovery is 28.2%.Generally speaking, chemosynthesis the most still uses at most, but synthesis is suitable only for medium
Length polypeptide (10-100 amino acid residue), reaction condition is complicated, reaction time length, product has racemization to produce and overall needs
Want the deficiencies such as Preservation tactics.
The second is enzyme process, and Chinese patent CN103525889 A discloses one and utilizes thioesterase to synthesize Cilengitide
Method.Compared with traditional chemical synthesis, the method catalysis time shortens 48 times, and total yield rises to 79.3%.To the greatest extent
Pipe chemical-enzymatic compares chemosynthesis, there is many advantages, but it often has certain selectivity to substrate, and the method starts to walk also
The most later, the scope therefore applied is the most extensive.
Recombinant DNA technology collection chemosynthesis and the advantage of chemical-enzymatic, based on this, propose the present invention.
Summary of the invention
For the above-mentioned technical problem of prior art, the invention provides a kind of RonaCare ring pentapeptide cosmetics of recombinating
Biological synthesis method, collection chemosynthesis and the advantage of Chemoenzymatic synthesis, easy control of reaction conditions, reaction time short and
Product purity, yield are high.
For reaching above-mentioned purpose, the present invention is achieved by the following technical solutions:
The biological synthesis method of a kind of RonaCare ring pentapeptide cosmetics of recombinating, comprises the following steps:
(1) RonaCare ring five peptide analogues is carried out molecular docking；
(2) linear RonaCare ring five peptide analogues is carried out gene design；
(3) acquisition with RonaCare cyclic peptide of fermenting of restructuring pTwin-RC5-DE3；
(4) detection to RonaCare cyclic peptide.
Further, the molecular docking of described step (1) comprises the steps:
A1, build little molecular database: generating cyclic peptide two-dimensional structure with ChemDraw Ultra 10.0, LigandFit is soft
Part can automatically generate three configurations of cyclic peptide in the calculation, and forms little molecular database；
A2, receptor protein file: according to the crystal structure of acquisition integrin albumen aIIbb3 in protein crystal number storehouse,
Little for part molecule is removed from the three dimensional structure of integrin aIIbb3, and confirms RonaCare ring five peptide analogues bound site
Point, builds and calculates network；
A3, carry out molecular docking by Ligandfit program: part RonaCare ring five peptide analogues and integrin aIIbb3
Docking, the ligand conformational obtained is further carried out ability optimization；
A4, utilize Ligandfit software that a series of ring five peptide analogues and integrin albumen aIIbb3 are carried out molecule pair
Connect, the binding pattern of the final interphase interaction confirming them.
Further, linear RonaCare ring five peptide analogues of described step (2) carries out gene design and includes walking as follows
Rapid:
The acquisition of B1, RC-5 gene order: use Primer primer 5 software design primer, for pTwin1 carrier
The SapI enzyme action insertion point of 6404 and 6437 in multiple clone site, design two complementary primers of synthesis, forward primer
RC5-F1:5 ' AAC CGT GGC GAT TTT GTG 3 ', downstream primer RC5-R1:5 ' GCA CAC AAA ATC GCC ACG
3’；By the method for primer degeneration renaturation, RC5-F1 and RC5-R1 annealing form two ends linear with the RC-5 of cohesive end
The gene of precursor；
The structure of B2, RC-5 recombinant expression carrier pTwin-RC5: clone is carried out bacterium colony PCR checking, to positive colony
Son checks order, positive colony correct to order-checking and pTwin bacterium extracting plasmid, carries out enzyme action with SapI enzyme action respectively, and 37
DEG C 2h, carries out agarose gel electrophoresis detection to digestion products, enzyme action fragment completely carries out glue and reclaims and quantitatively；By double enzymes
Cut back to close product linked system to connect overnight in 16 DEG C of metal baths, take connection product chemical transformation, product is converted extremely
In Rosseta, build recombinant pTwin-RC5-DE3.
Further, the fermentation of described step (3) restructuring pTwin-RC5-DE3 and the acquisition of RonaCare cyclic peptide include as
Lower step:
C1, pTwin-RC5-DE3 recombinant strains is inoculated on the LB solid plate of Amp activation, picking list bacterium colony
Be inoculated in LB+Amp fluid medium, shaken cultivation overnight, after be inoculated in fermentation liquid with the inoculum concentration of 4% and to be enlarged training
Support, until 0.5≤OD600≤1.0, add IPTG to final concentration of 0.5mM and carry out the abduction delivering of destination protein, with empty carrier
PTwin and do not induce as negative control, inductive condition is 16 DEG C, 120rpm, 6～10h, to maturing fermentation liquid with 4500rpm,
4 DEG C, 15min is centrifugal collects thalline, abandons supernatant；
C2, the addition abundant resuspended thalline of cell pyrolysis liquid Buffer B1, the homogeneous instrument of Precellys 24 crushes thalline bar
Part: 5000rpm/4 × 40s/30s, i.e. frequency are 5000rpm, every task 40s, work 3 times, every minor tick 30s, overall process
Operate in ice bath；4 DEG C afterwards, 12 000rpm, 10min are centrifugal, collect supernatant, simultaneously use resuspended to precipitation cell pyrolysis liquid
Detect in inclusion body；
C3, sample liquid upper prop: under the conditions of 4 DEG C, add 2mL chitin resin column material Chitin beads in Chitin post,
The Buffer B1 adding 10 times of column volumes carries out pretreatment, and gained supernatant in step C2 is added Chitin post, coutroi velocity
At 0.5-1.0mL/min；
C4, wash post: wash post with the Buffer B1 buffer of at least 20 times of bed volumes, due to CBD and chitin resin
Adhesion is very strong, and the flow velocity washing post can properly increase, and flow velocity can be at 1.5-2.0mL/min；
C5, the eluting of destination protein: make peptide bond fission with the buffer B uffer B2 of 4 times of bed volumes, cover cap,
Room temperature stands overnight, and the N end of release polypeptide is cut in the C end-grain cutting of induction intein 1；
C6, the cyclisation of destination protein: wash post with the flow velocity of 2mL/min with the Buffer B2 of 10 times of column volumes, with 3 times of posts
The quick post of crossing of volume Buffer B3, filler in making MESNA be uniformly distributed and soaking post, 4 DEG C of induction intein 2 cut and polypeptide
Cyclisation 16h；
C7, the eluting of desired polypeptides: with Buffer B3 eluting purpose cyclic peptide, 3-4 times of column volume eluent before collecting altogether；
C8, renaturation are tested: the thalline ultrasonication to generation inclusion body, and broken liquid abandons supernatant, add 5mL in precipitation
Buffer B1, collected after centrifugation precipitates, and adds carbamide, is stirred at room temperature and is completely dissolved to precipitation, and centrifugal collection supernatant, on gained
Clear liquid loads in the bag filter after activation, adds dialysis, and every 4-5h changes a buffer.
Further, the detection of described step (4) RonaCare cyclic peptide comprises the steps: to collect the albumen sample in each stage
Product, detect through row SDS-PAGE cellular lysate liquid, gradient eluent, purified concentration albumen, and try with BioRad protein quantification
Agent box is carried out quantitatively；Carry out lyophilization subsequently, add proper amount of methanol and again dissolve, after being centrifuged, product is carried out HPLC/
MS detects analysis.
Beneficial effects of the present invention is as follows:
The present invention recombinates the biological synthesis method of RonaCare ring pentapeptide cosmetics, collection chemosynthesis and Chemoenzymatic synthesis
Both advantages, easy control of reaction conditions, reaction time be short and product purity, yield are high.
Accompanying drawing explanation
Fig. 1 is restructuring pTwin-RC5 plasmid map；
Fig. 2 is the abduction delivering situation schematic diagram of restructuring pTwin-RC5-DE3；
Wherein: M, marker；1, cellular lysate liquid；2, wash post and flow through liquid；3, N end release destination proteins；4, RC5 cyclisation eggs
In vain.Arrow is corresponding albumen.
Fig. 3 is the HPLC collection of illustrative plates of restructuring pTwin-RC5-DE3 cyclisation 8h sample and purified product；Wherein；A is CBD-RC5-
CBD；B is RC5-CBD；C is cyclisation RC5；
Fig. 4 is that 4 DEG C of induction intein 2 cut and after polypeptide cyclisation 16h, RC5 restructuring pTwin-RC5-DE3 is cyclized 8h sample
HPLC collection of illustrative plates with purified product；
Fig. 5 is laser flying first mass spectrometric detection figure (cation mode) of RC5 sample after purification；Wherein, 575.9 is [M
+H]+。
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited to
This.
As Figure 1-5, the present invention recombinates the biological synthesis method of RonaCare ring pentapeptide cosmetics, including following step
Rapid:
(1), RonaCare ring pentapeptide analog molecules docking
Molecular docking experiment is to complete by commercial software LigandFit program (Accelery company, 3.5 versions).Base
This molecular docking flow process is:
A1, build little molecular database: generating cyclic peptide two-dimensional structure with ChemDraw Ultra 10.0, LigandFit is soft
Part can automatically generate three configurations of cyclic peptide in the calculation, and forms little molecular database.
A2, receptor protein file: according to protein crystal number storehouse (RCSB PDB, http://www.rcsb.org/
Pdb/home/home.do) in, (the numbered 3ZDZ of PDB, resolution is the crystal structure of acquisition integrin protein I Ib 3).Little for part molecule is removed from the three dimensional structure of integrin IIb 3, and confirms RonaCare ring five peptide analogues
Binding site, builds and calculates network.
A3, carry out molecular docking by Ligandfit program: part RonaCare ring five peptide analogues and integrin IIb 3
During docking, the conformation number of each ligand structure of selection is 100, and scoring functions is LigScore1, and energy networks is PLP,
To ligand conformational be further carried out ability optimization.
A4, utilize Ligandfit software that a series of ring five peptide analogues and integrin protein I Ib 3 are carried out molecule pair
Connect, the binding pattern of the final interphase interaction confirming them, it is believed that Arg-Gly-Asp-Phe-Val (being called for short RC-5) is the most
Peptide is more suitable for follow-up DNA restructuring catalysis and forms cyclic peptide.
(2), the gene design of linear RonaCare ring five peptide analogues
According to molecular docking result, RC-5 is carried out the degeneracy analysis of genetic codon, according to the net of prediction rare bases
Stand: http://nihserver.mbi.ucla.edu/RACC/ and http://www.genscript.com/cgi-bin/
Tools/rare_codon_analysis, the password Preference design gene order according to fermentation host e. coli is as follows:
CGT GGC GAT TTT GTG。
The acquisition of B1, RC-5 gene order
Use the Primer primer 5 following primer of software design.For in pTwin1 vector multiple cloning site 6404
SapI enzyme action insertion point with 6437, design two complementary primers of synthesis, forward primer RC5-F1:5 ' AAC CGT GGC
GAT TTT GTG 3 ', downstream primer RC5-R1:5 ' GCA CAC AAA ATC GCC ACG 3 '.By primer degeneration renaturation
Method, is formed the two ends gene with the RC5 linear precursor of cohesive end by RC5-F1 and RC5-R1 annealing.Reaction condition is such as
Under: in PCR instrument, 4 DEG C, 5min, slowly reduces temperature to 0 DEG C.PMD-18T carrier it is connected to by glue after PCR primer being reclaimed
On, by chemical conversion to DH5 obtains RC5-18T recombinant clone, linked system table 1.And coat containing 100g/mL ammonia
In the LB solid medium of benzylpcnicillin (Amp+).Being placed in 37 DEG C of incubators and cultivate 12h, picking monoclonal send invitrogen public
Department checks order.Sequencing result is carried out on NCBI (National Center of Biotechnology Information)
BLAST comparison determines fragment.
The structure of B2, RC-5 recombinant expression carrier pTwin-RC5
Clone is carried out bacterium colony PCR checking, positive colony is checked order.Positive colony correct to order-checking and
PTwin bacterium extracting plasmid, carries out enzyme action, 37 DEG C of 2h with SapI enzyme action respectively, and endonuclease reaction system is with reference to table 2.To digestion products
Carry out agarose gel electrophoresis detection, enzyme action fragment completely is carried out glue and reclaims and quantitative.Double digestion is reclaimed product according to
Table 3 linked system connects overnight in 16 DEG C of metal baths.Take connection product chemical transformation, product is converted to Rosseta
(DE3), in, recombinant pTwin-RC5-DE3 is built.Positive recombinant is screened by the LB solid plate containing Kan (50 μ g/mL),
With Ssp DnaB Intein Forward 5 ' ACT GGG ACT CCA TCG TTT 3 ' and Mxe Intein Reverse it is
5 ' GGC ACG ATG TCG GCG ATG 3 ' carry out bacterium colony PCR to clone, send order-checking company to detect positive colony, right
The correct clone that checks order is stored in LB+Amp (the 100 μ g/mI) fluid medium containing 20% glycerol, and-80 DEG C of storages.
The linked system that table 1RC5-18T recombinant expression carrier builds
| System | |
| Solution I | 5.0μL |
| PCR primer | 4.0μL |
| PMD18-T-carrier | 1.0μL |
| Amount to | 10.0μL |
The double digestion system that table 2RC5 recombinant expression carrier builds
| Enzyme action system | |
| Plasmid DNA | 1μg |
| NEBuffer2 | 5.0μL |
| Sap I | 1U |
| dH2O | To 50 μ L |
The linked system that table 3pTwin-RC5-DE3 recombinant expression carrier builds
| Linked system | |
| 10 × T4DNA ligase buffer | 2.0μL |
| PTwin1 carrier | 50ng |
| RC5 fragment | 135ng |
| T4 ligase | 1U |
| Amount to | 20.0μL |
(3), the restructuring fermentation of pTwin-RC5-DE3 and the acquisition of RC5 cyclic peptide
C1, pTwin-RC5-DE3 recombinant strains is inoculated on the LB solid plate of Amp (100 μ g/mL) activation,
Picking list colony inoculation in LB+Amp (100 μ g/mL) fluid medium, 37 DEG C, 250rpm shaken cultivation overnight.After with 4%
Inoculum concentration be inoculated in fermentation liquid (LB+Amp 100 μ g/mL) and be enlarged cultivating, 37 DEG C of 250rpm, until 0.5≤OD600
≤1.0.Add IPTG to final concentration of 0.5mM and carry out the abduction delivering of destination protein, using empty carrier pTwin and do not induce as
Negative control.Inductive condition is 16 DEG C, 120rpm, 6-10h.To maturing fermentation liquid with 4500rpm, 4 DEG C, 15min is centrifugal to be collected
Thalline, abandons supernatant.
C2, addition cell pyrolysis liquid Buffer B1 (20mM Tris-HCl, 500mM NaCl, 1mM EDTA, pH 8.0)
Abundant resuspended thalline.The homogeneous instrument of Precellys 24 crushes thalline condition: 5000rpm/4 × 40s/30s, i.e. frequency
5000rpm, every task 40s, work 3 times, every minor tick 30s, and overall process operates in ice bath.4 DEG C afterwards, 12 000rpm,
10min is centrifuged, and collects supernatant.Detect for inclusion body precipitation cell pyrolysis liquid is resuspended simultaneously.
C3, sample liquid upper prop: under the conditions of 4 DEG C, add 2mL chitin resin column material Chitin beads in Chitin post,
The Buffer B1 adding 10 times of column volumes carries out pretreatment.Gained supernatant in step (2) is added Chitin post, controls stream
Speed is at 0.5-1.0mL/min.
C4, wash post: wash post with the Buffer B1 buffer of at least 20 times of bed volumes, due to CBD and chitin resin
Adhesion is very strong, and the flow velocity washing post can properly increase, and flow velocity can be at 1.5-2.0mL/min.
C5, the eluting of destination protein: with buffer B uffer B2 (20mM Tris-HCl, the 500mM of 4 times of bed volumes
NaCl, 1mM EDTA, pH 7.0) make peptide bond fission, cover cap, room temperature stands overnight.The C end-grain cutting of induction intein 1 is cut and is released
Put the N end of polypeptide.
C6, the cyclisation of destination protein: wash post with the flow velocity of 2mL/min with the Buffer B2 of 10 times of column volumes, with 3 times of posts
Volume Buffer B3 (20mM Tris-HCl, 50mM NaCl, 1mM EDTA, 50mM MESNA, pH 8.5) is quick crosses post, makes
MESNA is uniformly distributed and soaks filler in post, and 4 DEG C of induction intein 2 cut and polypeptide cyclisation 16h.
C7, the eluting of desired polypeptides: with Buffer B3 eluting purpose cyclic peptide, 3-4 times of column volume eluent before collecting altogether.
C8, renaturation are tested: the thalline ultrasonication to generation inclusion body, and broken liquid abandons supernatant, add 5mL in precipitation
Buffer B1,4 DEG C of 10000rpm are centrifuged 2min, collect precipitation, add the carbamide of 5mL 8M, are stirred at room temperature 2～3h, to precipitation
Being completely dissolved, 4 DEG C of 10000rpm are centrifuged 10min, collect supernatant.Gained supernatant loads in the bag filter after activation, adds
15mL Buffer B1 dialyses about 20h, and every 4～5h change buffer
(4), the detection of RC5 cyclic peptide
Collect the protein sample in each stage: to cellular lysate liquid, gradient eluent, purified concentration albumen through row SDS-PAGE
Detection (table 4), and carry out quantitatively with BioRad protein quantification test kit.
Table 4SDS-PAGE component list
Carrying out lyophilization subsequently, add proper amount of methanol (chromatographically pure) and again dissolve, 12000rpm, centrifugal 10min is to instead
Answer product to carry out HPLC/MS detection to analyze.Liquid-phase condition: 4.6 × 250mm, VYDAC-C18.Solvent A:0.1% acetic acid
Water；Solvent B:100% acetonitrile；Gradient: B, 0～39min 40%-90%, 40min 90%；Detection wavelength:
220nm；Flow velocity: 0.2mL/min；Sample size: 10L.
Above-described embodiment is only used for illustrating the inventive concept of the present invention, rather than the restriction to rights protection of the present invention,
All changes utilizing this design that the present invention carries out unsubstantiality, all should fall into protection scope of the present invention.
Claims (5)
1. the biological synthesis method of RonaCare ring pentapeptide cosmetics of recombinating, it is characterised in that comprise the following steps:
(1) RonaCare ring five peptide analogues is carried out molecular docking；
(2) linear RonaCare ring five peptide analogues is carried out gene design；
(3) acquisition with RonaCare cyclic peptide of fermenting of restructuring pTwin-RC5-DE3；
(4) detection to RonaCare cyclic peptide.
2. the biological synthesis method of RonaCare ring pentapeptide cosmetics of recombinating as claimed in claim 1, it is characterised in that described step
Suddenly the molecular docking of (1) comprises the steps:
A1, build little molecular database: generating cyclic peptide two-dimensional structure with ChemDraw Ultra 10.0, LigandFit software exists
Calculating can automatically generate three configurations of cyclic peptide, and form little molecular database；
A2, receptor protein file: according to the crystal structure of acquisition integrin albumen aIIbb3 in protein crystal number storehouse, will join
The little molecule of body is removed from the three dimensional structure of integrin aIIbb3, and confirms RonaCare ring five peptide analogues binding site, structure
Build calculating network；
A3, carry out molecular docking by Ligandfit program: part RonaCare ring five peptide analogues and integrin aIIbb3 pair
Connecing, the ligand conformational obtained is further carried out ability optimization；
A4, utilize Ligandfit software that a series of ring five peptide analogues and integrin albumen aIIbb3 are carried out molecular docking,
Confirm the binding pattern of their interphase interaction eventually.
3. the biological synthesis method of RonaCare ring pentapeptide cosmetics of recombinating as claimed in claim 1, it is characterised in that described step
Suddenly linear RonaCare ring five peptide analogues of (2) carries out gene design and comprises the steps:
The acquisition of B1, RC-5 gene order: use Primer primer 5 software design primer, for many grams of pTwin1 carrier
The SapI enzyme action insertion point of 6404 and 6437 in grand site, design two complementary primers of synthesis, forward primer RC5-F1:
5 ' AAC CGT GGC GAT TTT GTG 3 ', downstream primer RC5-R1:5 ' GCA CAC AAA ATC GCC ACG 3 '；Pass through
The method of primer degeneration renaturation, is formed the two ends base with the RC-5 linear precursor of cohesive end by RC5-F1 and RC5-R1 annealing
Cause；
The structure of B2, RC-5 recombinant expression carrier pTwin-RC5: clone is carried out bacterium colony PCR checking, positive colony is entered
Row order-checking, positive colony correct to order-checking and pTwin bacterium extracting plasmid, carry out enzyme action with SapI enzyme action respectively, 37 DEG C of 2h,
Digestion products is carried out agarose gel electrophoresis detection, enzyme action fragment completely is carried out glue and reclaims and quantitative；Double digestion is returned
Receive product linked system to connect overnight in 16 DEG C of metal baths, take connection product chemical transformation, product is converted to Rosseta
In, build recombinant pTwin-RC5-DE3.
4. the biological synthesis method of RonaCare ring pentapeptide cosmetics of recombinating as claimed in claim 1, it is characterised in that described step
Suddenly the fermentation of (3) restructuring pTwin-RC5-DE3 and the acquisition of RonaCare cyclic peptide comprise the steps:
C1, pTwin-RC5-DE3 recombinant strains is inoculated on the LB solid plate of Amp activation, picking list colony inoculation
In LB+Amp fluid medium, shaken cultivation overnight, after be inoculated in fermentation liquid with the inoculum concentration of 4% and to be enlarged cultivating,
Until 0.5≤OD600≤1.0, add IPTG to final concentration of 0.5mM and carry out the abduction delivering of destination protein, with empty carrier
PTwin and do not induce as negative control, inductive condition is 16 DEG C, 120rpm, 6～10h, to maturing fermentation liquid with 4500rpm,
4 DEG C, 15min is centrifugal collects thalline, abandons supernatant；
C2, adding the abundant resuspended thalline of cell pyrolysis liquid Buffer B1, the homogeneous instrument of Precellys 24 crushes thalline condition:
5000rpm/4 × 40s/30s, i.e. frequency are 5000rpm, every task 40s, work 3 times, and every minor tick 30s, overall process is in ice
Bath operation；4 DEG C afterwards, 12 000rpm, 10min are centrifugal, collect supernatant, simultaneously resuspended for wrapping to precipitation cell pyrolysis liquid
Contain health check-up to survey；
C3, sample liquid upper prop: under the conditions of 4 DEG C, add 2mL chitin resin column material Chitin beads in Chitin post, add
The Buffer B1 of 10 times of column volumes carries out pretreatment, and gained supernatant in step C2 is added Chitin post, and coutroi velocity exists
0.5-1.0mL/min；
C4, wash post: wash post, due to the combination of CBD Yu chitin resin with the Buffer B1 buffer of at least 20 times of bed volumes
Power is very strong, and the flow velocity washing post can properly increase, and flow velocity can be at 1.5-2.0mL/min；
C5, the eluting of destination protein: make peptide bond fission with the buffer B uffer B2 of 4 times of bed volumes, cover cap, room temperature
Standing overnight, the N end of release polypeptide is cut in the C end-grain cutting of induction intein 1；
C6, the cyclisation of destination protein: wash post with the flow velocity of 2mL/min with the Buffer B2 of 10 times of column volumes, with 3 times of column volumes
The quick post of crossing of Buffer B3, filler in making MESNA be uniformly distributed and soaking post, 4 DEG C of induction intein 2 cut and polypeptide cyclisation
16h；
C7, the eluting of desired polypeptides: with Buffer B3 eluting purpose cyclic peptide, 3-4 times of column volume eluent before collecting altogether；
C8, renaturation are tested: the thalline ultrasonication to generation inclusion body, and broken liquid abandons supernatant, add 5mL Buffer in precipitation
B1, collected after centrifugation precipitates, and adds carbamide, is stirred at room temperature and is completely dissolved to precipitation, and centrifugal collection supernatant, gained supernatant loads
In bag filter after activation, adding dialysis, every 4-5h changes a buffer.
5. the biological synthesis method of RonaCare ring pentapeptide cosmetics of recombinating as claimed in claim 1, it is characterised in that described step
Suddenly the detection of (4) RonaCare cyclic peptide comprises the steps: to collect the protein sample in each stage, washes cellular lysate liquid, gradient
De-liquid, purified concentration albumen detect through row SDS-PAGE, and carry out quantitatively with BioRad protein quantification test kit；Carry out cold subsequently
Lyophilizing is dry, adds proper amount of methanol and again dissolves, and product carries out after being centrifuged HPLC/MS detection and analyzes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101990425A (en) * | 2008-04-08 | 2011-03-23 | 默克专利股份有限公司 | Compositions containing cyclic peptides and methods of use |
| CN103525889A (en) * | 2013-10-11 | 2014-01-22 | 宁波大学 | Synthetic method for Cilengitide by using thioesterase |
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2016
- 2016-04-18 CN CN201610243842.9A patent/CN105801671A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101990425A (en) * | 2008-04-08 | 2011-03-23 | 默克专利股份有限公司 | Compositions containing cyclic peptides and methods of use |
| CN103525889A (en) * | 2013-10-11 | 2014-01-22 | 宁波大学 | Synthetic method for Cilengitide by using thioesterase |
Non-Patent Citations (1)
| Title |
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| 高媛: "新型重组环状五肽结构类似物cyclo-(Cys-Arg-Lys-Asp-Val-Tyr)的研制", 《中国优秀博硕士学位论文全文数据库(硕士) 基础科学辑》 * |
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