CN117362451B - Fusion protein containing TEV protease, preparation method and application thereof - Google Patents
Fusion protein containing TEV protease, preparation method and application thereof Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
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- C12N15/09—Recombinant DNA-technology
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- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
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Abstract
The invention provides a fusion protein containing TEV protease, a preparation method and application thereof, and particularly relates to a method for preparing a fusion protein containing TEV protease, wherein a TEV protease beta chain is used as an expression promoting label, so that the expression of a target protein is promoted, the expression quantity of the target protein is increased, inclusion bodies are formed, and the target protein which is not easy to express originally can be expressed after the expression promoting label is added. The production process is favorable for the separation and purification of the downstream of the target protein and the large-scale industrial production of the medicinal polypeptide.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a fusion protein containing TEV protease, a preparation method and application thereof in preparation of protein aggregates.
Background
The recombinant protein or polypeptide expressed in the escherichia coli exists in a cell in a soluble or inclusion body form, the polypeptide with less than 100 amino acids is easy to be degraded by protease in the cell of the escherichia coli in the recombinant expression process, and the N end of the expressed polypeptide is added with a fusion promoting tag (inclusion body IB inducer) for promoting inclusion body formation so as to promote expression, and the expressed high-purity fusion protein exists in an insoluble inclusion body form, so that protein aggregate can be generated, the degradation of the fusion protein by the protease in host cells can be effectively reduced, and the purification of the target polypeptide is facilitated. Generally IB inhibitors are mostly hydrophobic amino acids forming β -sheets or α -helix short peptides forming α -helix bundles (alpha helix bundle) to promote polypeptide aggregate formation.
TEV Protease (TEV Protease) is an active domain of 27kDa in Nla Protease derived from Tobacco Etch Virus (TEV), has strong site specificity, and has sequence specificity far higher than that of Protease such as thrombin, factor Xa, enterokinase, etc. The wild-type TEV enzyme has certain defects in expression, solubility and the like. The wild-type TEV protease is extremely low in solubility, and if expressed in E.coli without a pro-lytic tag or chaperone (chaperone) co-expression, inclusion bodies are extremely likely to be formed in cells. TEV protease has been widely used as a tool enzyme in protein research and biopharmaceutical production, and is widely used for recombinant protein expression and purification and for cleavage and removal of expression-promoting or dissolution-promoting tags in fusion proteins.
Since TEV protease uses its cleavage property in many cases, it has not been studied as an expression-promoting tag for promoting expression of a target protein.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides application of TEV protease as an expression promotion tag. Specifically, the TEV protease is connected with the target protein to promote the formation of protein aggregates, thereby promoting the expression of the target protein.
In a first aspect of the present invention, there is provided a fusion protein comprising a TEV protease fragment and a protein of interest, the TEV protease fragment being SEQ ID NO: 1-5.
More preferably, the TEV protease fragment is linked to the N-terminus or the C-terminus of the protein of interest, and the TEV protease fragment is directly or indirectly linked to the protein of interest.
The target protein of the present invention includes proteins, and may include polypeptides, for example, polypeptides having 100 or less amino acid residues such as short peptides.
Preferably, the TEV protease fragment and the protein of interest are linked by a linker sequence.
Preferably, the linker sequence comprises a cleavage site for isolating the protein of interest. Such as bovine enterokinase cleavage sequence, SUMO protease cleavage sequence, immunoglobulin degradation cleavage sequence or 3C protease cleavage sequence.
More preferably, the amino acid sequence of the linker sequence is as shown in SEQ ID NO: 11.
Preferably, the TEV protease fragment promotes the formation of protein aggregates, thereby promoting the expression of the protein of interest, and the TEV protease fragment does not exert its cleavage activity.
Preferably, the nucleotide sequence encoding the TEV protease fragment comprises SEQ ID NO:6-10, a complement or degenerate sequence thereof, or a sequence complementary to any one of SEQ ID NOs: 6-10, and encodes a TEV protease fragment having the same function.
Preferably, the protein of interest comprises a β -sheet or an α -helical structure.
Preferably, the protein of interest includes, but is not limited to, IL-7, GLP1, GLP2, PTH, etc., more preferably, the protein of interest includes the sequence of SEQ ID NO: 13. 15-17.
In a second aspect of the invention, there is provided a protein aggregate comprising the fusion protein described above.
Preferably, the protein aggregate is insoluble in water.
In a third aspect of the present invention, there is provided a biomaterial comprising:
(A) A nucleic acid comprising a nucleic acid molecule encoding the fusion protein described above;
(B) A vector comprising the nucleic acid of (a);
(C) A host bacterium comprising the nucleic acid of (a) or the vector of (B).
Preferably, the biological material expresses a protein of interest.
In a specific embodiment, the protein of interest includes, but is not limited to, IL-7, GLP1, GLP2, PTH, etc., more preferably, the protein of interest includes the amino acid sequence of SEQ ID NO: 13. 15-17.
In a fourth aspect of the invention, there is provided the use of a biomaterial as described above in the preparation of a fusion protein or protein aggregate.
In a fifth aspect of the present invention, there is provided a method for preparing the above fusion protein, the method comprising:
1) Synthesizing a gene for encoding the fusion protein, constructing an expression vector, and transferring the expression vector into host bacteria;
2) Culturing host bacteria and expressing fusion protein.
In a sixth aspect of the present invention, there is provided a method for producing the protein aggregate described above, the method comprising:
1) Synthesizing a gene for encoding the fusion protein, constructing an expression vector, and transferring the expression vector into host bacteria;
2) Culturing a host bacterium and expressing the fusion protein;
3) Lysing host bacteria expressing the fusion protein to obtain protein aggregates, and separating the protein aggregates from the water-soluble content of the host bacteria.
Further, in the fifth and/or sixth aspect of the present invention,
preferably, the method of step 2) comprises culturing the host bacteria overnight, transferring the host bacteria to a culture medium, and adding an inducer for overnight culture.
Preferably, in the method of step 2), the bacterial liquid is: the transfer ratio of the culture medium is 1: (50-200), for example 1: (50, 70, 100, 120, 150, 170, 200). More preferably, the ratio is a mass ratio.
Preferably, the culture conditions in the method of step 2) are 24-37 ℃ (e.g. 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 ℃), 150-250rpm (e.g. 150, 180, 200, 210, 220, 230, 240, 250 rpm).
Preferably, the bacterial liquid is cultured until the OD600 is 1.0-2.0 (e.g., 1.0, 1.3, 1.5, 1.7, 1.9, 2.0), and then the inducer is added.
Preferably, the inducer includes, but is not limited to, IPTG, AHL, tetracycline, or arabinose.
Preferably, the inducer concentration is 0.1-1.0 mM (e.g., 0.1, 0.3, 0.5, 0.7, 0.9, 1.0 mM).
In one specific embodiment, the step 2) includes culturing the host bacteria in LB medium overnight according to the bacterial liquid: the culture medium was added to TB medium at a ratio of 1:100, incubated at 220rpm 37℃until OD600 was about 2.0, IPTG was added to a final concentration of 1mM, recombinant protein expression was induced, and overnight at 220rpm 37 ℃.
Preferably, the step 3) includes centrifugally collecting the cells, lysing the cells, and centrifugally separating protein aggregates.
Preferably, the method for lysing the bacterial cells comprises centrifugation, ultrasound, stirring or co-homogenizing of the bacterial cells.
Preferably, the host bacterium does not comprise a substrate for the TEV protease.
In a seventh aspect, the present invention provides an application of TEV protease in promoting expression of a target protein, wherein a nucleotide sequence encoding the TEV protease fragment is linked with a nucleotide sequence encoding the target protein, and the TEV protease fragment is transferred into a host bacterium, and a fusion protein comprising the TEV protease fragment and the target protein is expressed in the host bacterium, wherein the TEV protease fragment is SEQ ID NO: 1-5.
Preferably, the protein of interest comprises a β -sheet or an α -helical structure.
Preferably, the fusion protein forms protein aggregates.
Preferably, the protein aggregate is insoluble in water.
Preferably, the fusion protein is as described in the first aspect, or the protein aggregate as described in the second aspect is obtained.
The term "protein aggregate" according to the present invention is a process in which protein aggregates, which refer to misfolded protein molecules, form amorphous polymers by hydrophobic interactions, comprising inclusion bodies. Inclusion body formation is relatively complex, and is related to the rate of protein production within the cytoplasm, with higher concentrations of the newly produced polypeptide and insufficient time to fold, thereby forming amorphous, amorphous protein aggregates. The protein contained in the inclusion body is an aggregate in a non-folded state and has no biological activity.
The terms "comprising" or "includes" are used in this specification to be open-ended, having the specified components or steps described, and other specified components or steps not materially affected.
The invention has the beneficial effects that:
according to the method, the characteristic that the TEV protease is extremely hydrophobic and is easy to form inclusion bodies in escherichia coli is utilized, the TEV protease sequence is split into different fragments, a hydrophobic beta chain in the fragments is used as an inclusion body formation promotion tag, the expression of target proteins is promoted, the expression quantity of the target proteins is improved, and the inclusion bodies are formed. The polypeptide which is not easy to express originally can be expressed after the expression promoting label is added. The production process is favorable for the separation and purification of the downstream of the target protein and the large-scale industrial production of the medicinal polypeptide, especially the small molecular medicinal polypeptide.
Drawings
Fig. 1: AGGRESCAN software analyzes TEV protein sequence results.
Fig. 2: schematic representation of fusion proteins.
Fig. 3: SDS-PAGE analysis result of IL-7 related expression protein, wherein S is soluble protein, P is insoluble protein, and M is Marker.
Fig. 4: the gelAnalyzer software analyzed the results for the corresponding bands of FIG. 3.
Fig. 5: cell morphology under the IL-7 related expressed protein microscope, indicated by the arrow as inclusion bodies.
Fig. 6: SDS-PAGE analysis result of GLP1 related expression protein, wherein S is soluble protein, P is insoluble protein, and M is Marker.
Fig. 7: the gelAnalyzer software analyzed the results for the corresponding bands of FIG. 6.
Fig. 8: cell morphology under GLP 1-related expressed protein microscope, indicated by arrow is inclusion body.
Fig. 9: SDS-PAGE analysis results of GLP1, GLP2 and PTH related expression proteins, wherein S is soluble protein, P is insoluble protein, and M is Marker.
Fig. 10: the gelAnalyzer software analyzed the results for the corresponding bands of FIG. 9.
Fig. 11: cell morphology under GLP2 and PTH related expressed proteins microscope, indicated by arrows as inclusion bodies.
Fig. 12: SDS-PAGE analysis results of GLP1, GLP2 and PTH related expression proteins, wherein S is soluble protein, P is insoluble protein, and M is Marker.
Fig. 13: the gelAnalyzer software analyzed the results for the corresponding bands of FIG. 9.
Fig. 14: cell morphology under GLP2 and PTH related expressed proteins microscope, indicated by arrows as inclusion bodies.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 expression of a fusion protein comprising a TEV protease
1. Fusion protein design
This study first analyzed the TEV protein sequence for the formation of aggregated hot spots using AGGRESCAN software, and then the alpha-helix and beta-strand sequences were analyzed based on the known X-ray crystal structure of the TEV protease (amino acid sequence SEQ ID NO: 26). As shown in FIG. 1, the grey bolded amino acids are helix and the black bolded amino acids are beta-strand. The underlined sequences are TEV fragments as pro-expression tags, as detailed in Table 1.
TABLE 1 expression promoting tag sequences
The TEV protease protein sequence is split into 5 fragments containing beta-strand as polypeptide expression promotion tags, and the polypeptide expression sequences are connected through enzyme digestion sequences. If bovine enterokinase enzyme cutting sequence is selected (amino acid sequence is shown as SEQ ID NO: 11: DDDDK, nucleotide sequence is shown as SEQ ID NO: 12), the sequence is used for removing the expression promoting tag in the subsequent purification operation, and the specific structure of the fusion protein is shown as figure 2.
2. Construction and transformation of expression plasmids
The expression sequence is converted into a DNA sequence through the optimization of the escherichia coli codon, and is synthesized to serve as a target gene to be inserted into a vector pET11 plasmid of an escherichia coli table through NdeI and XhoI, and the expression plasmid is transformed into escherichia coli BL21 (DE 3) through a heat shock method. The expression strain is obtained through screening of ampicillin resistance.
3. Expression of fusion proteins
Shake flask expression
Each expression strain was cultured overnight in LB medium supplemented with ampicillin antibiotics, bacterial liquid: the culture medium was added to the ampicillin-added TB medium at a ratio of 1:100, incubated at 220rpm 37℃until OD600 was about 2.0, IPTG was added to a final concentration of 1mM, recombinant protein expression was induced, and overnight induction was carried out at 220rpm 37 ℃. Collecting the induced expression thalli, and observing the cell morphology by a microscope; and centrifuging to collect thalli, performing ultrasonic wall breaking, centrifuging, and performing SDS-PAGE electrophoresis analysis on soluble proteins and insoluble proteins.
The 5 TEV protease protein sequences are used as polypeptide inclusion body expression promotion labels to connect different polypeptides for expression test, and specific results are as follows:
(1) According to the above steps 1-3, IL-7 expression was tested before and after ligation of TEV fragments, and the IL-7 amino acid sequence was as shown in SEQ ID NO:13, the nucleotide sequence is shown as SEQ ID NO: 14. The result of SDS-PAGE electrophoresis analysis of the protein expression is shown in FIG. 3, and the relative expression amount of each protein was obtained by analyzing the corresponding bands using GelAnalyzer software on the result of SDS-PAGE electrophoresis, wherein the IL-7 band expressed without a tag was taken as 100%, and the results are shown in FIG. 4 and Table 2.
TABLE 2 expression of IL-7 related expression proteins
All TEV fragments were found to significantly increase IL-7 expression as pro-expression tags. The results of observing the expression of the fusion protein by a microscope are shown in FIG. 5. The cell morphology was observed microscopically, and inclusion bodies were not evident in intracellular microscopy expressing unlabeled IL7 after overnight induction, whereas dense inclusion bodies formed by stacking of numerous recombinant polypeptide expression were visible in other cells containing pro-expression tags. For a protein of interest of larger molecular weight, for example IL-7 has 152 amino acid residues, although there is a certain amount of expression in the host bacterium, increasing the tev protease fragment significantly increases the expression of the protein and the formation of protein aggregates inclusion bodies.
(2) According to the steps 1-3, the expression condition of GLP1 before and after the connection of the TEV fragment is tested, and the GLP1 amino acid sequence is shown as SEQ ID NO: 15. The result of SDS-PAGE analysis of protein expression is shown in FIG. 6, and the relative expression levels of the respective proteins were obtained by analyzing the corresponding bands using GelAnalyzer software on the result of SDS-PAGE, wherein TN-IL7 band was taken as 100%, and the results are shown in Table 3 and FIG. 7.
TABLE 3 expression of GLP1-associated expression proteins
The results of observing the expression of the fusion protein by a microscope are shown in FIG. 8. The results show that when no TEV protease fragment was ligated to GLP1, a short peptide of only 30 amino acid residues, the expression of the polypeptide could not be detected in the host cell, and the cell showed no inclusion bodies under the microscope. And after the TN, TE and TG fragments of the TEV protease are added as expression promoting labels, the recombinant GLP1 is expressed in an insoluble form, namely inclusion bodies, and especially the TN and TG expression promoting effect is more obvious. Coli cells expressing GLP1 with TN, TE and TG tags showed significant inclusion body formation under the microscope. However, TS and TA do not promote GLP1 expression for the short peptide GLP 1.
(3) According to steps 1-3 above, it was tested whether the TEV fragment TN has an expression promoting effect on GLP2 and alpha-helical polypeptides such as PTH similar to GLP1, and whether shortening the TN fragment into TNa and TNb portions would have an expression promoting effect on short peptides. In the expression situation before and after the TEV fragment is connected, the GLP2 amino acid sequence is shown as SEQ ID NO: shown at 16. PTH amino acid sequence is shown in SEQ ID NO: shown at 17. TNa amino acid sequence is shown in SEQ ID NO:18, the nucleotide sequence is shown as SEQ ID NO: shown at 20. TNb amino acid sequence is shown in SEQ ID NO:19, the nucleotide sequence is shown as SEQ ID NO: 21.
TABLE 4 expression of TN fragments GLP1, GLP2 and PTH-related expression proteins
The result of SDS-PAGE analysis of protein expression is shown in FIG. 9, and the relative expression levels of the respective proteins were obtained by analyzing the corresponding bands using GelAnalyzer software on the result of SDS-PAGE, wherein TN-GLP1 band was taken as 100%, and the results are shown in Table 4 and FIG. 10. The results of observing the expression of the fusion protein by a microscope are shown in FIG. 11.
TABLE 5 expression of TN fragments GLP1, GLP2 and PTH-related expression proteins
The results show that for polypeptides that are also relatively few amino acid residues, GLP2 (33 amino acid residues) or PTH (34 amino acid residues), TN can also promote expression of GLP2 and PTH in inclusion forms, and that inclusion bodies due to aggregation of large amounts of protein can be found by observing the cells under a microscope. TN fragments with a smaller number of amino acids (TNa and TNb) may not promote short peptide expression due to the reduced amino acids forming the β -sheet.
(4) According to steps 1-3 above, it was tested whether TEV fragment TG had an pro-expression effect on GLP2 and PTH similar to GLP1, and whether shortening the TG fragment to TGa and TGb had an pro-expression effect on short peptides. TGa amino acid sequence is shown in SEQ ID NO:22, the nucleotide sequence is shown as SEQ ID NO: shown at 24. TGb amino acid sequence is shown in SEQ ID NO:23, the nucleotide sequence of which is shown in SEQ ID NO: 25.
The results of SDS-PAGE analysis of protein expression are shown in FIG. 12, and the relative expression levels of the respective proteins were obtained by analyzing the corresponding bands using GelAnalyzer software on the SDS-PAGE results, wherein the TG-GLP1 band was taken as 100%, and the results are shown in Table 5 and FIG. 13. The results of observing the expression of the fusion protein by a microscope are shown in FIG. 14.
TABLE 6 prediction of expression of TG fragment GLP1, GLP2, PTH-related expression proteins
The results show that for polypeptides that are also relatively few amino acid residues, GLP2 (33 amino acid residues) or PTH (34 amino acid residues), TG can also promote expression of GLP2 and PTH in inclusion forms, and that inclusion bodies due to aggregation of a large number of proteins can be found by observing the cells under a microscope. TG fragments (TGa and TGb) with a smaller number of amino acids may not promote short peptide expression due to the reduced amino acids forming β -sheet.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (17)
1. A fusion protein comprising a TEV protease fragment and a protein of interest, wherein the TEV protease fragment is SEQ ID NO: 2-4.
2. The fusion protein of claim 1, wherein the protein of interest comprises a β -sheet or an α -helix.
3. The fusion protein of claim 2, wherein the protein of interest comprises IL-7, GLP1, GLP2, PTH.
4. A fusion protein according to claim 3, wherein the amino acid sequence of the protein of interest is as set forth in SEQ ID NO: 13. 15-17.
5. A fusion protein comprising a TEV protease and a protein of interest, wherein the TEV protease is SEQ ID NO:1 or 5, wherein the amino acid sequence of the target protein is shown as SEQ ID NO: shown at 13.
6. The fusion protein of any one of claims 1-5, wherein the TEV protease fragment is C-terminal or N-terminal to the protein of interest, and wherein the TEV protease fragment is directly or indirectly linked to the protein of interest.
7. The fusion protein of claim 6, wherein the indirect linkage comprises a linker sequence comprising a cleavage site for isolating the protein of interest.
8. The fusion protein of claim 7, wherein the cleavage site comprises a bovine enterokinase cleavage sequence, a SUMO protease cleavage sequence, an immunoglobulin degradation cleavage sequence, or a 3C protease cleavage sequence.
9. The fusion protein of claim 8, wherein the linker sequence has the amino acid sequence set forth in SEQ ID NO: 11.
10. A protein aggregate comprising the fusion protein of any one of claims 1-9.
11. The protein aggregate of claim 10, wherein the protein aggregate is insoluble in water.
12. A biomaterial, characterized in that the biomaterial comprises:
(A) A nucleic acid comprising a nucleic acid molecule encoding the fusion protein of any one of claims 1-9;
(B) A vector comprising the nucleic acid of (a);
(C) A host bacterium comprising the nucleic acid of (a) or the vector of (B).
13. Use of the biomaterial of claim 12 for the preparation of a fusion protein or protein aggregate.
14. A method for preparing the fusion protein of any one of claims 1-9, comprising:
1) Synthesizing a gene for encoding the fusion protein, constructing an expression vector, and transferring the expression vector into host bacteria;
2) Culturing host bacteria and expressing fusion protein.
15. A method of preparing the protein aggregate of any of claims 10-11, comprising:
1) Synthesizing a gene for encoding the fusion protein, constructing an expression vector, and transferring the expression vector into host bacteria;
2) Culturing a host bacterium and expressing the fusion protein;
3) Lysing host bacteria expressing the fusion protein to obtain protein aggregates, and separating the protein aggregates from the water-soluble content of the host bacteria.
16. The method according to claim 14 or 15, characterized in that the host bacterium does not comprise a substrate for TEV protease.
17. An application of TEV protease in promoting the expression of target protein, which is characterized in that a nucleotide sequence for encoding the TEV protease fragment is connected with a nucleotide sequence for encoding the target protein, and is transferred into host bacteria, and fusion protein containing the TEV protease fragment and the target protein is expressed in the host bacteria, wherein the TEV protease fragment is SEQ ID NO:1-5, said fusion protein being as defined in any one of claims 1-9, or obtaining a protein aggregate as defined in any one of claims 10-11.
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| CN106434745A (en) * | 2016-09-19 | 2017-02-22 | 马生武 | Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos |
| CN108884466A (en) * | 2015-11-19 | 2018-11-23 | 巴塞尔大学 | Protein delivery based on bacterium |
| CN111019926A (en) * | 2018-10-10 | 2020-04-17 | 上饶市康可得生物科技有限公司 | TEV protease variants, fusion proteins thereof, and methods of making and using |
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| CN108884466A (en) * | 2015-11-19 | 2018-11-23 | 巴塞尔大学 | Protein delivery based on bacterium |
| CN106434745A (en) * | 2016-09-19 | 2017-02-22 | 马生武 | Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos |
| CN111019926A (en) * | 2018-10-10 | 2020-04-17 | 上饶市康可得生物科技有限公司 | TEV protease variants, fusion proteins thereof, and methods of making and using |
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| 菌丝霉素MP1106融合蛋白的复性及纯化方法;王鹏举;谭焕波;苏文成;张文宇;邹培建;;生物技术通报(第09期);100-106 * |
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