CN102659928A - Synthetic signal peptide and application thereof - Google Patents
Synthetic signal peptide and application thereof Download PDFInfo
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- CN102659928A CN102659928A CN2012101186757A CN201210118675A CN102659928A CN 102659928 A CN102659928 A CN 102659928A CN 2012101186757 A CN2012101186757 A CN 2012101186757A CN 201210118675 A CN201210118675 A CN 201210118675A CN 102659928 A CN102659928 A CN 102659928A
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Abstract
The invention relates to synthetic signal peptide and application thereof. The amino acid sequence of the signal peptide is SEQIDNO:2. The signal peptide can be added to a mammalian cell expression vector and used for leading mammalian source protein secretion expression, and the foreign protein expression index of the signal peptide in mammalian cells can be improved.
Description
This case is dividing an application of following patented claim:
Application number: 201010284904.3;
The applying date: on September 17th, 2010;
Denomination of invention: a kind of signal peptide of synthetic and application thereof
Technical field
The present invention relates to biological technical field, be specifically related to be used for guiding of signal peptide and the application thereof of Mammals source protein at the mammalian cell secreting, expressing.
Technical background
Foreign gene efficiently expressing in host cell is the prerequisite of proteinic 26S Proteasome Structure and Function analysis, protein or polypeptide drug research and development.The expression system that is used for express recombinant protein has mikrobe, plant, yeast, insect cell and zooblast etc.Mammalian cell is to express to have the proteic best host of natural radioactivity; It is advantageous that can correct and effective synthetic, the processing and the secretion signal of ground identification eukaryotic protein; Intron in identification and the removal gene is processed into sophisticated mRNA through shearing again, can accomplish glycosylation, phosphorylation exactly; Form in the chain and translate the post-treatment processes with interchain disulfide bond and proteolyze etc.; Thereby the complete antibody that produces is the same with natural antibody, has BA, but not only can discern antigen but also activating complement system.Mammalian cell is prone to the DNA transfection that quilt is recombinated, and can obtain cell transformed through screening, has genetic stability and repeatability, and expressed products is secreted into and is easy to purifying in the substratum.Utilize the mammalian cell expression protein product to be widely used in biological products industry, like a large amount of preparations of virus vaccines, antibody, Interferon, rabbit, immunomodulator, hormone and growth factor etc.But zooblast exogenous protein expression efficient is low, and cost is high, therefore improves zooblast exogenous protein expression efficient, and reducing production costs is the most important thing in the work at present.
It is a lot of to influence the factor of exogenous protein in mammalian cell, like integration site on karyomit(e) of the stability of signal peptide, the number of transcribing and translate controlling elements, RNA processing (RNA Processing), gene copy, mRNA, foreign gene, recombinant protein to the genotoxic potential of host cell and the genome preference property (genetic properties) of host cell etc.Under the situation that expression vector has been confirmed, it is most important that the selection of signal peptide just seems.
The N-end of secretory protein is made up of the leader sequence of the 15-30Aa that can be sheared usually; This is called signal peptide (signal sequence) in proper order; At N-end or near N end place 2-3 polarity Aa arranged, and all be that a unique hydrophobic core perhaps is made up of a lot of hydrophobic Aa at the middle part of signal peptide.There is not other conservative order not have acidic-group in the signal peptide yet.With to lead peptide similar, signal peptide also is can be enough to any additional polypeptide is transported into target film.For example signal peptide is added in the N-end of globin, it is no longer stayed in the cytosol, but pass film and be secreted into outside the born of the same parents.
Signal peptide can make the rrna of translating be attached on the RER film.Rrna be through the function of signal peptide adhere to and synthesis secretion proteic.So and indifference between free rrna and the film binding ribosomal body own.Signal peptide is as the signal on a kind of ER of being attached to film identification, and this maybe be through the hydrophobic function of the N-termination several amino acid that begins to synthesize.Protein chain injects in the film then, and signal peptide is embedded in a kind of proteolytic enzyme in the film and shears at this moment rrna and accomplished translation, and albumen has prolonged leads peptide by way of passing film.The effect of signal peptide secretion foreign protein is following:
1) signal peptide can guide secretory protein or membranin to go out born of the same parents.
2) hydrophobic core of signal peptide determines proteinic secernment efficiency.
3) signal peptide can pilot protein matter in cell different zones or different organoid carry out correct positioning.
4) signal peptide can be secreted the secretion that enhanser strengthens foreign protein.
5) the glad molecule that helps reducing expression vector of short signal is put, and improves the stability that is incorporated into exogenous gene expression box on the host chromosome and transcribes efficient.
For secretory protein, select the appropriate signal peptide can its expression amount be significantly improved, no matter for industrial production or scientific research, its meaning all is very far-reaching.
Summary of the invention
The object of the invention provides a kind of signal peptide and application thereof that can guide external source mammalian cell albumen to efficiently express, to reduce the Mammals proteic production cost of originating.
What at first, the invention provides a kind of synthetic is used for that to guide the signal peptide of Mammals source protein at the mammalian cell secreting, expressing, the aminoacid sequence of said signal peptide be arbitrary among the SEQ ID NO:1-5.Wherein, the signal peptide of SEQ ID NO:1-5 has higher homology.
SEQ?ID?NO：1MDVLAFLLGLLLLWLPGVRC
SEQ?ID?NO：2MDVPAEFLGLLLLWLSGVRC
SEQ?ID?NO：3MRVLPEFLGLLLLWISGVRC
SEQ?ID?NO：4MDVPLQLLGLLLLWLSGVRC
SEQ?ID?NO：5MDVPAELLGLLLLWISGVRC
Described mammalian cell can be CHO, BHK, SP2/0, C127 etc.
Signal peptide of the present invention is used in the mammalian host cell guiding Mammals proteic secreting, expressing of originating.
The present invention also provides a kind of polynucleotide, and said polynucleotide encoding is said to be used for guiding the signal peptide of Mammals source protein at the mammalian cell secreting, expressing.
Further, the sequence of said many nucleic acids is selected from arbitrary among the SEQ ID NO:6-10:
ATGGATGTTCTTGCTTTTCTTCTGGGCTTGCTGCTTTTGTGGCTTCCCGGGGTGAGGTGC(SEQ?ID?NO：6)
ATGGATGTTCCTGCCGAATTTCTTGGATTGTTGTTGTTGTGGCTCTCCGGAGTGCGTTGC(SEQ?ID?NO：7)
ATGAGGGTTCTGCCTGAATTCCTGGGACTTTTGTTGTTGTGGATTTCCGGCGTGCGATGT(SEQ?ID?NO：8)
ATGGATGTACCACTTCAGCTTCTTGGCTTGTTGTTGCTTTGGCTTTCTGGCGTGAGATGT(SEQ?ID?NO：9)
ATGGATGTGCCTGCTGAACTTTTGGGCCTTCTTTTGTTGTGGATATCAGGAGTACGATGC(SEQ?ID?NO：10)
Polynucleotide of the present invention can be through conventional compound method preparation.
Polynucleotide of the present invention can make an addition to mammalian cell expression vector, are used to guide the Mammals proteic secreting, expressing of originating.
Utilize signal peptide of the present invention secreting, expressing Mammals in zooblast proteic method of originating to be: the polynucleotide of code book being invented signal peptide and coding are expressed the mammalian cell proteic polynucleotide of originating and are connected rear clone and go into mammalian cell expression vector, then with expressing target protein behind this recombinant mammalian cells expression vector transfection mammalian cell.
The present invention also provides a kind of mammalian cell expression vector, and said expression vector contains the polynucleotide of aforementioned code book invention signal peptide and the proteinic polynucleotide in encoding mammalian source.
In the said expression vector, the polynucleotide of code book invention signal peptide are right after the proteinic polynucleotide front end in said encoding mammalian source.
Further, in the said expression vector, the front end of the polynucleotide of said code book invention signal peptide is added with the Kozak sequence.
The promotor of said expression vector can be EF-1 α promotor, CMV promotor, SV40 late promoter or SV40 early promoter etc.
Enumerate like the embodiment of the invention; The pIRESneo3 of said expression vector for transforming; Among the pIRESneo3 of said transformation, people EF-1 α promoter sequence or people EF1-HTLV promoter sequence have been replaced the main immediate early promoter sequence of hCMV in the pIRESneo3 plasmid.
The present invention also provides a kind of mammalian host cell, and said host cell is the transfection of aforementioned expression vector institute.Described mammalian cell can be selected from CHO, BHK, SP2/0, C127 etc.
The present invention also further provides the method for producing protein in a kind of Mammals source; For being fit to express under the proteinic condition in said Mammals source; Cultivate the aforementioned mammal host cell, then from culture, adopt ordinary method to isolate the protein in said Mammals source.
The protein in said Mammals source can be the protein of molecular weight arbitrarily, like various pharmaceutical proteins: erythropoietin, various have that erythropoietin is active to change the structure erythropoietin, and antibody etc.
The present invention compares signal peptide of the present invention and protein self signal peptide; The mensuration result of exogenous protein expression amount shows that signal peptide of the present invention is specially adapted to Chinese hamster ovary celI, compares with existing signal peptide; Exogenous protein expression amount in zooblast obtains significantly to promote; Expression level has improved about 2～36 times, and its application helps screening the strain of high expression level monoclonal cell, has reducing production costs, is convenient to advantages such as subsequent purification.
Description of drawings
Fig. 1 shows the comparison (pEF carrier) of invention signal peptide and EPO self signal peptide
Fig. 2 shows the comparison (pEV carrier) of invention signal peptide and EPO self signal peptide
Embodiment
Following embodiment specifies the present invention, but its invention scope is not limited
The expression vector that the embodiment of the invention is cited is to be transformed by plasmid pIRESneo3 to obtain.Wherein, people EF-1 α promoter sequence, people EF1-HTLV promoter sequence have been replaced the main immediate early promoter of hCMV (the Human cytomegalovirus major immediate early promoter) sequence in the former plasmid respectively.
Synthesizing of embodiment 1 signal peptide
Shorter because of signal peptide, it is synthetic that signal peptide is pressed primer.5 ' end adds NheI restriction enzyme site and Kozak sequence, and 3 ' end adding purpose gene order part is so that add this signal peptide on goal gene.
Signalase 11:
TCGGAGCTAGCCACCATGGATGTTCTTGCTTTTCTTCTGGGCTTGCTGCTTTTGTGGCTTCCCGGGGTGAG GTGC
GCCCCACCACG(SEQ ID NO:11)
Signal peptide 2:
TCGGAGCTAGCCACCATGGATGTTCCTGCCGAATTTCTTGGATTGTTGTTGTTGTGGCTCTCCGGAGTGCG TTGC
GCCCCACCACG(SEQ ID NO:12)
Signal peptide 3:
TCGGAGCTAGCCACCATGAGGGTTCTGCCTGAATTCCTGGGACTTTTGTTGTTGTGGATTTCCGGCGT GCGATGT
GCCCCACCACG(SEQ ID NO:13)
Signal peptide 4:
TCGGAGCTAGCCACCATGGATGTACCACTTCAGCTTCTTGGCTTGTTGTTGCTTTGGCTTTCTGGCGTGAG ATGT
GCCCCACCACG(SEQ ID NO:14)
Signal peptide 5:
TCGGAGCTAGCCACCATGGATGTGCCTGCTGAACTTTTGGGCCTTCTTTTGTTGTGGATATCAGGAGTACG ATGC
GCCCCACCACG(SEQ ID NO:15)
The amplification of embodiment 2 people EF-1 α promotors
With plasmid pEF6/V5-HisA (available from Invitrogen company) is masterplate, and people EF-1 α promoter sequence is reference, and design primers F 01/R01 carries out the polymerase chain reaction, amplifies people EF-1 α promoter sequence, reaction conditions such as table 1.
F01：CATACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAG(SEQ?ID?NO：16)
R01：ACGGCTAGCTCCGAGCTCGGTACCAAGCTTACCTAGCCA(SEQ?ID?NO：17)
Table 1PCR reaction conditions
Gained PCR product is connected with the pUC57 (available from Fermentas company) that handles through SmaI, and the evaluation of checking order, and the result shows that sequence is as follows:
ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAGCTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGGTAAGCTTGGTACCGAGCTCGGAGCTAGC(SEQ?ID?NO：18)
The amplification of embodiment 3 people EF1-HTLV promotors
With plasmid pFUSE-CHIg-hG3 (available from InvivoGen company) is masterplate, and people EF1-HTLV promoter sequence is reference, and design primers F 02/R02 carries out the polymerase chain reaction, amplifies people EF1-HTLV promoter sequence, reaction conditions such as table 1.
F02：ACGACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGC(SEQ?ID?NO：19)
R02：ATCGCTAGCGTAGGCGCCGGTCACAGCT(SEQ?ID?NO：20)
Gained PCR product is connected with the pUC57 (available from Fermentas company) that handles through SmaI, and the evaluation of checking order, and the result shows that sequence is as follows:
ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGC(SEQ?ID?NO：21)
The structure of embodiment 4 reorganization pEF expression vectors
Product among the embodiment 2 and pIRESneo3 plasmid are carried out the SpeI/NheI double digestion be connected, obtain plasmid pEF.
Human erythropoietin gene (GeneBank accession number: AB463610 with codon optimization; Contain self signal peptide and 5 ' end and added the Kozak sequence) respectively after the NheI/BamHI double digestion is handled; Be connected to the plasmid pEF behind the NheI/BamHI double digestion, the recombinant plasmid called after pEF-EPO that is obtained.
With signalase 11, signal peptide 2, signal peptide 3, signal peptide 4, signal peptide 5 carry out the PCR splicing with erythropoietin (no signal peptide) respectively, and splicing product carries out the SpeI/NheI double digestion respectively with plasmid pEF and is connected.The recombinant plasmid that is obtained is called after pEF-EP01 respectively, pEF-EP02, pEF-EP03, pEF-EP04, pEF-EP05.
Embodiment 5 reorganization pEF expression vector transient transfection expression studies
The employing liposome method (lipofectamine LTX, invitrogen) with plasmid pEF-EPO, pEF-EP01, pEF-EP02, pEF-EP03, pEF-EP04, pEF-EP05 be transfection CHO-S cell respectively, detects the transient expression level of its erythropoietin.
According to lipofectamine LTX process specifications, with 3 μ gpEF-EPO, pEF-EP01, pEF-EP02, pEF-EP03, pEF-EP04, pEF-EP05 plasmid transfection respectively are incubated at the CHO-S cell (about 1.1 * 10 of 6 orifice plates (2.5ml/well, 10% foetal calf serum)
6Individual cell).Transfectional cell is cultivated in 37 ℃ the CO2gas incubator after 24 hours at 5%CO2, collects the nutrient solution supernatant, and expression product has the erythropoietin activity through evaluation, utilizes the ELISA method to measure its erythropoietin expression amount.PEF-EPO, pEF-EP01, pEF-EP02, pEF-EP03, pEF-EP04, the expression amount of pEF-EP05 are respectively 3ng/ml, 73ng/ml, 100ng/ml, 62ng/ml, 85ng/ml, 110ng/ml.The result shows that signal peptide of the present invention is with respect to erythropoietin self signal peptide, and under the same conditions, the EPO expression amount has improved 20～36 times.
The structure of embodiment 6 reorganization pEV expression vectors
Product among the embodiment 3 and pIRESneo3 plasmid are carried out the SpeI/NheI double digestion be connected, obtain plasmid pEF1-HTLV.
Human erythropoietin gene (GeneBank accession number: AB463610 with codon optimization; Contain self signal peptide and 5 ' end and added the Kozak sequence) respectively after the NheI/BamHI double digestion is handled; Be connected to the plasmid pEV behind the NheI/BamHI double digestion, the recombinant plasmid called after pEV-EPO that is obtained.
With signalase 11, signal peptide 2, signal peptide 3, signal peptide 4, signal peptide 5 carry out the PCR splicing with erythropoietin (no signal peptide) respectively, and splicing product carries out the SpeI/NheI double digestion respectively with plasmid pEV and is connected.The recombinant plasmid that is obtained is called after pEV-EP01 respectively, pEV-EP02, pEV-EP03, pEV-EP04, pEV-EP05.
Embodiment 7 reorganization pEV expression vector transient transfection expression studies
The employing liposome method (lipofectamine LTX, invitrogen) with plasmid pEV-EPO, pEV-EP01, pEV-EP02, pEV-EP03, pEV-EP04, pEV-EP05 be transfection CHO-S cell respectively, detects the transient expression level of its erythropoietin.
According to lipofectamine LTX process specifications, with 3 μ gpEV-EPO, pEV-EP01, pEV-EP02, pEV-EP03, pEV-EP04, pEV-EP05 plasmid transfection respectively are incubated at the CHO-S cell (about 1.1 * 10 of 6 orifice plates (2.5ml/well, 10% foetal calf serum)
6Individual cell).Transfectional cell is at 5%CO
2, cultivate in 37 ℃ the CO2gas incubator after 24 hours, collect the nutrient solution supernatant, expression product has the erythropoietin activity through evaluation, utilizes the ELISA method to measure its erythropoietin expression amount.PEV-EPO, pEV-EP01, pEV-EP02, pEV-EP03, pEV-EP04, the expression amount of pEV-EP05 are respectively 20ng/ml, 53ng/ml, 58ng/ml, 48ng/ml, 40ng/ml, 60ng/ml.The result shows that signal peptide of the present invention under the same conditions, has improved 2～3 times with the EPO expression amount with respect to erythropoietin self signal peptide.
Claims (13)
- A synthetic be used for guide the signal peptide of Mammals source protein at the mammalian cell secreting, expressing, the aminoacid sequence of said signal peptide is SEQ ID NO:2.
- 2. signal peptide is used at the mammalian host cell guiding Mammals proteic secreting, expressing of originating according to claim 1.
- 3. polynucleotide, the said signal peptide of said polynucleotide encoding claim 1.
- 4. like the said polynucleotide of claim 3, it is characterized in that the sequence of said many nucleic acids is SEQ ID NO:7.
- 5. mammalian cell expression vector, said expression vector contain the proteinic polynucleotide in claim 3 or 4 said polynucleotide and encoding mammalian source.
- 6. like the said mammalian cell expression vector of claim 5, it is characterized in that claim 3 or 4 said polynucleotide are right after the proteinic polynucleotide front end in said encoding mammalian source.
- 7. like the said mammalian cell expression vector of claim 5, it is characterized in that further, in the said expression vector, the front end of claim 3 or 4 said polynucleotide is added with the Kozak sequence.
- 8. like the said mammalian cell expression vector of claim 5, it is characterized in that the promotor of said expression vector is selected from EF-1 α promotor, CMV promotor, SV40 late promoter or SV40 early promoter.
- 9. like the said mammalian cell expression vector of claim 5, it is characterized in that the protein in said Mammals source is selected from erythropoietin, various have that erythropoietin is active to change the structure erythropoietin, and antibody.
- 10. mammalian host cell, said host cell are the transfection of the arbitrary said expression vector of claim 5-9 institute.
- 11., it is characterized in that said mammalian host cell is CHO, BHK, SP2/0 or C127 like the said mammalian host cell of claim 10.
- 12. the method for producing protein in a Mammals source; For being fit to express under the proteinic condition in Mammals source; Cultivate claim 10 or 11 said mammalian host cells, then from culture, isolate the protein in said Mammals source.
- 13. method for producing protein like the said Mammals of claim 12 source; It is characterized in that; The protein in said Mammals source is selected from erythropoietin, and various have that erythropoietin is active to change the structure erythropoietin, and antibody.
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| CN103254277A (en) * | 2013-05-03 | 2013-08-21 | 南京工业大学 | Strong secretory signal peptide enhanced small peptide module sequence and application thereof |
| CN104211776A (en) * | 2013-05-03 | 2014-12-17 | 南京工业大学 | Strong secretory signal peptide enhanced small peptide module sequence and application thereof |
| CN104974226A (en) * | 2014-04-01 | 2015-10-14 | 上海中信国健药业股份有限公司 | Signal peptide for protein expression |
| CN106478774A (en) * | 2015-08-25 | 2017-03-08 | 三生国健药业（上海）股份有限公司 | A kind of signal peptide for protein expression |
| CN106478773A (en) * | 2015-08-25 | 2017-03-08 | 三生国健药业（上海）股份有限公司 | A kind of novel signal peptide of synthetic |
| CN116769746A (en) * | 2023-05-12 | 2023-09-19 | 康彤(上海)生物研发有限公司 | Method for improving ability of pichia pastoris to secrete cyclodextrin glucosyltransferase |
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Cited By (9)
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| CN103254277A (en) * | 2013-05-03 | 2013-08-21 | 南京工业大学 | Strong secretory signal peptide enhanced small peptide module sequence and application thereof |
| CN104211776A (en) * | 2013-05-03 | 2014-12-17 | 南京工业大学 | Strong secretory signal peptide enhanced small peptide module sequence and application thereof |
| CN103254277B (en) * | 2013-05-03 | 2017-02-08 | 南京工业大学 | Strong secretory signal peptide enhanced small peptide module sequence and application thereof |
| CN104211776B (en) * | 2013-05-03 | 2017-11-28 | 南京工业大学 | Strong secretory signal peptide enhanced small peptide module sequence and application thereof |
| CN104974226A (en) * | 2014-04-01 | 2015-10-14 | 上海中信国健药业股份有限公司 | Signal peptide for protein expression |
| CN104974226B (en) * | 2014-04-01 | 2019-10-15 | 三生国健药业（上海）股份有限公司 | A kind of signal peptide for protein expression |
| CN106478774A (en) * | 2015-08-25 | 2017-03-08 | 三生国健药业（上海）股份有限公司 | A kind of signal peptide for protein expression |
| CN106478773A (en) * | 2015-08-25 | 2017-03-08 | 三生国健药业（上海）股份有限公司 | A kind of novel signal peptide of synthetic |
| CN116769746A (en) * | 2023-05-12 | 2023-09-19 | 康彤(上海)生物研发有限公司 | Method for improving ability of pichia pastoris to secrete cyclodextrin glucosyltransferase |
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