CN1100789C - Preparation process of gene recomibination calcitonin or calcitonin analog - Google Patents
Preparation process of gene recomibination calcitonin or calcitonin analog Download PDFInfo
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Abstract
The present invention relates to the technology for preparing gene recombination calcitonin or an analog of the calcitonin. A precursor gene segment of the calcitonin or the analog of the calcitonin is firstly chemically synthesized, a full-length gene is spliced, and the C terminal is lactamine, leucine or other amino acid codes which are hydrolyzed by carboxypeptidase-Y so that an expression product is in C terminal modification by the carboxypeptidase-Y; when modification is carried out, neighbouring nitrobenzene glycinamide is added for replacing a nucleophilic reagent so that the thirty-three position of a precursor of the calcitonin or the analog of the calcitonin is replaced by the neighbouring nitrobenzene glycinamide and then becomes a product of light sensitiveness. Linear calcitonin or the analog of the calcitonin is obtained by photolysis, and a product corresponding to the activity of natural calcitonin is obtained by renaturation.
Description
The present invention relates to medical technical field, is the preparation method of a kind of gene recombination thyrocalcitonin or calcitonin-like.
Thyrocalcitonin is a kind of endogenic calcium-regulating hormone that contains 32 amino-acid residues.Mainly act on bone.Now separated obtaining thyrocalcitonin (people, mouse, ox, pig, chicken, salmon, eel, the frog) from multiple animal, and chemosynthesis wherein 5 kinds or calcitonin-like.The clinical analogue that uses as salmon calcitonin see calcimar, human calcitonin, pig thyrocalcitonin and eel thyrocalcitonin.Existing thyrocalcitonin clinical indication mainly comprises: hypercalcemia, Paget's disease, osteoporosis, poisoning by vitamin D disease, bone pain, acute pancreatitis.
Because the practical value of thyrocalcitonin, many companies in the whole world and producer are are researching and producing the novel method or the novel process of thyrocalcitonin.At present, the commodity on the market are chemosynthesis, because its complex process requires harshness to hardware device, synthetic agent, production environment, make that the fixedly input of product cost costliness, large-scale production is huge, and easy contaminate environment.The particularly development of genetic engineering technique of 1980's biotechnology is for biosynthetic polypeptide, protein have been established solid basis.M.Yabuta (Appl Microbiol Bictechnol42:703-708) and Martha V.L.Ray (Bio/Technology, 1993,11:64-70) waiting by genetically engineered production thyrocalcitonin all is to obtain carboxyl terminal (C-terminal) by escherichia coli expression earlier to be the thyrocalcitonin precursor of glycine (Gly), (effect of α-AE) is processed as ripe reorganization thyrocalcitonin through α-amidating enzyme again.This is because the expression system of intestinal bacteria protokaryon does not possess the amidation modification of translation back, therefore the C-terminal of gained is the thyrocalcitonin precursor of being made up of 33 amino acid of Gly, must could modify C-terminal with α-AE and produce ripe thyrocalcitonin, but problem is α-AE not commercialization as yet, obtain α-AE complex process by biological extraction or genetically engineered, not only cause the thyrocalcitonin production cycle to prolong, improved cost again.Also there is research to realize that by the cell that use has the acetylize function C-terminal is amidated; (Protein Expression and Purification such as Silvia Merli for example; 1996; 7:347-354) with the human calcitonin gene clone on carrier for expression of eukaryon pRC/RSV; transfection has the mouse pituicyte (AtT-20) of amidation; through the G-418 resistance screening, obtain secreting the cell strain of ripe recombinant human calcitonin.But because productive rate is extremely low, so this amidation circuit can only stay in laboratory stage so far.Therefore, to prepare the key of thyrocalcitonin be the C-terminal amidation to genetically engineered.
The objective of the invention is to reform the preparation technology of said gene engineering, provide that a kind of technology is simple, with low cost, the production thyrocalcitonin that do not cause environmental pollution or the novel method of calcitonin-like.
The present invention utilizes genetically engineered to prepare thyrocalcitonin or calcitonin-like is mainly being done vital improvement aspect the C-terminal amidation:
1. making thyrocalcitonin that bacillus coli gene expresses or calcitonin-like precursor C-terminal is that leucine (Leu) or L-Ala (Ala) or other can be by the amino acid of carboxypeptidase-Y hydrolysis, so that use commercialization and cheap carboxypeptidase-Y to carry out the C-terminal amidation.
2. when adopting carboxypeptidase-Y to modify thyrocalcitonin or calcitonin-like precursor, add ortho-nitrophenyl G-NH2 (O-PNGA), the 33rd amino acids of thyrocalcitonin or calcitonin-like precursor is replaced by O-PNGA, become the photaesthesia intermediate, by photolysis, just obtaining C-terminal is NH again
2Linear peptide.This measure has improved amidation efficient, has reduced amidated cost.
The present invention utilizes genetically engineered to produce reorganization thyrocalcitonin or calcitonin-like, and key step comprises as shown in Figure 1:
1. synthesis of calcitonin or calcitonin-like precursor-gene fragment
For improving thyrocalcitonin or the expression amount of calcitonin-like in intestinal bacteria, aminoacid sequence according to thyrocalcitonin or calcitonin-like precursor, with colibacillary preference password, be encoded into some gene fragments, make be positioned at full-length gene 3 ' end can be for L-Ala (Ala) or leucine (Leu) or other by the amino acid whose codon of carboxypeptidase-Y hydrolysis, so that make the product that is gone out by escherichia coli expression, available carboxypeptidase-Y carries out the C-terminal amidation.Gene fragment adopts phosphorous acid diester method synthetic by ABI 391 dna synthesizers.
2. make up the engineering bacteria of the cloning vector that contains thyrocalcitonin or calcitonin-like precursor-gene
The synthetic gene fragment is used routinely " BASIC METHODS IN MOLECULARBIOLOGY " method be stitched together.At first with the fragment phosphorylation, behind the reheat to 96 ℃, be annealed to room temperature, be spliced into total length thyrocalcitonin or calcitonin-like precursor-gene, be connected with pGEM-7z cloning vector by the effect of ligase enzyme then through BamH1, Xho1 double digestion, become pGEM-thyrocalcitonin or calcitonin-like precursor cloning vector, transformed into escherichia coli, screening contains the positive colony engineering bacteria of pGEM-thyrocalcitonin or calcitonin-like precursor-gene according to the α complementary action.
3. make up the engineering bacteria of the fusion expression vector that contains thyrocalcitonin or calcitonin-like precursor-gene
To contain single bacterium colony of pGEM7z-thyrocalcitonin or calcitonin-like precursor-gene and contain single bacterium colony of pGEX-4T-3 plasmid respectively at increasing in the LB substratum, plasmid purification, use BamH1, the Xho1 double digestion, reclaim thyrocalcitonin or calcitonin-like precursor-gene and double digestion pGEX-4T-3 carrier segments completely by agarose gel electrophoresis again, by thyrocalcitonin or calcitonin-like precursor-gene: double digestion pGEX-4T-3 carrier segments completely is that 1: 3 molar weight ratio connects, form pGEX-4T-thyrocalcitonin or calcitonin-like precursor-gene, transformed into escherichia coli obtains engineering bacteria.Its plasmid DNA behind double digestion and dna sequencing evaluation insertion sequence, is carried out the evaluation of fusion rotein abduction delivering.
4. the high density fermentation of thyrocalcitonin or calcitonin-like engineering bacteria
Engineering bacteria is inoculated in the LB substratum, 37 ℃ of overnight incubation, as first order seed, next day first order seed is inoculated in the 2-YT substratum that contains 2% glycerine, 37 ℃ of joltings are cultivated, and (see Lee SY for details as secondary seed, TIBTECH, 1996,14:98-105), then secondary seed is inoculated in the fermentor tank of fermention medium and carries out fed batch cultivation.Whole fermentation process pH is controlled at about 7.0, and dissolved oxygen is about 35%, and stirring velocity increases gradually, and 800 commentaries on classics of maximum speed per minute (see M.Yabuta for details, Y.Suzuki, K.Ohsuye.Appl Microbiol Biotechnol.1995,42:703-708).
5. separation and purification thyrocalcitonin or calcitonin-like precursor
Adopt affinity chromatography purifying from the thalline that high density fermentation obtains to obtain the fusion rotein of glutathione sulfydryl transferase-thyrocalcitonin or calcitonin-like precursor routinely, through enzyme or chemical cracking, through ion exchange chromatography, anti-phase gel desalination, obtain the precursor of highly purified thyrocalcitonin or calcitonin-like again.
6. thyrocalcitonin or calcitonin-like precursor are through external amidation, and renaturation becomes ripe thyrocalcitonin or calcitonin-like
The solution that will contain 1mM EDTA, 200mM O-PNGA adds thyrocalcitonin or calcitonin-like precursor, carboxypeptidase-Y with 5N NaOH adjust pH to 6.0, in 35 ℃ of insulations, uses RP-C after reaction is finished
18Desalination and purifying, lyophilize obtains light activated intermediate thyrocalcitonin-O-PNGA or calcitonin-like-O-PNGA.Product is dissolved in 60mM NaHSO
3In the methanol-water solution, charged into argon gas 5 minutes, the beginning photodissociation can obtain linear thyrocalcitonin or calcitonin-like, uses RP-C after reaction is finished
18High performance liquid phase (HPLC) purifying is used in desalination again.The linear peptide that purifying obtains, folding through atmospheric oxidation, the reconstruction disulfide linkage [with dimethyl sulfoxide (DMSO), halfcystine, K
3(FeCN
6) or gsh be the system of medium], obtain the thyrocalcitonin or the calcitonin-like of biologically active.
6. measure active
By " Chinese pharmacopoeia method.With body weight is that the same source of 40-50g, same strain, other health of identity are greatly from mouse, be divided into 5 groups (control group, production sample high density group, production sample low concentration group, standard substance high density group, standard substance low concentration group) at random, every group 10, after the numbering, respectively in order, the thyrocalcitonin standard substance of tail vein injection 0.4ml respective concentration, production sample and blank diluent.After the administration accurate 1 hour, blood sampling respectively was with o-cresolphthalein complexone colorimetric method for determining blood calcium.Experimental result is by " the quantitative response parallel lines calculate in the Chinese pharmacopoeia appendix calibrating statistical technique, and design method is calculated and tired and experimental error at random.And carry out the statistics certificate authenticity.
Description of drawings:
Fig. 1 operational flowchart of the present invention
The splicing fragment sequence F1-F6 of Fig. 2 human calcitonin analogue precursor-gene
Fig. 3 human calcitonin analogue precursor-gene full length sequence
The electrophoretogram of Fig. 4 high density fermentation different time expressing quantity
The electrophoretogram of Fig. 5 glutathione sulfydryl transferase-human calcitonin analogue precursor affinity purification
Fig. 6 human calcitonin analogue precursor electron spray(ES)-mass spectrum is identified collection of illustrative plates
Fig. 7 human calcitonin analogue electron spray(ES)-mass spectrum is identified collection of illustrative plates
Fig. 8 salmon calcitonin see calcimar precursor-gene full length sequence
The gene fragment order F of Fig. 9 salmon calcitonin see calcimar precursor
1'-F
8'
Figure 10 salmon calcitonin see calcimar precursor electron spray(ES)-mass spectrum is identified collection of illustrative plates
Figure 11 salmon calcitonin see calcimar electron spray(ES)-mass spectrum is identified collection of illustrative plates
Now further set forth preparation method of the present invention with accompanying drawing in conjunction with the embodiments.
The preparation of gene recombination human calcitonin analogue
One. materials and methods
1. restriction enzyme BamH1, Xho1 be available from Biolab, T
4Ligase enzyme, T
4Kinases is available from Promaga.Zymoplasm, carboxypeptidase-Y are available from Sigma.
2. bacterial classification and plasmid pGEM-7z (+) are available from Promaga, and pGEX-4T-3 is available from Pharmacia Biotechs.DH5 α, TG1 are available from Promaga.
Radio isotope α-
32P-dATP Beijing inferior brightness Biomedicines, Inc..
4. agarose, biological agar are available from Sigma, and yeast powder, peptone are available from Oxid.
5. plasmid extraction, gel reclaim test kit available from Shanghai China Shun bio-engineering corporation, T7sequencing
TMKit is available from Pharmacia Biotechs.
6.Glutathione Sepharose 4B, SP-Sepharose Fast Flow resin, G-25 are available from Pharmacia Biotech, RP-C
18Available from Merk.
7. the preparation of plasmid DNA and segmental recovery are by magnificent Shun bio-engineering corporation test kit operational manual.The human calcitonin analogue gene sequencing is pressed T
7Sequencing
TMThe Kit operational manual.Other routine operation is pressed Molecular Cloning[1982].
8. high density fermentation is with reference to [M.Yabuta, Y.Suzuki, K.Ohsuye.ApplMicrobiol Biotechnol.1995,42:703-708].
9. the evaluation of tunning SDS-PAGE method.Dyeing glue scans with BioRad DENSITOMETER.
10. the purifying of fusion rotein is undertaken by Glutathione Sepharose 4B operational manual.
11. the renaturation of thyrocalcitonin or calcitonin-like is with reference to David Andreu, FernandoAlvericio, Nuria A.Sole Mark C.Munson, Mare Ferrer; And GeorgeBarany. (1994) in Methods in Molecular Biology.Vol.35:Peptide synthesis protocols (Pennington M.W.and Dunn B.M., Eds.), pp91-169, Humana Press Inc., Totowa, NJ.
12. activity identification is by " rat blood serum calcium decline degree after the bioassay method mensuration injection thyrocalcitonin of Chinese pharmacopoeia thyrocalcitonin.
Two. concrete operations
1. make up recombinant human calcitonin analogue precursor-gene
1.1. the segmental chemosynthesis of recombinant human calcitonin analogue precursor-gene
Recombinant human calcitonin analogue precursor-gene is divided into 6 fragment F1-F6 (seeing Fig. 2 for details), adopt phosphorous acid diester method synthetic by ABI 391 dna synthesizers, separate through 45 ℃ of ammonia that spend the night, product is with 12% polyacrylamide gel electrophoresis purifying, uses after the G-25 desalination standby again.Wherein fragment F3, F6 annealing back forms the encoding sequence of human calcitonin analogue precursor C end, contains the codon CTG of Leu.
1.2. the engineering bacteria that the full-length gene of splicing recombinant human calcitonin analogue precursor and structure contain cloning vector
In the ligation pipe, add above-mentioned 6 each 20pmol of gene fragment, ligase enzyme damping fluid 3 microlitres of 10 times of concentration, T4 kinases 10 units add water 19 microlitres again, make Tris-hydrochloric acid, MgCl
2, dithiothreitol (DTT) final concentration be respectively 50mM, 10mM, 10mM, put 37 ℃ 30 minutes, 96 ℃ of 2 minutes deactivation T4 kinases were annealed to about 20 ℃ through 30 minutes again.Get 10 microlitres annealing fragment solution, add pGEM-7z carrier soln, ligase enzyme 5 units of 6 microlitre BamH1, Xho1 double digestion, add ligase enzyme damping fluid 2 microlitres again, make Tris-hydrochloric acid, MgCl
2, dithiothreitol (DTT) final concentration be respectively 50mM, 10mM, 10mM, spend the night 14 ℃ of connections.Draw this connection carrier solution 10 microlitres, add 200 microlitre TG1 intestinal bacteria solution and (contain 10 approximately
6-10
7Individual competent cell), ice bath adds 800 microlitre LB substratum after 30 minutes 42 ℃ of 90 second, and coated plate after 45 minutes is cultivated in 37 ℃ of joltings.Use the α-Hu Bu screening positive clone, the size of its insertion sequence is identified by double digestion and dna sequencing.The human calcitonin analogue full-length gene that is spliced into is 120bp, as shown in Figure 3, comprising 5 ' BamH1 site (I), 3 ' Xho1 site (II), two termination codons (III) add GAA and constitute V8 cracking site (IV) between GST and hCTa encoding sequence, be in order further to obtain and the on all four aminoacid sequence of natural human thyrocalcitonin by enzyme processing later on, modify for ease of external amidation, add Leu codon CTG (V) at human calcitonin analogue full-length gene 3 ' end.
1.3. make up the engineering bacteria that contains recombinant human calcitonin analogue gene expression vector
Respectively picking contain single bacterium colony of pGEM7z-human calcitonin analogue precursor-gene and pGEX-4T-3 plasmid single bacterium colony each in 5 milliliters of LB substratum after the amplification, utilize plasmid extraction test kit plasmid purification, respectively get 10 microlitre plasmids, at 37 ℃ of double digestions after 2 hours, reclaim human calcitonin analogue precursor-gene and double digestion pGEX-4T-3 carrier segments completely through 4 BamH1 of unit, 4 Xho1 of unit with 1.2% agarose gel electrophoresis.Get human calcitonin analogue precursor-gene solution respectively and be 1: 3 mixing by the molar weight ratio, add 4 T of unit through the pGEX-4T-3 carrier soln of BamH1, Xho1 double digestion
4Ligase enzyme adds ligase enzyme damping fluid and water again, makes Tris-hydrochloric acid, MgCl
2, dithiothreitol (DTT) final concentration be respectively 50mM, 10mM, 10mM, spending the night at 4 ℃ is connected to form pGEX-4T-human calcitonin analogue precursor-gene, this product is reorganization human calcitonin analogue expression vector.Draw this carrier soln 10 microlitres, add 200 microlitre TG1 intestinal bacteria and (contain 10 approximately
6-10
7Individual competent cell), ice bath is after 30 minutes, 42 ℃ of 90 seconds of water-bath, adds 800 microlitre LB substratum, coated plate after 37 ℃ of joltings are cultivated 45 minutes.Positive engineering bacteria plasmid DNA is carried out the evaluation of fusion rotein abduction delivering routinely behind double digestion and dna sequencing evaluation insertion sequence.
2. high density fermentation
3 microlitre OD
600=0.7 engineering bacteria is inoculated in 37 ℃ of overnight incubation of 3 milliliters of LB substratum as first order seed, next day 3 milliliters of first order seeds is inoculated in that jolting is cultured to OD in 200 milliliters of 2-YT substratum that contain 2% glycerine
600=0.7 as secondary seed, routinely secondary seed is inoculated in then and carries out fed batch cultivation in the fermentor tank that contains 3 liters of fermention mediums.Whole fermentation process pH is controlled at about 7.0 and also constantly stirs, for keeping dissolved oxygen about 35%, need constantly to increase stirring velocity, revolution changes from initial per minute 50, strengthen gradually, reaching high speed per minute 800 about 7 hours changes, and supplies pure oxygen then, to keep colibacillary oxygen requirement.Batch culture after 5 hours cell density reach 5.3OD
600, enter 10 hours feed supplement cultivation stage subsequently, nearly 10 times of thalli growth.Cultivated 12 hours for 37 ℃, the isopropylthiogalactoside (IPTG) that adds 0.3mM induces target protein to express, and along with the target protein expression amount is increased to a certain degree, the specific growth rate of thalline obviously reduces, and growth tendency begins to slow down so stop fermentation.Fermentation thalline final concentration 53OD
600, weight 150 grams per liters, identify that through SDS-PAGE final fusion protein expression accounts for solvable tropina 25-30%.Induce the variation of rear fusion protein expression amount, (Fig. 4) can find out significantly by the SDS-gel electrophoresis spectrum, and the expression amount of target protein increases with the increase of induction time behind the adding IPTG.Swimming lane 1 is induced 0.5 hour tropina for IPTG among the figure, 2 for IPTG induces 1 hour tropina, and 3 for IPTG induces 1.5 hours tropinas, and 4 induce 2 hours tropinas for IPTG, 5 for IPTG induces 2.5 hours tropinas, and 6 induce 3 hours tropinas for IPTG.The band of molecular weight 30kDa is a target protein, i.e. glutathione sulfydryl transferase-human calcitonin analogue precursor fusion rotein.
3. purifying human calcitonin analogue precursor
The 100g coli somatic is placed the phosphoric acid buffer of 1000 milliliters of pH7.2, ultrasonication in the ice bath, add 20%Triton-X100 to final concentration 1%, left standstill 15 30 minutes, centrifugal 25 minutes of 000g, get supernatant liquor and mix, gentle jolting 30 minutes, centrifugal 5 minutes of 500g with 40 milliliters of GlutathioneSepharose 4B, to precipitate the dress post, phosphoric acid buffer is eluted to OD
280＜0.02, use 3 times of damping fluids (10mM glutathione again to column volume, 50mMTris-HCl, pH8.0) wash-out, elution samples the results are shown in Figure 5 with the SDS-gel electrophoresis analysis, the bacterial protein of solubility is through affine resin purification, and the purpose fusion rotein of acquisition (glutathione sulfydryl transferase-human calcitonin analogue precursor) purity is greater than 90%.Swimming lane 1 is a standard protein, and 2 is the purpose fusion rotein behind the purifying, and 3 is the bacterial protein of solubility before the purifying.The about 30kDa of molecular weight place is the purpose fusion rotein.Protein content with in the Bradford method mensuration elutriant adds 1 unit zymoplasm by the 100ug fusion rotein, cuts 20 hours in 25 ℃ of enzymes.Add the 1M dithiothreitol (DTT) to final concentration 10mM, transfer to pH2.0 with concentrated hydrochloric acid, cross SP-Sepharose Fast Flow post, (2M Urea 10mMHCl) is eluted to OD with No. 1 liquid
280＜0.02, change No. 2 liquid (2M Urea, 10mMHCl, 100mM NaCl) wash-out, detect down with spectrophotometer 220nm, collect the elutriant of absorption peak.Cross RP-C
18Desalting column is earlier with No. 3 liquid (5%CNCH
3: 95%H
2O:0.1%TFA) be eluted to OD
220After＜0.02, use liquid (50%CNCH again No. 4
3: 50%H
2O:0.1%TFA) wash-out is collected sample, frost drying.With high performance liquid phase (HPLC) assay products, condition is that first moving phase is 0.5%CNCH
3: 0.1%TFA, second moving phase is 90%CNCH
3: 0.1%TFA, flow velocity are 1 milliliter of per minute, linear gradient be in 30 minutes second moving phase by 0 to 60%.Analytical results shows that product purity is greater than 90%.See Fig. 6 with electrospray ionization mass spectrum identifier calcitonin-like precursor, the molecular weight of collection of illustrative plates explanation human calcitonin analogue precursor is 3805, and is in full accord with calculated value.
4. external amidation
The solution that will contain 1mM EDTA, 200mM O-PNGA adds the human calcitonin analogue precursor to final concentration 1mM with 5N NaOH adjust pH to 6.0, adds carboxypeptidase-Y30 microgram again, puts 35 ℃ of water-baths and uses RP-C after 90 minutes
18Desalination, lyophilize obtain photaesthesia intermediate human calcitonin analogue-O-PNGA.It is dissolved in 60mM NaHSO
3Methanol-water (methyl alcohol: water=1: 1) in the solution, carry out photodissociation after charging into argon gas, can obtain linear human calcitonin analogue, after reaction is finished, RP-C
18Desalination, the HPLC purified product is used in lyophilize, and condition is that first moving phase is 0.5%CNCH
3: 0.1%TFA, second moving phase is 90%CNCH
3: 0.1%TFA, 1 milliliter of flow velocity per minute, linear gradient is that second moving phase is by 0 to 60% in 30 minutes, the purity of the linear human calcitonin analogue that obtains is greater than 95%.
5. renaturation
The linear human calcitonin analogue of 1mg is dissolved in 1ml acetic acid-aqueous solution (acetate: water=1: 19), add (NH
4)
2CO
3Transfer to pH6.0, in 37 ℃ of insulations 4 hours, after renaturation is finished, carry out purifying with HPLC, condition is the same, and the purity of income earner's calcitonin-like is greater than 98%.With electrospray ionization mass spectrum identifier calcitonin-like, the results are shown in Figure 7, collection of illustrative plates explanation human calcitonin analogue molecular weight is 3691, and is in full accord with calculated value.
6. measure active
By " Chinese pharmacopoeia method.With the same source of body weight 40-50g, same strain, other health of identity greatly from mouse, be divided into 5 groups (control group, human calcitonin analogue sample high density group, human calcitonin analogue sample low concentration group, standard substance high density group, standard substance low concentration group) at random, every group 10, after the numbering, respectively in order, tail vein injection 1 * 10
-2Units per ml and 5 * 10
-2The thyrocalcitonin standard substance of two kinds of concentration of units per ml and human calcitonin analogue sample 0.4ml/, control group is only injected blank diluent 0.4ml/.After the administration accurate 1 hour, blood sampling respectively was with o-cresolphthalein complexone colorimetric method for determining blood calcium.Experimental result is by " the quantitative response parallel lines calculate in the Chinese pharmacopoeia appendix calibrating statistical technique, and design method is calculated and tired and experimental error at random.And carry out the statistics certificate authenticity.The results are shown in Table 1.
Table 1. human calcitonin analogue determination of activity result
Human calcitonin analogue sample standard product
0.01 0.05 unit of 0.05 0.01 unit of unit
7.31 5.85 7.52 6.59
6.99 5.84 7.58 5.53
6.24 6.59 7.55 6.03
7.04 6.20 6.81 5.28
7.15 5.85 7.16 5.58
7.38 5.61 7.37 5.99
8.51 6.30 7.58 6.15
7.16 5.89 6.62 5.66
6.63 5.81 6.87 5.59
7.26 5.46 7.37 6.12
Statistics: average fiducial limit (FL%)=23.890, tire: 180 international unit/milligrams.Table 1 and statistics show that human calcitonin analogue has significant blood droping calcium activity, and it is more consistent than living with natural human thyrocalcitonin unit to tire.
Genetically engineered is produced salmon calcitonin see calcimar
1. make up reorganization salmon calcitonin see calcimar precursor-gene
1.1 chemosynthesis reorganization salmon calcitonin see calcimar precursor-gene fragment
The salmon calcitonin see calcimar precursor-gene is divided into F
1'-F
8', 8 fragments are synthetic, see Fig. 8 for details, and operating process is with among the embodiment 1 1.1.Fragment F wherein
1'-F
8' annealing back forms the encoding sequence of salmon calcitonin see calcimar precursor C end, contains the codon GCT of Ala.
1.2. the worker that the full-length gene of splicing reorganization salmon calcitonin see calcimar precursor and structure contain cloning vector
The journey bacterium
Operation is with among the embodiment 1 1.2.The reorganization salmon calcitonin see calcimar precursor-gene full length sequence of splicing is seen Fig. 9, full-length gene 120bp, comprise 5 ' BamH1 site (I), add bromize fluoride crack site codon methionine ATG (VI) between 3 ' Xho1 site (II), two termination codons (III), glutathione sulfydryl transferase and the salmon calcitonin see calcimar precursor and be positioned at the L-Ala codon GCT (VII) that calcium gene 3 ' end falls in salmon for ease of later chemically modified.
1.3. make up the engineering bacteria that contains reorganization salmon calcitonin see calcimar precursor-gene expression vector
Operation is with among the embodiment 1 1.3.
2. high density fermentation
Operation is with among the embodiment 12.
3. purifying salmon calcitonin see calcimar precursor
3.1. the purifying of glutathione sulfydryl transferase-salmon calcitonin see calcimar precursor fusion rotein
Purge process is with among the embodiment 14.
3.2.S-sulfonated and bromize fluoride crack
With 55% saturated (NH
4)
2SO
4Precipitation glutathione sulfydryl transferase-salmon calcitonin see calcimar precursor fusion rotein, 15, centrifugal 15 minutes of 000g adds water and adjusts protein concentration to 10 mg/ml.Add 1/10 volume 1M Tris-hydrochloric acid and transfer to pH8.0, add 52 milligrams of Na by every milliliter of fusion rotein
2SO
3With 25 milligrams of Na
2S
4O
4Ratio add Na respectively
2SO
3And Na
2S
4O
4, lucifuge reaction 12 hours.Through (6 * 60cm) desalinations (using 10mMTris-hydrochloric acid in advance, the pH8.0 balance) of G-25 post.Collect the stream of elutriant and wear the peak, add urea, add concentrated hydrochloric acid, add cyanogen bromide lucifuge reaction 4 hours, the split products such as salmon calcitonin see calcimar precursor of must recombinating to final concentration 50mM to final concentration 8M.
3.3. split product purifying
Above-mentioned scission reaction liquid is added 4 times of volume water,, be eluted to OD with No. 1 liquid (2M urea, 10mM hydrochloric acid) through SP-Sepharose Fast Flow chromatography column
280＜0.02, change No. 2 liquid (2M urea, 10mM hydrochloric acid, 100mM NaCl), use spectrophotometer, detect with 220nm, collect the elutriant of this absorption peak.Collect liquid through RP-C
18Post is earlier with No. 3 liquid (5% acetonitriles: 95% water: 0.1% trifluoroacetic acid) be eluted to OD
220＜0.02, use 38%-49% acetonitrile gradient wash-out again, collect sample, the frost drying salmon calcitonin see calcimar precursor that obtains recombinating.The HPLC assay products, condition is that moving phase third is water: 0.1% trifluoroacetic acid, moving phase fourth are acetonitrile: 0.1% trifluoroacetic acid, flow velocity are 1 milliliter of per minute, linear gradient be 40 minutes fourth moving phase by 0 to 80%, gained reorganization salmon calcitonin see calcimar precursor product purity is greater than 95%.Identify the salmon calcitonin see calcimar precursor with electrospray ionization mass spectrum, see Figure 10, collection of illustrative plates shows that the molecular weight of salmon calcitonin see calcimar precursor is 3666, and is in full accord with Theoretical Calculation.
4. external amidation
The solution that will contain 1mM EDTA, 200mM O-PNGA adds the salmon calcitonin see calcimar precursor to final concentration 1mM with 5N NaOH adjust pH to 6.0, adds carboxypeptidase-Y150 microgram again, puts in 35 ℃ of water-baths and uses RP-C after 120 minutes
18Desalination, lyophilize obtains photaesthesia intermediate salmon calcitonin see calcimar-O-PNGA.It is dissolved in 60mM NaHSO
3Methanol-water (methyl alcohol: water=1: 1) in the solution, carry out photodissociation after charging into argon gas, can obtain linear salmon calcitonin see calcimar, after reaction is finished, RP-C
18Desalination, lyophilize is carried out purifying with HPLC, and condition is the same.The linear salmon calcitonin see calcimar purity that obtains is greater than 95%.
5. renaturation
The Modified Salmon Calcitonin1 milligram is dissolved in 1 milliliter of pH 8.0 contains in the solution of 0.1M Tris-hydrochloric acid and 5mM halfcystine, put 37 ℃ 5 minutes.The HPLC purified product, condition is the same.The salmon calcitonin see calcimar purity that obtains is greater than 99%.See Figure 11 with electrospray ionization mass spectrum evaluation salmon calcitonin see calcimar, collection of illustrative plates shows that the salmon calcitonin see calcimar molecular weight is 3432, and is in full accord with calculated value.
6. determination of activity
Operation is with among the embodiment 16.The results are shown in Table 2.
Table 2. is measured the activity of salmon calcitonin see calcimar
Salmon calcitonin see calcimar sample standard product
0.01 0.05 unit of 0.01 unit of 0.05 unit of unit
7.29 5.84 7.55 6.58
6.71 5.84 7.61 5.55
6.23 6.57 7.56 6.10
7.07 6.23 6.81 5.26
7.15 5.84 7.15 5.59
7.39 5.63 7.39 5.60
8.41 6.29 7.60 5.89
8.00 5.89 6.70 5.80
7.45 5.81 6.70 5.65
7.30 6.00 7.40 6.00
Statistics: average fiducial limit (FL%)=25.790, tire: 4100 international unit/milligrams.The reorganization salmon calcitonin see calcimar of table 2 and statistics explanation gene engineering expression has significant blood droping calcium activity, and it is more consistent than living with natural salmon calcitonin see calcimar unit to tire.
Advantage and good effect
The present invention compares with in the past preparation calcitonin or calcitonin-like method Advantage is that method is simple, with low cost, with short production cycle.
Claims (2)
1. the preparation method of gene recombination thyrocalcitonin or calcitonin-like comprises:
(1) makes up thyrocalcitonin or calcitonin-like precursor-gene and contain cloning vector pGEM-thyrocalcitonin or the engineering bacteria of calcitonin-like precursor-gene;
(2) make up the engineering bacteria that contains thyrocalcitonin or calcitonin-like precursor-gene fusion expression vector pGEX-thyrocalcitonin or calcitonin-like precursor-gene;
(3) fermentation of thyrocalcitonin or calcitonin-like engineering bacteria;
(4) separation, purifying thyrocalcitonin or calcitonin-like precursor;
(5) amidation of thyrocalcitonin or calcitonin-like precursor and renaturation become ripe thyrocalcitonin or calcitonin-like;
(6) the active mensuration of thyrocalcitonin or calcitonin-like;
When it is characterized in that making up thyrocalcitonin or calcitonin-like precursor-gene, at first press the aminoacid sequence of thyrocalcitonin or calcitonin-like precursor, become some gene fragments with colibacillary preference cryptography, and chemosynthesis gene fragment, be spliced into the full-length gene of thyrocalcitonin or calcitonin-like precursor again, the full-length gene 3 that is spliced into ' end can be by the amino acid whose codon of carboxypeptidase-Y hydrolysis for L-Ala (Ala) or leucine (Leu) or other, so that make the product that is gone out by escherichia coli expression, available carboxypeptidase-Y carries out the C-terminal amidation.
2. the preparation method of gene recombination thyrocalcitonin according to claim 1 or calcitonin-like, when the precursor that it is characterized in that thyrocalcitonin or calcitonin-like adopts carboxypeptidase-Y to carry out amidation, add ortho-nitrophenyl G-NH2 O-PNGA simultaneously, make the 33rd of thyrocalcitonin or calcitonin-like precursor to be replaced by O-PNGA, obtain light activated thyrocalcitonin-O-PNGA or calcitonin-like-O-PNGA, this product gets linear thyrocalcitonin or calcitonin-like through photodissociation, and can be folding by atmospheric oxidation, rebuild disulfide linkage, obtain biologically active thyrocalcitonin or calcitonin-like.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990008194A1 (en) * | 1989-01-17 | 1990-07-26 | Suntory Limited | PROCESS FOR PRODUCTION OF C-TERMINAL α-AMIDATED PEPTIDE |
| WO1995020039A2 (en) * | 1993-10-28 | 1995-07-27 | Calsberg A/S | Customized proteases with altered transacylation activity |
| CN1217724A (en) * | 1996-02-06 | 1999-05-26 | 柏内布拉斯加公司 | Recombination preparation of calcitonia fragments and use thereof in preparation of same and the like |
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2000
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990008194A1 (en) * | 1989-01-17 | 1990-07-26 | Suntory Limited | PROCESS FOR PRODUCTION OF C-TERMINAL α-AMIDATED PEPTIDE |
| WO1995020039A2 (en) * | 1993-10-28 | 1995-07-27 | Calsberg A/S | Customized proteases with altered transacylation activity |
| CN1217724A (en) * | 1996-02-06 | 1999-05-26 | 柏内布拉斯加公司 | Recombination preparation of calcitonia fragments and use thereof in preparation of same and the like |
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