CN104403005A - Novel fusion protein of glucagon-like peptide-1 (GLP-1) and human serum albumin as well as method for preparing fusion protein - Google Patents
Novel fusion protein of glucagon-like peptide-1 (GLP-1) and human serum albumin as well as method for preparing fusion protein Download PDFInfo
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- CN104403005A CN104403005A CN201410699879.3A CN201410699879A CN104403005A CN 104403005 A CN104403005 A CN 104403005A CN 201410699879 A CN201410699879 A CN 201410699879A CN 104403005 A CN104403005 A CN 104403005A
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Abstract
The invention provides a novel fusion protein of glucagon-like peptide-1 (GLP-1) analogue and human serum albumin as well as a method for preparing the fusion protein. The composition frame of the novel fusion protein is T-S-X-G-G-H, wherein the protein sequence represented by T is a biological affinity tag; the protein sequence represented by S is a protease enzyme cutting site; X represents an amino acid; G represents GLP-1 analogue protein; H represents human serum albumin. According to the design, a GLP-1 analogue sample, of which the N end is homogeneous, is obtained through biological preparation and enzyme digestion in sequence, so that the homogeneity and activity of a biological sample are improved. The invention further provides a Chinese hamster ovary cell (CHO) stable expressing cell strain capable of stably expressing the fusion protein. The novel fusion protein is higher in biological activity and better in vivo and vitro stability, can be used for treating 2-type diabetes mellitus and other diseases capable of being treated through fasting plasma glucose reduction, can be used for treating 1-type diabetes mellitus by inducing cells to differentiate into Beta pancreatic cells, and can be further used for treating other diseases capable of being treated through nervous system irritation causing satiety generation and peristole inhibition.
Description
Technical field
The present invention utilizes genetic engineering means, the analogue of futuramic GLP-1 and HSA.Novel recombinant protein utilizes the efficient expression plasmid pMH of novel structure
3, and express in Chinese hamster cell (CHO).Obtain the cell strain (CHO/pMH of high reactivity and high expression level
3/ TSXGGH).The bacterial strain productive rate obtained is up to 9.45 μ g*d
-1* 10
6cells.Fermentation expression output is high, and purifying process is convenient, and the product that purifying obtains meets the requirement of bulk drug.This medicine is used for the treatment of diabetes and relative disease.
Background technology
Diabetes B, is referred to as non-insulin-dependent diabetes mellitus again, and low because of β cell function, Regular Insulin lacks and insulin resistant relatively.In recent years, the morbidity of diabetes B increases gradually, and according to World Health Organization's prediction, to the year two thousand thirty, will there be 300,000,000 diabetes B patients in the whole world.GLP-1 medicine can stimulate differentiation and the increment of β cell, and promotes the ability of the excreting insulin of β cell, and becomes the critical medication for the treatment of diabetes B.
The A Bilutai (Albugon) that the medicine of GLP-1 analogue patent medicine has Glaxo Smith Kline company to produce and the Exenatide (exendin-4) that Eli lily company produces.Domestic long-acting GLP-1 analogue also goes on the market without any the approval of a medicine by CFDA.The A Bilutai (Albugon) that the medicine that external approval is gone on the market has Glaxo Smith Kline company to produce and the Exenatide (exendin-4) that Eli lily company produces, but they have defect clearly in the middle of application.A Bilutaiyin merges with lipid acid and becomes depot drug product because of its preparation method and cause the units activity of medicine to lose more than 100 times, and the transformation period of medicine only has 4-6 about h, and unit taking dose is large and the side effect of patient is strengthened.Exenatide is single protein drug, and biological preparation method makes quality product control very well, but the transformation period of its medicine only has 2 h and patient will inject twice every day, adds misery and the side effect of patient.
Human serum albumin and the GLP-l analogue recombinant protein of present research report are expressed in yeast, do not consider N-terminal consistence and improve the highly active problem of medicine.Biosimulation calculating and experimental result display N-terminal be the active region of GLP-1 medicine, for ensureing the consistence of N-terminal, the present invention adds His-tag label before the N-terminal of novel recombinant protein, and after His-tag label, add protease site (enteropeptidase DDDDK).The recombinant protein 6*His-DDDDK-X-GGH expressed, cuts through enteropeptidase enzyme after purifying and obtains recombinant protein (fusion protein).The N-terminal of the recombinant protein obtained is consistent, and active high.
Summary of the invention
The present invention devises a kind of recombinant protein and in the middle preparation method of Chinese hamster ovary cell (chinese hamster ovary, CHO), ensure that the consistence of N-terminal and obtain highly active pharmaceutical protein.The summary of the invention mainly comprised comprise following some.
(1) the recombinant protein design of novel GLP-1 analogue.GLP-1 (A2G)-GLP-1 (A2G)-HSA albumen is abbreviated as GGH respectively, design XGGH albumen (X represents an amino acid or nothing).Holding conforming target protein for expressing N, adding the biomarkers such as His-tag (being called for short T) at the N end of albumen, and after biomarker, add the protease cleavage site such as enteropeptidase DDDDK (being called for short S).The gene fragment of TSXGGH is obtained by genetic engineering means.SEQ ID NO:1 GLP-1 (A2G)-GLP-1 (A2G)-HSA gene order in accompanying drawing, SEQ ID NO:2 GLP-1 (A2G)-GLP-1 (A2G)-HSA protein sequence.Other GLP-1 analogue sequence by that analogy.
(2) efficient expression plasmid (pMH
3/ TSXGGH) structure.The height built voluntarily is utilized to start active, the plasmid pMH of high GC sequence and poly A sequence
3(international patent: WO/2008/091276).The recombinant protein of expressing is T(affinity tag)-S (protease site)-X-GLP-1m-GLP-1m-HSA, is called for short TSXGGH by the recombinant protein of expressing.Warp
ecor I He
noti enzyme cuts pcr amplification product and and pMH
3carrier connects.Construction expression cell expression plasmid pMH
3/ TSXGGH.
(3) screening of stably express Chinese hamster ovary celI strain.The adherent culture base (D001) of Chinese hamster ovary celI used and suspension medium (B001) are the substratum after optimization.The plasmid built, after extracting plasmid without intracellular toxin test kit, turns through electricity and obtains expression strain with gradient pressure screening.Obtain after repeating low density bed board and phenotypic screen four circulation and stablize and the cell strain of high expression level.Stable cell line can be more than cultured continuously 20 generation, and expression amount is without considerable change.
(4) suspension of stably express Chinese hamster ovary celI strain is tamed and fermentation.The cell strain of 8 strain stably express of screening, in B001, cultured continuously cultivates 2 months, makes Chinese hamster ovary celI adapt to suspension culture.Cultivate suspension cell according to 1:3 scale amplifying, finally in 10 L bio-reactors, fermentation culture is continuously fermented 12 d.Protein yield reaches 4.58-9.43 μ g*d
-1* 10
6cells.
(5) pharmaceutical protein purifying and Activity determination.First transfer the albumen containing HSA by Blue affinity column and remove most foreign protein.After diluting soln is identical to affinity column sample solutions such as conductance and Ni ion columns, then transfer the albumen of N end containing His-tag by affinity columns such as Ni ions.Through desalting column displacement damping fluid, the albumen of acquisition is cut by enteropeptidase and is waited proteolytic enzyme enzyme to cut the rear N of acquisition eventually to hold homogeneous target protein.Pharmaceutical protein detects through In vitro and in vivo activity, all shows good drug effect.
Accompanying drawing explanation
Fig. 1: the Dot blot of cell strain expressing protein screens figure; The monoclonal cell selected selects the clone of high expression level through Dot blot.
Fig. 2: the Western blot of cell strain expressing protein screens figure; The Western blot of the GLP-1 monoclonal antibody of the 8 Expression of Plant Height cell strains selected schemes, and swimming lane 1 is positive in product GGH.
Fig. 3: the Western blot of cell strain expressing protein screens figure; The Western blot of the HSA monoclonal antibody of the 8 Expression of Plant Height cell strains selected schemes, and swimming lane 1 is positive in product GGH.
Fig. 4: 10 L fermentor tank process monitorings of Chinese hamster ovary celI strain.
Fig. 5: the external activity experiment of pharmaceutical protein is expressed in Chinese hamster ovary celI strain; Detect at the CHO/GLP-1R/CRE-fLUC cell model built.
Fig. 6: the intracorporeal active experiment of pharmaceutical protein is expressed in Chinese hamster ovary celI strain; Dosage is 3 mg/kg.
Embodiment
Specific implementation method of the present inventionly prepares one of approach, and the present invention can also be implemented by other approach and apply, and the present invention can carry out modifying and improving not running counter under spirit of the present invention.
The stable strain clone of specific implementation method 1:GLP-1 recombinant protein in CHO and preparation
Experiment material
Chinese hamster ovary cell (CHO-S); Geneticin G418(sigma); The expression vector pMH that oneself builds
3; The substratum of oneself configuration, adherent culture base (D001), suspension medium (B001), fed-batch medium (F001); Other reagent used is all analytical reagent.
Laboratory apparatus
Electroporation; Cell culture incubator; Cell shaking table; 10 L bio-reactors; Conventional medical disposable material.
Experimental procedure
(1) plasmid extraction
Solution preparation: solution I: 50mM glucose; 25mM Tris-cl (pH 8.0); 10mM EDTA (pH 8.0), prepares rear sterilizing and is placed on 4 DEG C.Solution II: 0.2 mol/L NaoH; SDS 1%(m/L), matching while using.0.4 mol/L NaoH and 2%(m/L) SDS equal-volume mixing.Solution III (100 mL): 5mol/L KAc 60 mL; Glacial acetic acid 11.5 ml; Water 28.5 mL.
Operation steps.Cultivate the fresh bacterium liquid 6-8 mL of 12-16 h, 12000 rpm 1min, sop up remaining medium at the bottom of pipe.Solution I 300 μ L, makes thalline fully resuspended with pipettor piping and druming.Solution II 300 μ L, careful upset EP pipe, after liquid change is limpid, leaves standstill 1 min.Solution III 300 μ L, careful mixing, has a large amount of white flock to separate out (liquid is transparent).Centrifugal 15 min of 12000 rpm, move in the new EP pipe of supernatant to, repeat.Add Virahol to 1.5 mL ,-20 DEG C, 10min.Centrifugal 25 min of 12000 rpm.In pipe, add the 75% ethanol rinse precipitation of 1mL, outwell ethanol, dry.Add ultrapure water to dissolve, add appropriate RNA enzyme, 37 DEG C of 1 h.1:1 adds phenol by volume, acutely mixes, and leaves standstill 2 min.Centrifugal 10 min of 12000 rpm, the careful new EP of supernatant to that draws manages.1:1 adds chloroform by volume, acutely mixes, and leaves standstill 2 min.Centrifugal 10 min of 12000 rpm, the careful new EP of supernatant to that draws manages.In supernatant, add the NaAc of 75 μ L3M, add Virahol to 1.5 mL ,-20 DEG C, 10min.Centrifugal 25 min of 12000 rpm, outwell supernatant, add 75% ethanol rinse of 1mL.Centrifugal 2 min of 12000 rpm, outwell ethanol, dry.Be dissolved in (100 μ about L) in appropriate ultrapure water.
(2) cell transfecting
Prepare high density without intracellular toxin plasmid, collect Chinese hamster ovary celI.Configuration electricity turns reaction system: 200 μ L cell suspension+20 μ g plasmid+10 μ g salmon sperm dna (raising transfection efficiency), add after mixing in 2 mm pole cups.By pole cup ice bath 1 min, 250V, 12 mS shock by electricity once, ice bath 1 min.Respectively add the substratum (D001 is containing 10% FBS) of 10 mL in two dish, the suspension in pole cup is divided equally in two dish.
(3) colony screening
After dish Growth of Cells two days is adherent, change liquid (D001+6%FBS) pressurization, adding G418 concentration is 1.2-1.8mg/mL.There is a large amount of dead cell directly to change liquid not pressurize, treat that clone grows.Cell monoclonal grows to suitable size, prepares to choose clone.Clone is chosen in 96 orifice plates.Put in incubator and cultivate, treat that clone grows to and be paved with at the bottom of 80% hole, by Dot blot (accompanying drawing 1) and WB(accompanying drawing 2, Fig. 3) detect expression.Select positive colony to continue to amplify adherent culture, be placed on pressurization in dish and cultivate after continuing to say positive clone digestion, clone to be grown can see below and to 96 orifice plates, detect expression according to choosing by Dot blot and WB.
(4) cell suspension domestication
Select 8 positive colonies, cell is transferred in T75 culturing bottle and cultivates, time until about cell confluency degree to 90%, by two bottles of T75 cell dissociations, merge and be transferred in 40mL shaking flask, add 30mL suspending nutrient solution B001 suspension domestication.Cultured continuously 2 months, selects the cell strain of final adaptation suspension culture as engineering strain.
(5) Dot blot detects
Collect sample, be generally cell culture supernatant; Point film, use NC film or pvdf membrane (need steep methyl alcohol), according to sampfe order point film, 5uL/ point, arranges negative control (empty nutrient solution, ghost nutrient solution) and positive control (dilution gradient), and carries out mark with ballpoint pen; Film is put 37 DEG C of oven dry, about 10-15min; Close, prepare 5% skim-milk with TBST, room temperature shaker closes 1h; TBST washing 1-2 time; Primary antibodie (TBST preparation) hatches 1h; TBST washs three times, each 10min; 1h is hatched in two anti-(TBST preparations); TBST washs three times, each 10min; ECL develops.
(6) Western blot detects
Collect sample, be generally cell culture supernatant.Sample preparation, adds the Loading buffer of respective amount, boils 5min; SDS-PAGE electrophoresis; Transferring film, after electrophoresis is complete, carefully peels glue, and install by correct order with filter paper, fiber mat, nitrocellulose membrane ,-20 DEG C are soaked in transfering buffering liquid 40 min; Install electrophoretic blotting box, put into rotor, ice chest, connect power supply, 50-100V constant voltage transfer 1h on magnetic stirring apparatus; Remove transfer device after having shifted, take film off, marked pros and cons (film and sticker one side be front), put into ponceau dye liquor dyeing 5-10 min, take out, deionized water washes away loose colour, observes transfer effect, and ponceau dyeing can be done and can not do; Add confining liquid, 4 DEG C are spent the night, or room temperature 1h; TBST washes film 1-2 time; Dilute primary antibodie with TBST, be placed in incubated at room 1 h on horizontal shaker; TBST washs three times, each 10min; Resist with TBST dilution two, be placed in incubated at room 1 h on horizontal shaker; TBST washs three times, each 10minECL colour developing.
(7) 10 L bio-reactor fermentation expressions of stable cell line
Liquid amount is 6 L, prepares 1 L seed liquor, and density is 4.0 × 10
6individual/mL.Rotating speed is that 110 rpm, pH control to be 7.2, and culture temperature is 37 DEG C.Ensure that dissolved oxygen controls between 30 ~ 60% by regulating the air flow of oxygen and carbonic acid gas.Treat that cell density length is 9.0 × 10
6after individual/mL, reduce temperature flow and add F001 continuation cultivation 10 d(Fig. 4).Result shows, and first three day cell density double growth every day in fermentor tank, within the 3rd day, cell density is up to 8.78*10
6cell/mL.Change in cell density of lowering the temperature afterwards for 3rd day is little, and cell starts to produce target protein in a large number, and fermentation later stage cell mortality increases, and protein accumulation amount reduces, and final protein concentration can reach 487 mg/L.
(8) purifying of recombinant protein
Fermented liquid 8000 rpm, the centrifugal acquisition supernatant liquor of 10 min, degerming with the membrane filtration of 0.45 μm.With the ultrafiltration instrument ultrafiltration and concentration 5 ~ 12 times of molecular weight cut-off 10 kDa.The first step selects Blue Sepharose, 0.1 mol/L NaCl(pH 6.0 ~ 9.0) balance, use concentrated solution loading, drip washing balances.2 mol/L NaCl(pH 6.0 ~ 9.0) be B liquid, with 100%B liquid gradient elution.The elutriant that second dilution is collected, through Ni Sepharose affinity chromatography, collects the elutriant of 100 mmol/L imidazoles.Final step is through G25 Sepharose ion chromatography displacement damping fluid.Purifying protein after displacement after enterokinase cleavage, then wears through Ni Sepharose affinity chromatography stream the sample that liquid is final preparation.
Whole purification process step is connected better, and the elutriant of previous step chromatography only needs adjustment a little just to can be used as next step chromatography sample solution.Reduce intermediate process steps, be convenient to the pollution that industrial operation brings with minimizing intermediary operation.
(9) Activity determination of recombinant protein
Add 1.5 μ g/mL GLP-1R plasmids in In vitro cell model Activity determination: liposome is clockwise: optiwcly 100 μ L and 0.5 μ g/mL CRE/Luc puts 15min, add in HEK293 cell, overnight incubation.Cell is laid on (80*10 in 96 orifice plates
4individual/mL), incubated overnight to 90%-95% degree of converging, every hole 100 μ L.Substratum is siphoned away, adds each 100 μ L of stimulation fluid of DMEM and medicine, hatch 4h.Siphon away substratum, every hole adds 50 μ L lysates, in horizontal shaker concussion 30min.Draw 30 μ L lysates to detect liquid with the 15 μ L prepared and mix, add 96 orifice plates, shaking table concussion 15min, detects OD
560.The pharmaceutical protein that result display CHO expresses can stimulate the cAMP content in born of the same parents promote and stimulate the raising of fluorescin (Luc), the results are shown in accompanying drawing 5.
Activity in vivo detects: get body weight 22-25 g male KM mouse 50, be divided at random: normal group, Exendin-4 group (0.125mg/Kg), GGH high dose group (6.0 mg/kg) and low dose group (3 mg/kg).After mouse fasting (can't help water) is spent the night, respectively organize gastric infusion, normal group is to physiological saline.3 respectively group mouse stomaches give the GGH of tail vein injection Exendin-4 and various dose at once after 1.5 g/kg glucose solutions.The blood glucose value of 20,40,60 min, 80 min, 100min after injection.Result shows, and relative to control group, the glucose level of experimental group is controlled for a long time, the results are shown in accompanying drawing 6.
SEQ ID NO:1
1 CACCACCACC ATCACCATGA TGACGATGAC AAGCACGGTG AAGGTACTTT CACTTCTGAT
61 GTTTCTTCTT ACTTGGAAGG TCAAGCTGCT AAGGAGTTCA TTGCTTGGTT GGTTAAGGGT
121 AGACACGGTG AAGGTACTTT CACTTCTGAT GTTTCTTCTT ACTTGGAAGG TCAAGCTGCT
181 AAGGAGTTCA TTGCTTGGTT GGTTAAGGGT AGAGATGCAC ACAAGAGTGA GGTTGCTCAT
241 CGATTTAAAG ATTTGGGAGA AGAAAATTTC AAAGCCTTGG TGTTGATTGC CTTTGCTCAG
301 TATCTTCAGC AGTGTCCATT TGAAGATCAT GTAAAATTAG TGAATGAAGT AACTGAATTT
361 GCAAAAACAT GTGTTGCTGA TGAGTCAGCT GAAAATTGTG ACAAATCACT TCATACCCTT
421 TTTGGAGACA AATTATGCAC AGTTGCAACT CTTCGTGAAA CCTATGGTGA AATGGCTGAC
481 TGCTGTGCAA AACAAGAACC TGAGAGAAAT GAATGCTTCT TGCAACACAA AGATGACAAC
541 CCAAACCTCC CCCGATTGGT GAGACCAGAG GTTGATGTGA TGTGCACTGC TTTTCATGAC
601 AATGAAGAGA CATTTTTGAA AAAATACTTA TATGAAATTG CCAGAAGACA TCCTTACTTT
661 TATGCCCCGG AACTCCTTTT CTTTGCTAAA AGGTATAAAG CTGCTTTTAC AGAATGTTGC
721 CAAGCTGCTG ATAAAGCTGC CTGCCTGTTG CCAAAGCTCG ATGAACTTCG GGATGAAGGG
781 AAGGCTTCGT CTGCCAAACA GAGACTCAAG TGTGCCAGTC TCCAAAAATT TGGAGAAAGA
841 GCTTTCAAAG CATGGGCAGT AGCTCGCCTG AGCCAGAGAT TTCCCAAAGC TGAGTTTGCA
901 GAAGTTTCCA AGTTAGTGAC AGATCTTACC AAAGTCCACA CGGAATGCTG CCATGGAGAT
961 CTGCTTGAAT GTGCTGATGA CAGGGCGGAC CTTGCCAAGT ATATCTGTGA AAATCAAGAT
1021 TCGATCTCCA GTAAACTGAA GGAATGCTGT GAAAAACCTC TGTTGGAAAA ATCCCACTGC
1081 ATTGCCGAAG TGGAAAATGA TGAGATGCCT GCTGACTTGC CTTCATTAGC TGCTGATTTT
1141 GTTGAAAGTA AGGATGTTTG CAAAAACTAT GCTGAGGCAA AGGATGTCTT CCTGGGCATG
1201 TTTTTGTATG AATATGCAAG AAGGCATCCT GATTACTCTG TCGTGCTGCT GCTGAGACTT
1261 GCCAAGACAT ATGAAACCAC TCTAGAGAAG TGCTGTGCCG CTGCAGATCC TCATGAATGC
1321 TATGCCAAAG TGTTCGATGA ATTTAAACCT CTTGTGGAAG AGCCTCAGAA TTTAATCAAA
1381 CAAAATTGTG AGCTTTTTGA GCAGCTTGGA GAGTACAAAT TCCAGAATGC GCTATTAGTT
1441 CGTTACACCA AGAAAGTACC CCAAGTGTCA ACTCCAACTC TTGTAGAGGT CTCAAGAAAC
1501 CTAGGAAAAG TGGGCAGCAA ATGTTGTAAA CATCCTGAAG CAAAAAGAAT GCCCTGTGCA
1561 GAAGACTATC TATCCGTGGT CCTGAACCAG TTATGTGTGT TGCATGAGAA AACGCCAGTA
1621 AGTGACAGAG TCACCAAATG CTGCACAGAA TCCTTGGTGA ACAGGCGACC ATGCTTTTCA
1681 GCTCTGGAAG TCGATGAAAC ATACGTTCCC AAAGAGTTTA ATGCTGAAAC ATTCACCTTC
1741 CATGCAGATA TATGCACACT TTCTGAGAAG GAGAGACAAA TCAAGAAACA AACTGCACTT
1801 GTTGAGCTCG TGAAACACAA GCCCAAGGCA ACAAAAGAGC AACTGAAAGC TGTTATGGAT
1861 GATTTCGCAG CTTTTGTAGA GAAGTGCTGC AAGGCTGACG ATAAGGAGAC CTGCTTTGCC
1921 GAGGAGGGTA AAAAACTTGT TGCTGCAAGT CAAGCTGCCT TAGGCTTATA A
SEQ ID NO:2
1 HisHisHisHisHisHisAspAspAspAspLysHisGlyGluGlyThrPheThrSerAsp
21 ValSerSerTyrLeuGluGlyGlnAlaAlaLysGluPheIleAlaTrpLeuValLysGly
41 ArgHisGlyGluGlyThrPheThrSerAspValSerSerTyrLeuGluGlyGlnAlaAla
61 LysGluPheIleAlaTrpLeuValLysGlyArgAspAlaHisLysSerGluValAlaHis
81 ArgPheLysAspLeuGlyGluGluAsnPheLysAlaLeuValLeuIleAlaPheAlaGln
101 TyrLeuGlnGlnCysProPheGluAspHisValLysLeuValAsnGluValThrGluPhe
121 AlaLysThrCysValAlaAspGluSerAlaGluAsnCysAspLysSerLeuHisThrLeu
141 PheGlyAspLysLeuCysThrValAlaThrLeuArgGluThrTyrGlyGluMETAlaAsp
161 CysCysAlaLysGlnGluProGluArgAsnGluCysPheLeuGlnHisLysAspAspAsn
181 ProAsnLeuProArgLeuValArgProGluValAspValMETCysThrAlaPheHisAsp
201 AsnGluGluThrPheLeuLysLysTyrLeuTyrGluIleAlaArgArgHisProTyrPhe
221 TyrAlaProGluLeuLeuPhePheAlaLysArgTyrLysAlaAlaPheThrGluCysCys
241 GlnAlaAlaAspLysAlaAlaCysLeuLeuProLysLeuAspGluLeuArgAspGluGly
261 LysAlaSerSerAlaLysGlnArgLeuLysCysAlaSerLeuGlnLysPheGlyGluArg
281 AlaPheLysAlaTrpAlaValAlaArgLeuSerGlnArgPheProLysAlaGluPheAla
301 GluValSerLysLeuValThrAspLeuThrLysValHisThrGluCysCysHisGlyAsp
321 LeuLeuGluCysAlaAspAspArgAlaAspLeuAlaLysTyrIleCysGluAsnGlnAsp
341 SerIleSerSerLysLeuLysGluCysCysGluLysProLeuLeuGluLysSerHisCys
361 IleAlaGluValGluAsnAspGluMETProAlaAspLeuProSerLeuAlaAlaAspPhe
381 ValGluSerLysAspValCysLysAsnTyrAlaGluAlaLysAspValPheLeuGlyMET
401 PheLeuTyrGluTyrAlaArgArgHisProAspTyrSerValValLeuLeuLeuArgLeu
421 AlaLysThrTyrGluThrThrLeuGluLysCysCysAlaAlaAlaAspProHisGluCys
441 TyrAlaLysValPheAspGluPheLysProLeuValGluGluProGlnAsnLeuIleLys
461 GlnAsnCysGluLeuPheGluGlnLeuGlyGluTyrLysPheGlnAsnAlaLeuLeuVal
481 ArgTyrThrLysLysValProGlnValSerThrProThrLeuValGluValSerArgAsn
501 LeuGlyLysValGlySerLysCysCysLysHisProGluAlaLysArgMETProCysAla
521 GluAspTyrLeuSerValValLeuAsnGlnLeuCysValLeuHisGluLysThrProVal
541 SerAspArgValThrLysCysCysThrGluSerLeuValAsnArgArgProCysPheSer
561 AlaLeuGluValAspGluThrTyrValProLysGluPheAsnAlaGluThrPheThrPhe
581 HisAlaAspIleCysThrLeuSerGluLysGluArgGlnIleLysLysGlnThrAlaLeu
601 ValGluLeuValLysHisLysProLysAlaThrLysGluGlnLeuLysAlaValMETAsp
621 AspPheAlaAlaPheValGluLysCysCysLysAlaAspAspLysGluThrCysPheAla
641 GluGluGlyLysLysLeuValAlaAlaSerGlnAlaAlaLeuGlyLeu
SEQ ID NO:1
1 CACCACCACC ATCACCATGA TGACGATGAC AAGCACGGTG AAGGTACTTT CACTTCTGAT
61 GTTTCTTCTT ACTTGGAAGG TCAAGCTGCT AAGGAGTTCA TTGCTTGGTT GGTTAAGGGT
121 AGACACGGTG AAGGTACTTT CACTTCTGAT GTTTCTTCTT ACTTGGAAGG TCAAGCTGCT
181 AAGGAGTTCA TTGCTTGGTT GGTTAAGGGT AGAGATGCAC ACAAGAGTGA GGTTGCTCAT
241 CGATTTAAAG ATTTGGGAGA AGAAAATTTC AAAGCCTTGG TGTTGATTGC CTTTGCTCAG
301 TATCTTCAGC AGTGTCCATT TGAAGATCAT GTAAAATTAG TGAATGAAGT AACTGAATTT
361 GCAAAAACAT GTGTTGCTGA TGAGTCAGCT GAAAATTGTG ACAAATCACT TCATACCCTT
421 TTTGGAGACA AATTATGCAC AGTTGCAACT CTTCGTGAAA CCTATGGTGA AATGGCTGAC
481 TGCTGTGCAA AACAAGAACC TGAGAGAAAT GAATGCTTCT TGCAACACAA AGATGACAAC
541 CCAAACCTCC CCCGATTGGT GAGACCAGAG GTTGATGTGA TGTGCACTGC TTTTCATGAC
601 AATGAAGAGA CATTTTTGAA AAAATACTTA TATGAAATTG CCAGAAGACA TCCTTACTTT
661 TATGCCCCGG AACTCCTTTT CTTTGCTAAA AGGTATAAAG CTGCTTTTAC AGAATGTTGC
721 CAAGCTGCTG ATAAAGCTGC CTGCCTGTTG CCAAAGCTCG ATGAACTTCG GGATGAAGGG
781 AAGGCTTCGT CTGCCAAACA GAGACTCAAG TGTGCCAGTC TCCAAAAATT TGGAGAAAGA
841 GCTTTCAAAG CATGGGCAGT AGCTCGCCTG AGCCAGAGAT TTCCCAAAGC TGAGTTTGCA
901 GAAGTTTCCA AGTTAGTGAC AGATCTTACC AAAGTCCACA CGGAATGCTG CCATGGAGAT
961 CTGCTTGAAT GTGCTGATGA CAGGGCGGAC CTTGCCAAGT ATATCTGTGA AAATCAAGAT
1021 TCGATCTCCA GTAAACTGAA GGAATGCTGT GAAAAACCTC TGTTGGAAAA ATCCCACTGC
1081 ATTGCCGAAG TGGAAAATGA TGAGATGCCT GCTGACTTGC CTTCATTAGC TGCTGATTTT
1141 GTTGAAAGTA AGGATGTTTG CAAAAACTAT GCTGAGGCAA AGGATGTCTT CCTGGGCATG
1201 TTTTTGTATG AATATGCAAG AAGGCATCCT GATTACTCTG TCGTGCTGCT GCTGAGACTT
1261 GCCAAGACAT ATGAAACCAC TCTAGAGAAG TGCTGTGCCG CTGCAGATCC TCATGAATGC
1321 TATGCCAAAG TGTTCGATGA ATTTAAACCT CTTGTGGAAG AGCCTCAGAA TTTAATCAAA
1381 CAAAATTGTG AGCTTTTTGA GCAGCTTGGA GAGTACAAAT TCCAGAATGC GCTATTAGTT
1441 CGTTACACCA AGAAAGTACC CCAAGTGTCA ACTCCAACTC TTGTAGAGGT CTCAAGAAAC
1501 CTAGGAAAAG TGGGCAGCAA ATGTTGTAAA CATCCTGAAG CAAAAAGAAT GCCCTGTGCA
1561 GAAGACTATC TATCCGTGGT CCTGAACCAG TTATGTGTGT TGCATGAGAA AACGCCAGTA
1621 AGTGACAGAG TCACCAAATG CTGCACAGAA TCCTTGGTGA ACAGGCGACC ATGCTTTTCA
1681 GCTCTGGAAG TCGATGAAAC ATACGTTCCC AAAGAGTTTA ATGCTGAAAC ATTCACCTTC
1741 CATGCAGATA TATGCACACT TTCTGAGAAG GAGAGACAAA TCAAGAAACA AACTGCACTT
1801 GTTGAGCTCG TGAAACACAA GCCCAAGGCA ACAAAAGAGC AACTGAAAGC TGTTATGGAT
1861 GATTTCGCAG CTTTTGTAGA GAAGTGCTGC AAGGCTGACG ATAAGGAGAC CTGCTTTGCC
1921 GAGGAGGGTA AAAAACTTGT TGCTGCAAGT CAAGCTGCCT TAGGCTTATA A
SEQ ID NO:2
1 HisHisHisHisHisHisAspAspAspAspLysHisGlyGluGlyThrPheThrSerAsp
21 ValSerSerTyrLeuGluGlyGlnAlaAlaLysGluPheIleAlaTrpLeuValLysGly
41 ArgHisGlyGluGlyThrPheThrSerAspValSerSerTyrLeuGluGlyGlnAlaAla
61 LysGluPheIleAlaTrpLeuValLysGlyArgAspAlaHisLysSerGluValAlaHis
81 ArgPheLysAspLeuGlyGluGluAsnPheLysAlaLeuValLeuIleAlaPheAlaGln
101 TyrLeuGlnGlnCysProPheGluAspHisValLysLeuValAsnGluValThrGluPhe
121 AlaLysThrCysValAlaAspGluSerAlaGluAsnCysAspLysSerLeuHisThrLeu
141 PheGlyAspLysLeuCysThrValAlaThrLeuArgGluThrTyrGlyGluMETAlaAsp
161 CysCysAlaLysGlnGluProGluArgAsnGluCysPheLeuGlnHisLysAspAspAsn
181 ProAsnLeuProArgLeuValArgProGluValAspValMETCysThrAlaPheHisAsp
201 AsnGluGluThrPheLeuLysLysTyrLeuTyrGluIleAlaArgArgHisProTyrPhe
221 TyrAlaProGluLeuLeuPhePheAlaLysArgTyrLysAlaAlaPheThrGluCysCys
241 GlnAlaAlaAspLysAlaAlaCysLeuLeuProLysLeuAspGluLeuArgAspGluGly
261 LysAlaSerSerAlaLysGlnArgLeuLysCysAlaSerLeuGlnLysPheGlyGluArg
281 AlaPheLysAlaTrpAlaValAlaArgLeuSerGlnArgPheProLysAlaGluPheAla
301 GluValSerLysLeuValThrAspLeuThrLysValHisThrGluCysCysHisGlyAsp
321 LeuLeuGluCysAlaAspAspArgAlaAspLeuAlaLysTyrIleCysGluAsnGlnAsp
341 SerIleSerSerLysLeuLysGluCysCysGluLysProLeuLeuGluLysSerHisCys
361 IleAlaGluValGluAsnAspGluMETProAlaAspLeuProSerLeuAlaAlaAspPhe
381 ValGluSerLysAspValCysLysAsnTyrAlaGluAlaLysAspValPheLeuGlyMET
401 PheLeuTyrGluTyrAlaArgArgHisProAspTyrSerValValLeuLeuLeuArgLeu
421 AlaLysThrTyrGluThrThrLeuGluLysCysCysAlaAlaAlaAspProHisGluCys
441 TyrAlaLysValPheAspGluPheLysProLeuValGluGluProGlnAsnLeuIleLys
461 GlnAsnCysGluLeuPheGluGlnLeuGlyGluTyrLysPheGlnAsnAlaLeuLeuVal
481 ArgTyrThrLysLysValProGlnValSerThrProThrLeuValGluValSerArgAsn
501 LeuGlyLysValGlySerLysCysCysLysHisProGluAlaLysArgMETProCysAla
521 GluAspTyrLeuSerValValLeuAsnGlnLeuCysValLeuHisGluLysThrProVal
541 SerAspArgValThrLysCysCysThrGluSerLeuValAsnArgArgProCysPheSer
561 AlaLeuGluValAspGluThrTyrValProLysGluPheAsnAlaGluThrPheThrPhe
581 HisAlaAspIleCysThrLeuSerGluLysGluArgGlnIleLysLysGlnThrAlaLeu
601 ValGluLeuValLysHisLysProLysAlaThrLysGluGlnLeuLysAlaValMETAsp
621 AspPheAlaAlaPheValGluLysCysCysLysAlaAspAspLysGluThrCysPheAla
641 GluGluGlyLysLysLeuValAlaAlaSerGlnAlaAlaLeuGlyLeu
Claims (9)
1. a method of design for GLP-1 analogue protein, is characterized by: protein sequence structure is T-S-X-GGH; Wherein the protein sequence of T representative is biological affinity tag; The protein sequence of S representative is protease cleavage site; X represents an amino acid; G represents GLP-1 analogue albumen; H representative serum albumin.
2. method of design as claimed in claim 1, it is characterized by: the protein sequence of T representative is affinity tag protein sequence, be preferably the one in His-tag, His-flag, GST-tag, MBP-tag, Strep-tag, but be not only confined to cited rabphilin Rab label.
3. method of design as claimed in claim 1, it is characterized by: the protein sequence of S representative is the protein sequence of protease cleavage site, be preferably the one in enterokinase cleavage site DDDDK, zymoplasm restriction enzyme site LVPRGS, factor Xa and IE/DGR, tobacco etch virus protease ENLYFQ, but be not only confined to cited protease cleavage site.
4. method of design as claimed in claim 1, is characterized by: the amino acid of X representative is the one in Gly, Pro, Arg, Val, Phe, Trp, Met, Lys, Ala and Tyr; One in prioritizing selection Gly, Pro, Lys, Ala.
5. the method for claim 1, is characterized by, and it is following any one that designed recombinant protein comprises sequence: T-S-GGH, T-S-X-GGH, X-GGH and GGH; T-S-GGH is the albumen that T-S and GGH directly merges, and T-S-X-GGH is the albumen that T-S, X and GGH merge according to right 1 order, and X-GGH is the albumen that X and GGH directly merges, and GGH is the diad of GLP-1 analogue albumen and the fusion rotein of human serum albumin.
6. the method for claim 1, is characterized in that, designed recombinant protein is expressed in Chinese hamster ovary cell CHO, and preferably in CHO-S cell, suspension culture is expressed.
7. method as claimed in claim 6, is characterized in that, by efficient expression plasmid pMH
3be incorporated in CHO genome and express.
8. method as claimed in claim 6, is characterized by: the substratum of culture expression is the substratum of high expression after optimizing, and the adherent culture base of Chinese hamster ovary celI is D001, and suspension medium is B001, and fed-batch medium is F001; Cultivate suspension cell according to 1:3 scale amplifying, finally continuously ferment 12 d in 10 L bio-reactors, and protein yield is up to 4.58-9.43 μ g*d
-1* 10
6cells; The recombinant protein that fermentation expression obtains, first by Blue affinity column Ni chromatography, then through desalting column displacement damping fluid, then holds homogeneous recombinant protein by obtaining N after enterokinase cleavage.
9. the method as described in right 8, is characterized in that: the recombinant protein prepared can be used for diabetes B and the treatment by reducing plasma glucose and benefited Other diseases; Can be used for the treatment that Cell differentiation inducing activity is the type 1 diabetes of β pancreatic cell; Can be used for exciting nerve system generation satiety and suppressing peristole and benefited other diseases.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894196A (en) * | 2015-05-28 | 2015-09-09 | 中国药科大学 | Novel method for preparing recombinant exenatide or derivative thereof |
| CN106282131A (en) * | 2016-09-30 | 2017-01-04 | 广东温氏大华农生物科技有限公司 | A kind of keratinase expression system and its preparation method and application |
| CN107922961A (en) * | 2015-07-13 | 2018-04-17 | 生命技术公司 | System and method for transient protein expression improved in Chinese hamster ovary celI |
| US11498949B2 (en) | 2012-05-02 | 2022-11-15 | Life Technologies Corporation | System and method for high-yield transient expression in mammalian cells |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11498949B2 (en) | 2012-05-02 | 2022-11-15 | Life Technologies Corporation | System and method for high-yield transient expression in mammalian cells |
| CN104894196A (en) * | 2015-05-28 | 2015-09-09 | 中国药科大学 | Novel method for preparing recombinant exenatide or derivative thereof |
| CN107922961A (en) * | 2015-07-13 | 2018-04-17 | 生命技术公司 | System and method for transient protein expression improved in Chinese hamster ovary celI |
| CN106282131A (en) * | 2016-09-30 | 2017-01-04 | 广东温氏大华农生物科技有限公司 | A kind of keratinase expression system and its preparation method and application |
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