CN105218633B - Formic acid cleavage site peptide and its relevant biological material and they production calcitonin in application - Google Patents
Formic acid cleavage site peptide and its relevant biological material and they production calcitonin in application Download PDFInfo
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- CN105218633B CN105218633B CN201410231041.1A CN201410231041A CN105218633B CN 105218633 B CN105218633 B CN 105218633B CN 201410231041 A CN201410231041 A CN 201410231041A CN 105218633 B CN105218633 B CN 105218633B
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- fusion protein
- formic acid
- calcitonin
- protein
- cutting
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Abstract
The invention discloses formic acid cleavage site peptide and its relevant biological material and they production calcitonin in application.Formic acid cleavage site peptide provided by the present invention, entitled CA3, amino acid sequence peptide as shown in SEQ ID No.2.The fusion protein constructed using formic acid cleavage site Peptide C A3 of the invention as formic acid cleavage site, cutting rate under following formic acid cutting condition is 85.3%: formic acid (cutting agent) concentration is 40% (percent by volume), cutting temperature is 43 DEG C, clipping time 2.5h.Fusion protein containing formic acid cleavage site Peptide C A3 of the invention carries out formic acid cutting production destination protein, formic acid clipping time is short and cutting efficiency is high, effectively improve the cutting efficiency of fusion protein, the purifying cost of destination protein is saved, new thinking is opened for the industrial production of Protein reconstitution, provides technical support for the production of protein product.
Description
Technical field
The present invention relates to formic acid cleavage site peptide and its relevant biological material in field of biotechnology and they production drop
Application in calcium element.
Background technique
Recombinant expression has many advantages, such as expression, and speed is fast, at low cost, high production efficiency becomes the convenient of Peptide systhesis
Method.The key technology of Protein reconstitution expression is the purifying of destination protein, and recombinant expression generally uses the expression side of fusion protein
Method, therefore, it is the key that recombinant expression that destination protein, which cuts from fusion protein and separates, is merged for protein product
The cutting of albumen and separative efficiency seem increasingly important.The cleavage reaction of fusion protein mainly has chemical cleavage and digestion to cut at present
Two kinds.
Comparatively reaction condition is milder for the method for enzymatic hydrolysis, it is often more important that, it is commonly used to the protease of this purposes all
Specificity with height.Site-specific protease (such as fibrin ferment, enterokinase, Xa factor etc.) is usually used to cutting and melts
Hop protein.Fibrin ferment is that activity specific is highest in these three enzymes, can effectively cut quality than be only 1:2000 egg
It is white, but digesting efficiency is very low, and only 10% enzyme can play cutting function when for cutting.On the specificity of enterokinase is
It states best in three kinds, but seems more expensive due to cutting efficiency low (usually requiring that mass ratio reaches 1:10).Xa factor
Seem that the sequence around for point of contact is very sensitive, often will appear that specific position cutting is undesirable but to be occurred other site and cut
The case where cutting.More importantly protease itself can be mixed into destination protein, cause new pollution, improve the complexity of purifying.
It is cut relative to zymetology, chemical cleavage is more efficient in the cutting of fusion protein, and it is low in cost, easy to operate,
And there is no new albumen to introduce in fusion protein cutting, Sample Purification on Single is simpler, therefore has become a hot topic of research.Mesh
Before, widely applied chemical cleavage method mainly include the following types:
Hydrogen bromide (CNBr) patterning method, CNBr separate fusion protein by cutting Met residue c-terminus.Advantage is at low cost
And effectively；Disadvantage CNBr in the presence of Met can scinderin, cracking site specificity it is low, destination protein may be produced
Raw unnecessary modification.
Azanol (NH2OH) patterning method, NH2OH can specificity cutting peptide bonds in polypeptides Asn-Gly.Advantage is will not to bring
Heterologous pollution, advantage of lower cost.The disadvantage is that azanol is unstable, easy decomposition, and it is more toxic.
Based on Ca2+Peptide bond Asp-Pro patterning method, advantage is that clipping time is fast, can complete to cut in 30 minutes；It lacks
Point is to require cutting system strictly, and cutting efficiency is substantially reduced in the presence of other ions in addition to required ion in cutting solution,
It is at high cost, it is complex for operation step.
The formic acid patterning method of peptide bond D-P (Asp-Pro).In acid condition, D-P key be cut open be nucleophillic attack result
(Maria Thuveson,Erik Fries,2000).Benjamin D. etc. (Benjamin D, 1989) has been proved in an experiment
What is be broken in acid condition is only D-P key, and the side links of Pro can protonate when principle is low pH, and excite Asp
β carbon-based group on residue can initiate nucleophillic attack to its α carbon-based group, so that D-P key be made to disconnect.Asp-Pro formic acid patterning method
Advantage has following three: (1) it only in acid condition could complete cut, and low pH can restrictively cut D-P key, it will not
Other peptide bonds are had an impact；(2) probability that D-P occurs in most albumen is very low；(3) to solution environmental require compared with
Low, the presence of other ions does not influence cutting efficiency in solution, can increase cutting efficiency by properly increasing temperature.It is main
Include two o'clock in place of shortcoming and deficiency: (1) clipping time is relatively long, and all cutting need to be 30 hours or so for general completion；(2)
Low pH may influence the activity and stability of albumen.
Osteoporosis seriously threatens human health, and calcitonin (calcitonin, CT) is current treatment one line of osteoporosis
Drug, clinical application nearly 30 years, the annual sales amount of international market was up to 700,000,000 dollars at present.Expensive is that influence calcitonin is wide
The main reason for general application, the calcitonin in China injection is 5200 yuan/milligram at present, and raw medicine price is also up to
Ten thousand yuan/gram of 0.8-1.0.Calcitonin it is expensive the reason is that: the calcitonin clinically applied is mainly chemical synthesis at present
Production, have by-product mostly and is difficult to the problem of being mass produced.What gene engineering research was expressed in Escherichia coli or yeast
Calcitonin, since 32 amino acids of the end C- are unable to amidation, biological activity is very low, and every milligram is only had 100-200 international unit,
It can not be applied to clinic, two above reason causes the market price of current calcitonin very high.The principle of oil body expression system is
So that destination protein is inserted in the oil body surface of seed, constitutes fusion protein, the specifically expressing on the oil body of transgenic plant seed, benefit
With the hydrophobic characteristic of oil body lipophilic, seed crushing-centrifugation-recycling upper oil phase-is deoiled, can be obtained fusion protein.It should
The advantages of method has extraction process simple, is easily isolated purifying, is also greatly improved the added value of agricultural and sideline product.
Summary of the invention
A kind of formic acid short technical problem to be solved by the invention is to provide formic acid clipping time and that cutting efficiency is high is cut
Cut site Peptide C A3 and its application.The formic acid cleavage site peptide effectively improves the formic acid cutting efficiency of fusion protein, saves purpose egg
White purifying cost.The best formic acid cutting condition of fusion protein containing CA3 is that formic acid concn is 40% (percent by volume), is cut
Cutting temperature is 43 DEG C, clipping time 2.5h.
Formic acid cleavage site peptide provided by the present invention, entitled CA3 is amino acid sequence as shown in SEQ ID No.2
Peptide.
Wherein, SEQ ID No.2 is made of 6 amino acid residues.
Fusion protein containing CA3 also belongs to protection scope of the present invention.The above-mentioned fusion protein containing CA3 is made with CA3
The cutting separation destination protein from the fusion protein is used for for formic acid cleavage site.
In above-mentioned fusion protein, the fusion protein is calcitonin fusion protein G-sCT-SO, and the calcitonin merges egg
White G-sCT-SO is that glycoprotein extracellular domain and calcitonin are connected to the fusion protein G-sCT to be formed to connect shape with sesame oil body protein
At protein；The fusion protein G-sCT is by formic acid cleavage site peptide by the glycoprotein extracellular domain and the calcitonin
Connect the fusion protein formed；The amino acid sequence of the formic acid cleavage site peptide is SEQ ID No.2.
The amino acid sequence of the calcitonin fusion protein G-sCT-SO concretely SEQ ID No.4.
Following biomaterials relevant to formic acid cleavage site Peptide C A3 or biomaterial relevant with the fusion protein
Also belong to protection scope of the present invention:
Any one of biomaterial relevant to formic acid cleavage site Peptide C A3 is B1)-B12):
B1 the nucleic acid molecules of formic acid cleavage site Peptide C A3) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules；
B4) contain B2) recombinant vector of the expression cassette；
B5) contain B1) recombinant microorganisms of the nucleic acid molecules；
B6) contain B2) recombinant microorganism of the expression cassette；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) contain B4) recombinant microorganism of the recombinant vector；
B9) contain B1) transgenic animals of the nucleic acid molecules or transgenic plant cells system；
B10) contain B2) transgenic animals of the expression cassette or transgenic plant cells system；
B11) contain B3) transgenic animals of the recombinant vector or transgenic plant cells system；
B12) contain B4) transgenic animals of the recombinant vector or transgenic plant cells system；
Any one of biomaterial relevant to the fusion protein is C1)-C12):
C1) the nucleic acid molecules of encoding said fusion protein；
C2) contain C1) expression cassettes of the nucleic acid molecules；
C3) contain C1) recombinant vectors of the nucleic acid molecules；
C4) contain C2) recombinant vector of the expression cassette；
C5) contain C1) recombinant microorganisms of the nucleic acid molecules；
C6) contain C2) recombinant microorganism of the expression cassette；
C7) contain C3) recombinant microorganism of the recombinant vector；
C8) contain C4) recombinant microorganism of the recombinant vector；
C9) contain C1) transgenic animals of the nucleic acid molecules or transgenic plant cells system；
C10) contain C2) transgenic animals of the expression cassette or transgenic plant cells system；
C11) contain C3) transgenic animals of the recombinant vector or transgenic plant cells system；
C12) contain C4) transgenic animals of the recombinant vector or transgenic plant cells system.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also
To be RNA, such as mRNA or hnRNA.
In above-mentioned biomaterial, B1) concretely coded sequence is SEQ ID No.3 in sequence table for the nucleic acid molecules
The cDNA molecule or DNA molecular of 112-129 nucleotide (i.e. the 1580-1597 nucleotide of SEQ ID No.5).C1)
Any one of the nucleic acid molecules concretely 1) -4):
1) coded sequence is that the cDNA molecule of the 956-1696 nucleotide of SEQ ID No.5 or DNA divide in sequence table
Son；
2) sequence is the cDNA molecule or DNA molecular of SEQ ID No.5 in sequence table；
1) or 2) 3) there is 75% or 75% or more identity, and encoding said fusion protein with the nucleotide sequence limited
CDNA molecule or genomic DNA molecule；
4) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and the cDNA of encoding said fusion protein points
Son or genomic DNA molecule.
Wherein, SEQ ID No.3 is by formic acid cleavage site Peptide C A3 gene by salmon's calcitonin gene and glycoprotein film
The nucleotide sequence for the fusion protein F P3 gene that outskirt Gene Fusion obtains, SEQ ID No.3 are made of 1491 nucleotide,
Its coded sequence is 10-1476 of SEQ ID No.3, encodes FP3 shown in SEQ ID No.1.In SEQ ID No.3,
1-9 for I site BamH and protection base, 13-111 be salmon calcitonin (sCT) coded sequence, 112-129
For the coded sequence of CA3,130-135 are I site Sac, and 160-1476 are glycoprotein extracellular domain
(Glycoprotein) coded sequence, 1483-1491 are I site Xho and protection base.
SEQ ID No.4 is the amino acid sequence of salmon calcitonin fusion protein G-sCT-SO, by 247 amino acid residues
Composition.Salmon calcitonin fusion protein G-sCT-SO is that glycoprotein extracellular domain and salmon are dropped calcium by formic acid cleavage site Peptide C A3
The fusion protein G-sCT that element connection is formed connect the salmon calcitonin fusion protein G-sCT-SO to be formed with sesame oil body protein.
The amino acid sequence of fusion protein G-sCT-SO is in SEQ ID No.4, SEQ ID No.4, and 8-150 are sesame oil body egg
White sequence, the outer region sequence of the 153-208 films for glycoprotein, 209-214 are Asp-Pro formic acid cleavage site peptide
The sequence of CA3, the 215-247 sequences for salmon calcitonin.
SEQ ID No.5 is salmon calcitonin fusion protein G-sCT-SO expression casette sequence, by 2163 nucleotide
Composition, coded sequence is 956-1696 of SEQ ID No.5, encodes salmon calcitonin shown in SEQ ID No.4 and melts
Hop protein G-sCT-SO.In SEQ ID No.5,1-9 are III site Hind and protection base, and 10-949 are Canola oil
The sequence of body protein promoter (rape oleosin promoter), the 977-1405 sequences for sesame oil body protein, the
The sequence of the 1412-1579 film outskirts for glycoprotein, the 1580-1597 sequences for Asp-Pro formic acid cleavage site Peptide C A3
Column, the 1598-1696 sequences for salmon calcitonin, the 1715-2154 sequences for terminator.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid.Identity can with the naked eye or
Computer software is evaluated.Using computer software, the identity between two or more sequences can use percentage (%)
It indicates, can be used to evaluate the identity between correlated series.
The stringent condition can hybridize at 68 DEG C and wash film 2 times, every time in 2 × SSC, the solution of 0.1%SDS
5min, but in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and washes film 2 times, each 15min.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.
In above-mentioned biomaterial, the carrier can be plasmid, clay, bacteriophage or viral vectors.
In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi, such as Escherichia coli.
In above-mentioned biomaterial, the transgenic plant cells system and transgenetic animal cell system do not include propagation material.
The present invention also provides the methods for preparing the fusion protein.
The method provided by the present invention for preparing the fusion protein, including by the encoding gene of the fusion protein in life
The step of obtaining the fusion protein is expressed in object cell；The biological cell is microbial cell, plant cell or non-
People's zooblast.
In the above method, the microorganism can be yeast or bacterium or algae or fungi, such as Escherichia coli.
The present invention also provides a kind of methods for preparing destination protein by the fusion protein.It is provided by the present invention by institute
The method that fusion protein prepares destination protein is stated, including formic acid is carried out to the fusion protein and cuts to obtain the destination protein；
The formic acid cutting condition is that the concentration of formic acid is 40% (percent by volume), and cutting temperature is 43 DEG C, clipping time 2.5h.
The present invention also provides the methods containing calcitonin fusion protein transgene rape of cultivation.
Method of the cultivation containing calcitonin fusion protein transgene rape provided by the present invention, the method includes to by
The encoding gene that calcitonin fusion protein G-sCT-SO is imported in body rape, obtains the transgenic rape containing calcitonin fusion protein
Dish；
The calcitonin fusion protein G-sCT-SO is that glycoprotein extracellular domain is connected the fusion protein to be formed with calcitonin
G-sCT connect the protein to be formed with sesame oil body protein；The fusion protein G-sCT is will be described by formic acid cleavage site peptide
Glycoprotein extracellular domain connects the fusion protein to be formed with the calcitonin；The amino acid sequence of the formic acid cleavage site peptide is
SEQ ID No.2。
In the above method, the amino acid sequence of the calcitonin fusion protein G-sCT-SO is SEQ ID No.4；The drop
The amino acid sequence of calcium element is the 215-247 of SEQ ID No.4.
It is obtained using above-mentioned cultivation containing the method for calcitonin fusion protein transgene rape the present invention also provides a kind of
The method for preparing calcitonin of the transgene rape containing calcitonin fusion protein.
The method provided by the present invention for preparing calcitonin, including contain calcitonin fusion protein transgenosis from above-mentioned cultivation
The oil body that the transgene rape is separated in the transgene rape containing calcitonin fusion protein that the method for rape obtains, to institute
Oil body progress formic acid is stated to cut to obtain the calcitonin；The formic acid cutting condition is that the concentration of formic acid is 40% (volume basis
Than), cutting temperature is 43 DEG C, clipping time 2.5h.
The more commercially available Calcitonin Salmon activity of calcitonin for preparing the method preparation of calcitonin is high.
The fusion protein constructed using formic acid cleavage site Peptide C A3 of the invention as formic acid cleavage site, in following first
Cutting rate under sour cutting condition is 85.3%: formic acid (cutting agent) concentration is 40% (percent by volume), cutting temperature 43
DEG C, clipping time 2.5h.Fusion protein containing formic acid cleavage site Peptide C A3 of the invention carries out formic acid cutting production purpose
Albumen, formic acid clipping time is short and cutting efficiency is high, effectively improves the cutting efficiency of fusion protein, has saved the pure of destination protein
Chemical conversion originally, for the industrial production of Protein reconstitution opens new thinking, provides technical support for the production of protein product.
Detailed description of the invention
Fig. 1 is psCA132a(+)、psCA232a(+)、psCA332a (+) and psCA4The map of 32a (+).
Fig. 2 is prokaryotic expression bacterial protein electrophoresis；1: albumen pre-dyed Marker；2: Escherichia coli Rosetta/pET32a
(+) induces bacterial protein without IPTG；3: Escherichia coli Rosetta/pET32a (+) induces bacterial protein through IPTG；4:
Escherichia coli Rosetta/psCA132a (+) induces bacterial protein through IPTG；5: Escherichia coli Rosetta/psCA232a(+)
Bacterial protein is induced through IPTG；6: Escherichia coli Rosetta/psCA332a (+) induces bacterial protein through IPTG；7: large intestine
Bacillus Rosetta/psCA432a (+) induces bacterial protein through IPTG.
Fig. 3 is that the existence form of target protein is analyzed；1: albumen pre-dyed Marker；2: Escherichia coli Rosetta/
psCA132a (+) induces bacterial cell disruption supernatant through IPTG；3: Escherichia coli Rosetta/psCA132a (+) is through IPTG induction bacterium
The broken precipitating of body；4: Escherichia coli Rosetta/psCA232a (+) induces bacterial cell disruption supernatant through IPTG；5: Escherichia coli
Rosetta/psCA232a (+) is through IPTG induction bacterial cell disruption precipitating；6: Escherichia coli Rosetta/psCA332a (+) is through IPTG
Induce bacterial cell disruption supernatant；7: Escherichia coli Rosetta/psCA332a (+) is through IPTG induction bacterial cell disruption precipitating；8: large intestine
Bacillus Rosetta/psCA432a (+) induces bacterial cell disruption supernatant through IPTG；9: Escherichia coli Rosetta/psCA432a(+)
Through IPTG induction bacterial cell disruption precipitating.
Fig. 4 is prokaryotic expression mycoprotein electrophoresis；1: albumen pre-dyed Marker；2: Escherichia coli Rosetta/pET32a
(+) is without IPTG induction bacterial cell disruption precipitating；3: Escherichia coli Rosetta/pET32a (+) is heavy through IPTG induction bacterial cell disruption
It forms sediment；4: Escherichia coli Rosetta/psCA132a (+) is through IPTG induction bacterial cell disruption precipitating；5: Escherichia coli Rosetta/
psCA232a (+) is through IPTG induction bacterial cell disruption precipitating；6: Escherichia coli Rosetta/psCA332a (+) induces thallus through IPTG
Broken precipitating；7: Escherichia coli Rosetta/psCA432a (+) is through IPTG induction bacterial cell disruption precipitating；8: Escherichia coli
Rosetta/psCA332a (+) induces bacterial cell disruption supernatant through IPTG.
Fig. 5 is that the Western-Blot of fusion protein is analyzed；1: albumen pre-dyed Marker；2: Escherichia coli Rosetta/
PET32a (+) is without IPTG induction bacterial cell disruption precipitating；3: Escherichia coli Rosetta/pET32a (+) is broken through IPTG induction thallus
Broken precipitating；4: Escherichia coli Rosetta/psCA132a (+) is through IPTG induction bacterial cell disruption precipitating；5: Escherichia coli Rosetta/
psCA232a (+) is through IPTG induction bacterial cell disruption precipitating；6: Escherichia coli Rosetta/psCA332a (+) induces thallus through IPTG
Broken precipitating；7: Escherichia coli Rosetta/psCA432a (+) is through IPTG induction bacterial cell disruption precipitating；8: Escherichia coli
Rosetta/psCA332a (+) induces bacterial cell disruption supernatant through IPTG.
Fig. 6 is SDS-PAGE analysis after fusion protein purification desalination；1: albumen pre-dyed Marker；2: from Escherichia coli
Rosetta/psCA1The fusion protein of 32a (+) purification desalination；3: from Escherichia coli Rosetta/psCA232a (+) purification desalination
Fusion protein；4: from Escherichia coli Rosetta/psCA3The fusion protein of 32a (+) purification desalination；5: from Escherichia coli
Rosetta/psCA4The fusion protein of 32a (+) purification desalination.
Fig. 7 is the fusion protein Western-Blot analysis of purifying；1: albumen pre-dyed Marker；2: from Escherichia coli
Rosetta/psCA1The fusion protein of 32a (+) purification desalination；3: from Escherichia coli Rosetta/psCA232a (+) purification desalination
Fusion protein；4: from Escherichia coli Rosetta/psCA3The fusion protein of 32a (+) purification desalination；5: from Escherichia coli
Rosetta/psCA4The fusion protein of 32a (+) purification desalination.
Fig. 8 is formic acid acid cutting analysis after four kinds of fusion protein purifications；1: albumen pre-dyed Marker；2: formic acid cutting
FP1；3: the FP1 without formic acid cutting；4: the FP2 of formic acid cutting；5: the FP2 without formic acid cutting；6: the FP3 of formic acid cutting；
7: the FP3 without formic acid cutting；8: the FP4 of formic acid cutting；9: the FP4 without formic acid cutting.
Fig. 9 is fusion protein difference formic acid concn cutting analysis；L1: fusion protein F P1；L2: fusion protein F P2；L3: melt
Hop protein FP3；L4: fusion protein F P4.
The cutting temperature of fusion protein when Figure 10 is 40% formic acid concn；L1: fusion protein F P1；L2: fusion protein F P2；
L3: fusion protein F P3；L4: fusion protein F P4.
Figure 11 is the analysis of formic acid clipping time；1: albumen pre-dyed Marker；2:0h；3:0.5h；4:1.0h；5:1.5h；6:
2.0h；7:2.5h；8:3.0h；9:3.5h.
Figure 12 is the analysis of formic acid clipping time gray scale scanning.
Figure 13 is the physical map of pBOGSC3301.
Figure 14 is the rape PCR detection after Basta screening；Marker2-10:9 plants of I/Hind of 1:Lamda DNA EcoR III
T0 turns pBOGSC3301 rapeseed plants for the Basta positive；11: wild type rape, i.e., double 4 in the cabbage type rape of non-transgenosis
Number.
Figure 15 is to turn the Basta screening of fusion protein G-sCT-SO vector for transgenic rape the T1 generation in crop field；Left side plant is T1 generation
Turn fusion protein G-sCT-SO vector for transgenic rape；Right side plant is wild type rape, i.e., double 4 in the cabbage type rape of non-transgenosis
Number.
Figure 16 is the PAGE gel electrophoresis detection after the cutting of rapeseed oil body formic acid；1. protein molecular weight
Marker；2-4 is the oil body formic acid cleaved products for turning fusion protein G-sCT-SO vector for transgenic rape seed；5-6 wild type rapeseed oil
Body formic acid cleaved products.
Figure 17 is the PAGE gel electrophoresis detection of the cation-exchange chromatography eluting peak of oil body formic acid cleaved products.1
It is salmon calcitonin standard for protein molecular weight Marker, 2；3-5 contains salmon for what cation-exchange chromatography purified
The solution of fish calcitonin.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
PET32a (+), Escherichia coli Rosetta-gami in following embodimentsTM2(DE3)pLysS、pCAMBIA3301
It is Beijing Baeyer enlightening biology Co., Ltd product with agrobacterium tumefaciens lba4404.In cabbage type rape in following embodiments
Double No. 4 are oil plant institute, the Chinese Academy of Agricultural Sciences product.
Embodiment 1, the Asp-Pro for being suitable for Asp-Pro formic acid patterning method (the formic acid patterning method of peptide bond D-P (Asp-Pro))
The screening of formic acid cleavage site peptide
The present embodiment devises four kinds of Asp-Pro formic acid cleavage site peptides in table 1, and title is respectively CA1, CA2, CA3
And CA4, the fusion protein being made up of salmon calcitonin with glycoprotein are screened from these four Asp-Pro formic acid cleavage site peptides
The high Asp-Pro formic acid cleavage site Peptide C A3 of cutting efficiency.
Salmon calcitonin is merged with the film outskirt of glycoprotein respectively by these four Asp-Pro formic acid cleavage site peptides
To four kinds of fusion protein F P1, FP2, FP3 and FP4, the Asp-Pro formic acid cleavage site peptide in FP1 is CA1, the Asp- in FP2
Pro formic acid cleavage site peptide is CA2, and the Asp-Pro formic acid cleavage site peptide in FP3 is CA3 (amino acid sequence such as SEQ ID
No.2), the Asp-Pro formic acid cleavage site peptide in FP4 is CA4.The amino acid sequence of FP3 is SEQ ID No.1；FP1 be by
CA3 shown in the 35-40 amino acids residue of SEQ ID No.1 replaces with the amino acid sequence Asp-Pro-Asp- of CA1
Pro-Asp-Pro, and keep the constant obtained fusion protein of other amino acid of SEQ ID No.1；FP2 is by SEQ ID
CA3 shown in the 35-40 amino acids residue of No.1 replaces with the amino acid sequence Asp-Asp-Asp-Asp-Pro- of CA2
Ile, and keep the constant obtained fusion protein of other amino acid of SEQ ID No.1；FP4 is by the 35- of SEQ ID No.1
CA3 shown in 40 amino acids residues replaces with the amino acid sequence Ile-Ile-Val-Asp-Pro-Asn-Pro-Thr of CA4,
And keep the constant obtained fusion protein of other amino acid of SEQ ID No.1.
The sequence of table 1, four kind of Asp-Pro formic acid cleavage site peptide
| Fusion protein Asp-Pro formic acid cleavage site peptide | Amino acid sequence |
| CA1 | Asp-Pro-Asp-Pro-Asp-Pro |
| CA2 | Asp-Asp-Asp-Asp-Pro-Ile |
| CA3 | Asp-Pro-Pro-Asp-Pro-Pro |
| CA4 | Ile-Val-Asp-Pro-Asn-Pro |
1, prepare the fusion protein F P1 containing these four Asp-Pro formic acid cleavage site peptides of CA1, CA2, CA3 and CA4,
FP2, FP3 and FP4
The preparation of 1.1 expression FP1, FP2, FP3 or FP4 recombinant bacteriums
Artificial synthesized FP1 gene, FP2 gene, FP3 gene and FP4 gene.The sequence of FP3 gene such as SEQ ID No.3,
Its coded sequence is 10-1476 of SEQ ID No.3, encodes FP3 shown in SEQ ID No.1.In SEQ ID No.3,
1-9 for I site BamH and protection base, 13-111 be salmon calcitonin (sCT) coded sequence, 112-129
For the coded sequence of CA3,130-135 are I site Sac, and 160-1476 are glycoprotein extracellular domain
(Glycoprotein) coded sequence, 1483-1491 are I site Xho and protection base.
The sequence of FP1 gene is the coding that the 112-129 CA3 coded sequences of SEQ ID No.3 are replaced with to CA1
The sequence that other invariant nucleotides of sequence GATCCAGATCCTGATCCA, SEQ ID No.3 obtain.
The sequence of FP2 gene is the coding that the 112-129 CA3 coded sequences of SEQ ID No.3 are replaced with to CA2
The sequence that other invariant nucleotides of sequence GACGATGACGATCCAATT, SEQ ID No.3 obtain.
The sequence of FP4 gene is the coding that the 112-129 CA3 coded sequences of SEQ ID No.3 are replaced with to CA4
The sequence that other invariant nucleotides of sequence ATCGTTGATCCAAACCCA, SEQ ID No.3 obtain.
FP3 gene and carrier pET32a (+) shown in BamH I and XhoI difference double digestion SEQ ID No.3, by digestion
Product is attached, and is obtained the sequence between the BamH I and the site XhoI by pET32a (+) and is replaced with SEQ ID No.3 4-
The recombinant vector of DNA fragmentation shown in 1488 (keeping the other sequences of pET32a (+) constant), which is named as
psCA332a(+)。psCA3Fusion protein F P3 shown in 32a (+) expression SEQ ID No.3.By psCA332a (+) imports large intestine
Bacillus Rosetta-gamiTMIn 2 (DE3) pLysS, obtain containing psCA3The recombinant bacterium of 32a (+), is named as Escherichia coli
Rosetta/psCA332a(+)。
With BamH I and XhoI difference double digestion FP1 gene and carrier pET32a (+), digestion products are attached, are obtained
Sequence between the BamH I and the site XhoI of pET32a (+) is replaced with into FP1 gene and (keeps the other sequences of pET32a (+) not
Become) recombinant vector, which is named as psCA132a(+)。psCA132a (+) expressed fusion protein FP1.It will
psCA132a (+) imports Escherichia coli Rosetta-gamiTMIn 2 (DE3) pLysS, obtain containing psCA1The recombination of 32a (+)
Bacterium is named as Escherichia coli Rosetta/psCA132a(+)。
With BamH I and XhoI difference double digestion FP2 gene and carrier pET32a (+), digestion products are attached, are obtained
Sequence between the BamH I and the site XhoI of pET32a (+) is replaced with into FP2 gene and (keeps the other sequences of pET32a (+) not
Become) recombinant vector, which is named as psCA232a(+)。psCA232a (+) expressed fusion protein FP2.It will
psCA232a (+) imports Escherichia coli Rosetta-gamiTMIn 2 (DE3) pLysS, obtain containing psCA2The recombination of 32a (+)
Bacterium is named as Escherichia coli Rosetta/psCA232a(+)。
With BamH I and XhoI difference double digestion FP4 gene and carrier pET32a (+), digestion products are attached, are obtained
Sequence between the BamH I and the site XhoI of pET32a (+) is replaced with into FP4 gene and (keeps the other sequences of pET32a (+) not
Become) recombinant vector, which is named as psCA432a(+)。psCA432a (+) expressed fusion protein FP4.It will
psCA432a (+) imports Escherichia coli Rosetta-gamiTMIn 2 (DE3) pLysS, obtain containing psCA4The recombination of 32a (+)
Bacterium is named as Escherichia coli Rosetta/psCA432a(+)。
Empty carrier-pET32a (+) is imported into Escherichia coli Rosetta-gamiTM2 (DE3) pLysS are obtained containing pET32a
The recombinant bacterium of (+) is named as Escherichia coli Rosetta/pET32a (+), as empty vector control bacterium.
psCA132a(+)、psCA232a(+)、psCA332a (+) and psCA4The map of 32a (+) as shown in Figure 1,
psCAxX in 32a (+) represents 1,2,3 or 4.
The inducing expression of 1.2 fusion protein F P1, FP2, FP3 or FP4
By Escherichia coli Rosetta/pET32a (+), Escherichia coli Rosetta/psCA132a (+), Escherichia coli
Rosetta/psCA232a (+), Escherichia coli Rosetta/psCA332a (+) and Escherichia coli Rosetta/psCA432a (+) point
It is not inoculated in individually containing ampicillin (100mM/L), kanamycins (50mg/L), chloramphenicol (34mg/L), tetracycline
In the LB liquid medium of (12.5mg/L) and streptomysin (50mg/L), 37 DEG C, 200rpm shake culture overnight after by 1:100
Inoculum concentration switching is primary, and switching culture volume is 20ml, 37 DEG C of shake cultures to OD600=0.4-0.6, and each strain takes respectively
10ml bacterium solution continues to cultivate as negative control, remaining bacterium solution is separately added into IPTG (final concentration of 1mM/L), Fiber differentiation 4h.4
Above-mentioned culture thallus DEG C is collected by centrifugation respectively, 8000rpm is centrifuged 10min, the phosphate buffer of the thallus pH7.4 of collection
(20mM/L sodium phosphate, 0.5M/L NaCl, 30mM/L imidazoles) washes twice, and the thallus finally collected uses the phosphoric acid of pH7.4 respectively
Buffer resuspension is placed on ice.The thallus of collection carries out ultrasonic disruption respectively, the separately sampled SDS-PAGE electrophoresis of disruption solution,
After electrophoresis, gel is removed, coomassie brilliant blue staining simultaneously decolourizes to analyze result (Fig. 2).
Protein electrophoresis contains the expression strain Escherichia coli Rosetta/pET32a of empty carrier pET32a (+) as the result is shown
(+) induces through IPTG and forms with the strain protein not induced without significant change, Escherichia coli Rosetta/psCA1It is 32a (+), big
Enterobacteria Rosetta/psCA232a (+), Escherichia coli Rosetta/psCA332a (+) and Escherichia coli Rosetta/
psCA432a (+) is after IPTG is induced in the theoretical value slightly larger than one apparent band of appearance at 70kD, with fusion protein
It is in the same size, about 73.51kD, without all occurring without apparent specific band in the bacterial strain by IPTG induction.
The existence form of 1.3 fusion proteins is analyzed
Ultrasonication carries out the Escherichia coli Rosetta/psCA of IPTG induction according to step 1.2132a (+), Escherichia coli
Rosetta/psCA232a (+), Escherichia coli Rosetta/psCA332a (+) and Escherichia coli Rosetta/psCA432a (+) bacterium
Body, 4 DEG C of broken solution, 7000rpm are centrifuged 10min, collect the supernatant precipitating of bacterial cell disruption solution respectively, and precipitating uses
A small amount of phosphate buffer is resuspended, and is placed in spare on ice.
Configure PAGE gel, resolving gel concentration 15%, concentration gum concentration be 5%, before ready supernatant
Precipitation solution is mixed with bromophenol blue buffer respectively boil 10min after loading, coomassie brilliant blue staining is analyzed after transformation electrophoresis, electricity
Swimming result is shown in Fig. 3.It is by analytical electrophoresis result it is found that not apparent at 73.51kD in the fragment supernatant of four kinds of bacterial strains
Band occurs, and has apparent specific band to occur in precipitating, and stripe size meets fusion protein molecule amount, illustrates that four kinds melt
Hop protein mainly exists in the form of inclusion body when inducing expression in Escherichia coli.
The fusion protein Western-Blot of 1.4 inducing expressions is detected
In the case where above-mentioned experiment determines that fusion protein molecule amount size meets expected situation, further detection fusion albumen
Correctness.The Escherichia coli Rosetta/pET32a (+) that obtains according to step 1.2 and 1.3 is induced through IPTG and is not induced
Thallus ultrasonic disruption precipitating, according to the Escherichia coli Rosetta/ for the progress IPTG induction that step 1.2 and 1.3 obtain
psCA132a (+), Escherichia coli Rosetta/psCA232a (+), Escherichia coli Rosetta/psCA332a (+) and Escherichia coli
Rosetta/psCA4The ultrasonic disruption product centrifugation of 32a (+) thallus and supernatant, it is separately sampled at two pieces identical poly- third
Acrylamide gel SDS-PAGE electrophoresis, is concentrated glue 5%, separation gel 14%, and glue 80V, separation gel 120V is concentrated in electrophoretic voltage.Electrophoresis
After take one of gel coomassie brilliant blue staining to decolourize, see that Fig. 4, gel shows the Escherichia coli only through IPTG induction
Rosetta/psCA132a (+), Escherichia coli Rosetta/psCA232a (+), Escherichia coli Rosetta/psCA332a (+) and
Escherichia coli Rosetta/psCA4There is the appearance of 73kD purpose band in the ultrasonic disruption product centrifugation of 32a (+) thallus；Separately
One clotting glue is detected for Western-Blot, and filter paper, gel and nitrocellulose filter are placed on half-dried transferring film instrument in order
On, 50mA transferring film 1 hour.Nitrocellulose filter is removed after the completion of transferring film, standing is dried.Nitrocellulose filter uses after drying
The TBS-T solution room temperature of 5% alipoidic milk power is closed 1 hour, is hybridizing 2 using containing rabbit-anti salmon calcitonin antibody solution room temperature
Hour, hybridized 2 hours after washing film using goat anti-rabbit antibody secondary antibody room temperature, uses NBT-BCIP (the chloro- 3- Yin of the bromo- 4- of 5- after washing film
Diindyl phosphoric acid/nitroblue tetrazolium) Color Appearance System is protected from light colour developing, WB is analyzed as a result, seeing Fig. 5.The large intestine that WB is induced through IPTG as the result is shown
Bacillus Rosetta/psCA132a (+), Escherichia coli Rosetta/psCA232a (+), Escherichia coli Rosetta/psCA332a
(+) and Escherichia coli Rosetta/psCA4There is item in the corresponding swimming lane of 32a (+) thallus ultrasonic disruption product centrifugation
Band, size are slightly larger than 70kD, and the analysis from SDS-PAGE is it is found that the Escherichia coli Rosetta/psCA induced through IPTG132a
(+), Escherichia coli Rosetta/psCA232a (+), Escherichia coli Rosetta/psCA332a (+) and Escherichia coli Rosetta/
psCA4The centrifugation of 32a (+) thallus ultrasonic disruption product is only broken with empty vector control bacterium and induction at 73.51kD
Supernatant is had any different band, illustrates the fusion protein expressed for the purpose of the band occurred on nitrocellulose filter in WB experiment.
The affinitive layer purification of 1.5 inclusion bodys
Using the phosphate buffer (20mM/L sodium phosphate, 0.5M/L NaCl, 30mM/L imidazoles) of pH7.4 from Escherichia coli
Middle separation, washing inclusion body remove impurity protein, then contain the phosphate buffer dissolution inclusion body of 8M urea with pH7.4, and 16 DEG C
Shaking table shakes 1h to solution clear.Solution is transferred to 4 DEG C of centrifuge tube, 5000rpm centrifugation 10min, supernatant is collected, used
0.22um membrane filtration removes impurity, spare.GE company protein purification prepacked column HisTrapTMHP is connected to AKTA Prime
On plus purification system, sterile water (0.22um membrane filtration) rinse-system and prepacked column are first used, equilibration buffer is used
(pH7.4, sodium phosphate 50mM/L, NaCl0.5M/L, imidazoles 30mM/L and urea 8M/L) balances pillar, flat to ultraviolet monitoring curve
After weighing apparatus is stablized, the ready sample containing 8M urea sample-loading buffer of preceding step is with 0.5ml/min loading.After completion of the sample
Unadsorbed sample is washed away with the equilibration buffer containing 8M urea, flow velocity 1-2ml/min, flat spreader is sub again.It is washed after balance
De- buffer (pH7.4, sodium phosphate 50mM/L, NaCl0.5M/L, imidazoles 500mM/L) elution, is washed away with elution buffer
The sample of absorption, flow velocity 1-2ml/min, 2ml/ pipe collect eluting peak, the entire affinity chromatography process ultraviolet real-time prison of 280nm
It surveys, the results showed that when being observed that Escherichia coli Rosetta/pET32a (+) bacterial cell disruption liquid elutes from ultraviolet monitoring curve
There is no apparent eluting peaks to occur, and Escherichia coli Rosetta/psCA132a (+), Escherichia coli Rosetta/psCA232a
(+), Escherichia coli Rosetta/psCA332a (+) and Escherichia coli Rosetta/psCA4When 32a (+) bacterial cell disruption solution elutes
As the increase of eluent injection rate starts apparent eluting peak occur when close to 100%, start to occur using at eluting peak
The centrifuge tube of 2.0ml starts to collect elution solution, obtains the protein solution containing FP1, the protein solution containing FP2, contains FP3
Protein solution and protein solution containing FP4.
The fusion protein desalination of 1.6 purifying and detection and analysis
The protein solution containing FP1, the albumen containing FP2 of the collection of AKTA Prime plus system purification step 1.5 are molten
Liquid, the protein solution containing FP3, the protein solution containing FP4, are transferred in the 10kD super filter tube of 20ml, respectively as centrifugation
4 DEG C on machine, 5000rpm be centrifuged 20-30min.It is removed when solution remains 2-3ml in filter core in the container of super filter tube top, outwells filter
Under waste liquid, be centrifuged, so repeat 3-5 times, so that albumen is molten again after the distilled water trim of filtering is added in the filter of upper layer
Salt ionic concentration in liquid, which is preferably minimized, avoids influencing follow-up test reaction.Solution after desalination collects protein solution 2- respectively
3ml is transferred in the sterile centrifuge tube of 2.0ml, is placed in freezing and is drained in machine freezing and drain, the protein powder of freeze-drying can store
It is spare in -80 DEG C of refrigerators, respectively obtain fusion protein F P1, FP2, FP3 and FP4 of purifying.4 kinds of fusion protein powder difference
Small part is taken to be re-dissolved with distilled water, separately sampled liquid and 5X bromophenol blue buffer mix, and 100 DEG C are boiled 8-10min, centrifugation
It is spare afterwards.Two pieces of identical sds pages are configured, aforementioned ready solution draws supernatant point sample electrophoresis respectively,
Loading sequence deposition condition is identical.One of gel coomassie brilliant blue staining analysis (Fig. 6), another clotting glue after electrophoresis
It is analyzed for Western-Blot, method is same as above the half-dried transferring film method of Western Blot, is protected from light colour developing post analysis experimental result, sees
Fig. 7.Comprehensive analysis SDS-PAGE and Western-Blot is it is found that salt ionic concentration is very low to electricity in albumen after super filter tube desalination
The swimming noiseless phenomenon of result, and purified albumen only has an obvious band, illustrates that His-Tag label uses ni-sepharose purification
Effect is ideal；It can be found that only have an apparent band to occur on cellulose membrane by Western-Blot testing result,
Illustrate that antibody screen selects destination protein, both the expressed albumen purified was purpose albumen, can carry out follow-up test.
1.7 fusion protein formic acid cutting analysis
Following experiments are all provided with to be repeated three times.
1.7.1 formic acid is cut
The fusion protein powder of these four purifying of fusion protein F P1, FP2, FP3 and the FP4 for taking step 1.6 to purify respectively,
It is put into centrifuge tube and is separately added into 1ml distilled water soluble protein, 88% (percent by volume) is added in protein solution
Solution is shaken and is mixed, is placed in 50 DEG C of water-baths, in above-mentioned condition to final concentration of 50% (percent by volume) by formic acid solution
12h is cut in lower heat preservation.The 2.0ml centrifuge tube containing solution is respectively placed in quick-frozen in liquid nitrogen, unlatching low temperature freeze-drying after completing cutting
Protein solution freezing after cutting is drained to albumen completion and becomes powder by machine.The protein powder for completing freeze-drying is used on a small quantity respectively
Distilled water re-dissolves, and four kinds of fusion proteins and its cleaved products are separately added into bromophenol blue buffer and boil, and then carries out SDS-
PAGE (concentration glue 5%, separation gel 14%), observes cutting result (Fig. 8) after coomassie brilliant blue staining, decoloration, analysis is respectively melted
The cutting effect of hop protein.By SDS-PAGE result it is found that containing 4 kinds of fusion eggs of different Asp-Pro formic acid cleavage site peptides
It is white under the conditions of 50% formic acid, 50 DEG C by 12 hours cutting after, the fusion protein of the cleavage site Peptide C of formic acid containing Asp-Pro A1
FP1 has obvious protein band to occur at 50kD and 24kD, and band is relatively weak, and still has band appearance at 73kD；Contain
The fusion protein of the fusion protein F P2 and the cleavage site Peptide C of formic acid containing Asp-Pro A3 of Asp-Pro formic acid cleavage site Peptide C A2
FP3 has obvious protein band to occur at 50kD and 24kD, and band is most obvious, and occurs at 73kD without obvious band；But
Only there is an obvious band after above-mentioned condition is handled at 73kD in fusion protein F P4.The above results illustrate in 50% first
Acid cut lower FP2 and FP3 by 12 hours under the conditions of 50 DEG C and can be cut completely, the Asp-Pro formic acid in two kinds of fusion proteins
Cleavage site peptide cutting efficiency with higher, the cutting efficiency of formic acid cleavage site is lower in FP1, the formic acid cleavage of FP4
Point cutting efficiency is worst, least sensitive to formic acid.
1.7.2 formic acid concn gradient is cut
The fusion protein powder of these four purifying of fusion protein F P1, FP2, FP3 and the FP4 for taking step 1.6 to purify respectively,
It is separately added into distilled water dissolution, obtains the fusion protein solution that fusion protein concentration is 11.2mg/ml, respectively takes a small amount of fusion egg
White solution is separately added into formic acid and configures different gradients in 2.0ml centrifuge tube, and formic acid final concentration is respectively as follows: 25% (volume hundred
Point ratio), 30% (percent by volume), 35% (percent by volume), 40% (percent by volume), 45% (percent by volume),
50% (percent by volume), 55% (percent by volume), solution reaction system react 12h in 50 DEG C of water-baths.All fusions
After Protein cleavage 12h respectively using liquid nitrogen carry out it is quick-frozen, it is quick-frozen after centrifuge tube using narrow meshed film wrap up centrifugation nozzle set
It is drained in machine in cryogenic freezing, after freeze-dried white powder, is separately added into that a small amount of water re-dissolves and to carry out SDS-PAGE (dense
Contracting glue 5%, separation gel 14%) analysis, Band Leader3.0 software gray scale scanning quantitative analysis of protein is utilized after dyeing, decoloration
Content, gray scale scanning value is averaged after repeating three times calculates cutting rate (the sum of 50kD and 24kD band gray value cut/be somebody's turn to do
The total gray value of swimming lane) make curve, see Fig. 9.By tracing analysis it is found that 4 kinds of fusion protein maximum cutting rates are 86% or so,
But required formic acid concn is different, and formic acid concn needed for fusion protein F P3 completes maximum cutting rate is minimum, and about 40%,
Cutting rate is without significant change after acidity increase；Acidity ratio FP3 needed for FP1 completes maximum cutting rate is slightly larger, and about 41%；FP2
With acidity highest needed for FP4 more than 50%, thereby determine that formic acid cleavage site is most sensitive to formic acid in FP1 and FP3, it is complete
It is lower at formic acid concn needed for maximum cutting rate, and formic acid concn highest needed for FP2 and FP4, it is low to the susceptibility of formic acid,
Screening minimum formic acid cutting concentration with this is 40% needed for FP3.
1.7.3 formic acid cutting temperature screens
The fusion protein powder of these four purifying of fusion protein F P1, FP2, FP3 and the FP4 for taking step 1.6 to purify respectively,
It is separately added into distilled water dissolution, is separately added into formic acid, making fusion protein concentration is 11.2mg/ml, and the concentration of formic acid is
40% (percent by volume), respectively obtains four kinds of formic acid cleavage reaction liquid, and configured four kinds of formic acid cleavage reaction liquid moves respectively
Enter in 2.0ml centrifuge tube, four kinds of cutting system temperatures are respectively 30 DEG C, 35 DEG C, 37 DEG C, 40 DEG C, 42 DEG C, 45 DEG C, 47 DEG C, 50
℃.Centrifuge tube liquid nitrogen flash freezer is taken out after cutting 10h, freezing is then moved into and drains in machine and be lyophilized, to albumen at powdered, takes out weight
It newly dissolves and carries out SDS-PAGE (concentration glue 5%, separation gel 14%), carry out gray scale scanning quantitative analysis egg after dyeing, decoloration
Bai Hanliang, gray scale scanning value be averaged after repeating three times calculate cutting rate (the sum of 50kD and 24kD band gray value cut/
The total gray value of the swimming lane) make curve, see Figure 10.Known to quantitative result when formic acid concn is 40%, four kinds of fusion proteins
As the increase of reaction temperature can be close to or up to maximum cutting efficiency, but the maximum cutting rate completed is different, speed and institute
Minimum temperature is needed also to be not quite similar.FP4 is minimum about in 50 DEG C of maximum temperature maximum cutting rates achieved of experiment condition
66%；FP2 equally reaches maximum cutting rate, about 78% at 50 DEG C, and has the space further increased with the former；FP1
All reach maximum cutting rate, about 85% when being lower than 50 DEG C with FP3, is differed with cutting rate maximum under conventional high formic acid concn
It is very few, but temperature required for FP3 will be significantly lower than FP1, and maximum cutting can be completed at 43 degrees Celsius, thus filter out anti-
The minimum cutting temperature answered.Hence, it can be determined that the Asp-Pro formic acid cleavage site peptide in FP3, is completed needed for maximum cutting rate
The formic acid concn wanted it is minimum 40% when required minimum temperature be 43 DEG C.
1.7.4FP3 best formic acid clipping time screening
The fusion protein F P3 powder for taking step 1.6 to purify is separately added into distilled water dissolution, is separately added into formic acid mixing, makes
FP3 concentration is 11.2mg/ml, and the concentration of formic acid is 40% (percent by volume), obtains FP3 formic acid cleavage reaction liquid, configures
FP3 formic acid cleavage reaction liquid move into 2.0ml centrifuge tube in, be placed in 43 DEG C of water-baths, respectively 0h, 0.5h, 1.0h, 1.5h,
2.0h, 2.5h, 3.0h, 3.5h sampling liquid, which move into freeze after liquid nitrogen flash freezer in 2.0ml centrifuge tube, to be drained.The protein sample powder of freeze-drying
End is dissolved with sterile water respectively, is carried out SDS-PAGE (concentration glue 5%, separation gel 14%) electrophoresis, through coomassie brilliant blue staining, is taken off
Gel result after color is shown in that Figure 11, gel carry out gray scale scanning quantitative analysis of protein content, and gray scale scanning value takes after repeating three times
Mean value calculation cutting rate (the sum of 50kD and 24kD band gray value the cut/total gray value of the swimming lane) makees curve, sees figure
12.The result shows that existing three protein bands of FP3 cleaved products occur, and FP3 band color is as time increases when 0.5h
And weaken, existed without obvious band 73kD at when arriving 3h, only two apparent protein bands, size be respectively 50kD with
24kD illustrates that fusion protein has been fully completed cutting at this time.Gray scale scanning curve is more exact to be shown in 3.0h and 3.5h
When cutting rate be respectively 86% and 85.7%, and the very close maximum cutting rate of the cutting rate in 2.5h is
85.3%, cutting efficiency has no significant change as time increases, in the case where considering economic cost, will complete maximum
The time of cutting rate is set to 2.5h.Therefore, the highest Asp-Pro formic acid cleavage site peptide of the cutting efficiency filtered out is amino acid
The best formic acid cutting condition of the sequence CA3 as shown in SEQ ID No.2, CA3 are that formic acid (cutting agent) concentration is 40% (volume
Percentage), cutting temperature is 43 DEG C, clipping time 2.5h.
Embodiment 2 cultivates production salmon calcitonin transgene rape using Asp-Pro formic acid cleavage site Peptide C A3
The building of 2.1 fusion protein G-sCT-SO expression vectors
Salmon calcitonin is connected with the film outskirt of glycoprotein by Asp-Pro formic acid cleavage site Peptide C A3 and is merged
Protein G-sCT connects sesame in one end of fusion protein G-sCT to express that fusion protein G-sCT in the oil body of rape
Oil body protein obtains fusion protein G-sCT-SO.The amino acid sequence of fusion protein G-sCT-SO is SEQ ID No.4, SEQ ID
In No.4, the 8-150 sequences for sesame oil body protein, the outer region sequence of the 153-208 films for glycoprotein, 209-
214 sequences for Asp-Pro formic acid cleavage site Peptide C A3, the 215-247 sequences for salmon calcitonin.
Fusion protein G-sCT-SO expression casette shown in artificial synthesized SEQ ID No.5, SEQ ID No.5 is salmon
Calcitonin fusion protein G-sCT-SO expression casette sequence, is made of, coded sequence is SEQ ID 2163 nucleotide
956-1696 of No.5 encode salmon calcitonin fusion protein G-sCT-SO shown in SEQ ID No.4.SEQ ID
In No.5,1-9 are III site Hind and protection base, and 10-949 are rape oil body protein promoter (rape
Oleosin promoter) sequence, 977-1405 be sesame oil body protein sequence, 1412-1579 for sugar egg
The sequence of white film outskirt, the 1580-1597 sequences for Asp-Pro formic acid cleavage site Peptide C A3,1598-1696
For the sequence of salmon calcitonin, the 1715-2154 sequences for terminator.
Fusion protein G-sCT-SO expression casette shown in double digestion SEQ ID No.5 is distinguished with EcoR I and Hind III
With carrier pCAMBIA3301, digestion products are attached, obtaining will be between III site the EcoR I of pCAMBIA3301 and Hind
Sequence replace with SEQ ID No.5 4-2160 shown in the DNA fragmentation other sequences of pCAMBIA3301 (keep constant)
Recombinant vector, which is named as pBOGSC3301 (Figure 13).PBOGSC3301 is expressed shown in SEQ ID No.4
Fusion protein G-sCT-SO.
The acquisition of 2.2 turns of fusion protein G-sCT-SO vector for transgenic rape
After pBOGSC3301 electrization is converted agrobacterium tumefaciens lba4404, the recombination root containing pBOGSC3301 is obtained
Cancer Agrobacterium, is named as LBA4404/pBOGSC3301.
(1) the activation of LBA4404/pBOGSC3301
1. LBA4404/pBOGSC3301 is inoculated into (Kan100mg/L+Rif50mg/ in 5ml YEB fluid nutrient medium
L), 200rpm28 DEG C of overnight incubation.
2. taking 1ml bacterium solution, it is inoculated into 50ml YEB fluid nutrient medium in (Kan100mg/L+Rif50mg/L),
200rpm28 DEG C of shaken cultivation is 0.3-0.4 to OD600, and 5000rpm is centrifuged 5min.
3. thallus is washed once with M1 (MS0+6BA1.0mg/L) fluid nutrient medium, with the training of M2 (MS0+100mg/L AS) liquid
It is outstanding to support base weight, makes OD600 0.15-0.25, for infecting.
(2) the genetic transformation of rape
1. taking double No. 4 seeds in appropriate cabbage type rape, sterilize (75% ethyl alcohol 2min, 20% sodium hypochlorite 10min), it will
Seed is seeded in MS after drying0On culture medium, illumination cultivation.
2. the cotyledon of double No. 4 5d in clip cabbage type rape, in pre-culture medium YP (MS0+NAA0.2mg/L+6BA2.0mg/
L 3d is cultivated on).
3. infecting 10min with OD600 for the LBA4404/pBOGSC3301 bacterium solution of 0.15-0.25, after infecting, cotyledon is turned
It moves to and co-cultures base GP (MS0+ NAA1.0mg/L+6BA4.0mg/L) on dark culture 3d.
4. washing cotyledon 2-3 times with M3 (MS0+CB500mg/L) after co-culturing, after drying, it is transferred into bud differentiation
It is cultivated on culture medium SP (MS0+6BA2mg/L+NAA0.2mg/L+Basta6mg/L+CB500mg/L).
5. culture 4 weeks or so, when the budlet wait differentiate is about 1cm, moves it to root media SG (MS0+
Basta6mg/L+CB100mg/L it on), takes root within 2 weeks or so, obtains T0 for the Basta positive and turn pBOGSC3301 Brassica campestris L seedling.
The genomic DNA that 9 plants of T0 turn pBOGSC3301 rapeseed plants for the Basta positive is extracted, with primer sCT5
(CTCTTCGATCTCTCTCA), sCT3 (TCCTGGAGTTCCAGA) carries out PCR amplification detection, PCR program are as follows: 95 DEG C of 5min；
94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, totally 32 recycle；72℃10min.The size of electrophoresis detection PCR product, as a result as schemed
Shown in 14,9 plants of T0 turn to expand in the genomic DNA of pBOGSC3301 rapeseed plants for the Basta positive obtains the PCR of 780bp
Product illustrates that fusion protein G-sCT-SO gene has been transferred in rape, which turns pBOGSC3301 rape for the Basta positive
To turn fusion protein G-sCT-SO vector for transgenic rape.In harvest T0 generation, turns the kind that fusion protein G-sCT-SO vector for transgenic rape plant is tied
Son, obtain T1 generation turn fusion protein G-sCT-SO vector for transgenic rape seed.
Wherein, 5. middle Basta resistance screening concentration determines step as follows: taking double 4 in appropriate cabbage type rape
Number seed sterilizes (75% ethyl alcohol 2min, 20% sodium hypochlorite 10min), is seeded in MS after seed is dried0On culture medium, light
According to culture, the cotyledon of clip culture 5d, in pre-culture medium YP (MS0+ NAA0.2mg/L+6BA2.0mg/L) on cultivate 3d.It will be pre-
Cotyledon after culture is put on the Basta resistance screening culture medium of various concentration, and Basta concentration is respectively 10mg/ in culture medium
L, 8mg/L, 6mg/L, 4mg/L, 0mg/L, each concentration repeat 3 culture dishes, and cotyledon number is 13 in each culture dish, i.e., often
Totally 39 plants of a concentration, culture is observed after 3 weeks.The Basta of experimental result discovery 6mg/L can kill whole non-transgenics just
Rape can be used as the concentration when Basta resistance screening of rape genetic transformation.
In T1 generation, is turned into fusion protein G-sCT-SO vector for transgenic rape seed in field seeding, it is dilute with Basta in the rape 4-5 leaf phase
Liquid (PPT concentration is 68mg/L) sprinkling rape is released to be screened, it is each primary every sprinkling in 3 days, it sprays 3 times, observes after a week altogether
Experimental result.Experimental result such as Figure 15.Experiment is sprayed by Basta it is found that T1 generation turns fusion protein G-sCT-SO vector for transgenic rape
Blade thickens, energy normal growth, and non-transgenic wild type rape, i.e., double No. 4 blades in the cabbage type rape of non-transgenosis
Bleach death.The experimental results showed that T1 generation turns in fusion protein G-sCT-SO vector for transgenic rape, fusion protein G-sCT-SO gene is
Through being integrated into the genome of rape, and transcriptional expression.
The biological activity of salmon calcitonin in 2.3 turns of fusion protein G-sCT-SO vector for transgenic rape seeds
The oil body and wild type rape for turning fusion protein G-sCT-SO vector for transgenic rape seed of extraction step 2.2 respectively, i.e., not
The oil body of double No. 4 vegetable seed in the cabbage type rape of transgenosis, is dissolved with the formic acid solution of 40% (percent by volume), is put at 43 DEG C
It sets 2.5h and completes formic acid cutting, 15000rpm, is centrifuged 30min, removes upper layer oil body, remove clear liquid, freezing is drained by 4 DEG C.It is lower clear
Liquid SDA-PAGE gel electrophoresis result shows the oil body for turning to extract in fusion protein G-sCT-SO vector for transgenic rape seed, cuts through formic acid
After cutting, the differential protein band of an about 3.45kD can release, do not find this band (Figure 16) in wild type rape seed.
After lower clear liquid freezing after formic acid is cut is drained, is re-dissolved with redistilled water, cross 0.22um filter membrane.It utilizes
HiTrapTM1 × 5ml of SPFF prepacked column (internal diameter × height of bed 16/25mm；Column volume: 5ml；Average grain: 90UM) it is purified,
Elution carries out linear gradient elution with 0-1M NaCl solution (solvent is 20mM aqueous citric acid solution, pH3.0), totally 20 cylinders
Product, NaCl concentration pace of change are at the uniform velocity.The eluting peak eluted with 350mM NaCl solution is collected, purifying is obtained containing rape table
The solution of the salmon calcitonin reached, SDS-PAGE detection show that the peak is the eluting peak (figure of target product salmon calcitonin
17).Salmon calcitonin standard in Figure 17 is the chemically synthesized product in BeiJing HuaDa protein Research Center Co., Ltd.
It drains the solution low temperature freeze-drying instrument freezing for the salmon calcitonin containing rape expression that purifying obtains to obtain oil
The salmon calcitonin powder of dish expression, 0.9% physiology salt of salmon calcitonin powder for the rape expression that above-mentioned purifying is obtained
Water re-dissolves, and obtains the salmon calcitonin injection of rape expression, is equivalent to the calcitonin of an international unit (IU)
The standard calcitonin active ingredient of 0.0008mg is 1IU, utilizes the biological activity of mouse tail vein injection method test sample.It takes
The SPF/VAF grade mouse [strain CD-1 (ICR)] mouse 30 of 20 ± 2g of weight, random point 3 groups, i.e. physiological saline group, close
The salmon calcitonin group of lid breath group and rape expression, every group 10, distilled water 19-21h, every mouse injection are given in fasting before injecting
100ul.It is negative control, commercially available Calcitonin Salmon injection (salmon calcitonin injection) for positive control using 0.9% physiological saline.
Every mouse tail vein injection 100ul0.9% physiological saline of physiological saline group；Every mouse tail vein injection of Calcitonin Salmon group
The commercially available Calcitonin Salmon injection of 100ul, injection dosage are that every mouse injects 5IU；Rape is expressed salmon calcitonin group every small
The salmon calcitonin injection of tail vein injection 100ul rape expression, injection dosage are that every mouse injects 5IU.After injection
1h is injected intraperitoneally with 10% chloraldurate, and eye is plucked after mouse anesthesia and takes blood, and blood plasma stands 2 hours in 4 DEG C, 3000 × g centrifugation
15min takes serum spare.Utilize blood calcium in o-cresolphthalein complex copper colorimetric determination mouse blood.The experimental results showed that
The blood calcium of physiological saline group mouse does not change, and compared with physiological saline group, the blood calcium of Calcitonin Salmon group mouse has dropped 20%, and
The blood calcium of the salmon calcitonin group mouse of rape expression has dropped 23% (table 2 and table 3), the salmon that this explanation is expressed using rape
The salmon calcitonin that calcitonin fusion protein G-sCT-SO is obtained after being cut by formic acid has the biology for reducing blood calcium concentration
Activity.
Blood calcium concentration (mmol/L) after 2, three groups of mouse injections of table
Claims (9)
1. amino acid sequence peptide as shown in SEQ ID No.2.
2. a kind of fusion protein, it is characterised in that: the fusion protein is calcitonin fusion protein G-sCT-SO, the calcitonin
Fusion protein G-sCT-SO is that glycoprotein extracellular domain and calcitonin are connected to the fusion protein G-sCT to be formed and sesame oil body protein
Connect the protein formed；The fusion protein G-sCT is by formic acid cleavage site peptide by the glycoprotein extracellular domain and described
Calcitonin connects the fusion protein to be formed；The amino acid sequence of the formic acid cleavage site peptide is SEQ ID No.2.
3. fusion protein according to claim 2, it is characterised in that: the amino of the calcitonin fusion protein G-sCT-SO
Acid sequence is SEQ ID No.4.
4. biomaterial relevant to peptide described in claim 1 or biology relevant with fusion protein described in Claims 2 or 3
Material:
Any one of biomaterial relevant to peptide described in claim 1 is B1)-B5):
B1 the nucleic acid molecules of peptide described in claim 1) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules；
B4) contain B1) recombinant microorganisms of the nucleic acid molecules；
B5) contain B1) the transgenetic animal cell systems of the nucleic acid molecules；
Any one of biomaterial relevant to fusion protein described in Claims 2 or 3 is C1)-C5):
C1 the nucleic acid molecules of fusion protein described in Claims 2 or 3) are encoded；
C2) contain C1) expression cassettes of the nucleic acid molecules；
C3) contain C1) recombinant vectors of the nucleic acid molecules；
C4) contain C1) recombinant microorganisms of the nucleic acid molecules；
C5) contain C1) the transgenetic animal cell systems of the nucleic acid molecules.
5. the method for preparing fusion protein described in Claims 2 or 3, including by the volume of fusion protein described in claim 2 or 3
Code gene is expressed the step of obtaining fusion protein described in Claims 2 or 3 in biological cell；The biological cell is micro-
Biological cell, plant cell or non-human animal cell.
6. the method that the fusion protein as described in Claims 2 or 3 prepares destination protein, including merged described in Claims 2 or 3
Albumen carries out formic acid and cuts to obtain the destination protein；The formic acid cutting condition is that the concentration of formic acid is 40%(volume basis
Than), cutting temperature is 43 DEG C, and clipping time is 2.5 h.
7. a kind of method of the cultivation containing calcitonin fusion protein transgene rape, the method includes importing into receptor rape
The encoding gene of calcitonin fusion protein G-sCT-SO, obtains the transgene rape containing calcitonin fusion protein；
The calcitonin fusion protein G-sCT-SO is that glycoprotein extracellular domain is connected to the fusion protein G-sCT to be formed with calcitonin
The protein to be formed is connect with sesame oil body protein；The fusion protein G-sCT is by formic acid cleavage site peptide by the sugared egg
Tunica albuginea outskirt connects the fusion protein to be formed with the calcitonin；The amino acid sequence of the formic acid cleavage site peptide is SEQ ID
No.2。
8. according to the method described in claim 7, it is characterized by: the amino acid sequence of the calcitonin fusion protein G-sCT-SO
Column are SEQ ID No.4；The amino acid sequence of the calcitonin is 215-247 of SEQ ID No.4.
9. the method for preparing calcitonin, including containing calcitonin fusion protein transgenic rape from cultivation described in claim 7 or 8
The transgene rape containing calcitonin fusion protein that the method for dish obtains separates the oil body of the transgene rape, to the oil
Body carries out formic acid and cuts to obtain the calcitonin；The formic acid cutting condition is that the concentration of formic acid is 40%(percent by volume), it cuts
Cutting temperature is 43 DEG C, and clipping time is 2.5 h.
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Non-Patent Citations (7)
| Title |
|---|
| 15.5 kDa oleosin [Sesamum indicum];GenBank: AAB58402.1;《GenBank: AAB58402.1》;20011030;全文 * |
| Production and isotope labeling of antimicrobial peptides in Escherichia coli by means of a novel fusion partner that enables high-yield insoluble expression and fast purification;Verica Vidovic等;《J. Pept. Sci.》;20090202;第15卷;278-284 * |
| salm-calcotinin [synthetic construct];GenBank: CAA00272.1;《GenBank: CAA00272.1》;19930407;全文 * |
| sCT纯化方法的建立和油体表达体系的优化;李梅;《中国优秀硕士学位论文全文数据库 基础科学辑》;20091015(第9期);A006-110 * |
| Sequence 29 from patent US 7910718;GenBank: AED75735.1;《GenBank: AED75735.1》;20110430;全文 * |
| Verica Vidovic等.Production and isotope labeling of antimicrobial peptides in Escherichia coli by means of a novel fusion partner that enables high-yield insoluble expression and fast purification.《J. Pept. Sci.》.2009,第15卷278-284. * |
| 植物油体表达体系的建立及Profilin2维管束特异表达启动子的区段缺失分析;刘昱辉;《中国优秀博硕士学位论文全文数据库 (博士) 基础科学辑》;20020615(第1期);A006-51 * |
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