CN109971777B - Method for enhancing protein expression in prokaryotic system - Google Patents
Method for enhancing protein expression in prokaryotic system Download PDFInfo
- Publication number
- CN109971777B CN109971777B CN201910234754.6A CN201910234754A CN109971777B CN 109971777 B CN109971777 B CN 109971777B CN 201910234754 A CN201910234754 A CN 201910234754A CN 109971777 B CN109971777 B CN 109971777B
- Authority
- CN
- China
- Prior art keywords
- protein
- asn
- sequence
- expression
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for improving the expression of a target protein in a prokaryotic system, which is characterized in that the expression of the target protein is guided by using a signal peptide sequence of a specific protein or a mutant sequence of the signal peptide sequence or a chimeric sequence of the signal peptide sequence or a polypeptide sequence designed by artificial simulation, so that the expression quantity of the target protein in the expression system is increased and/or the solubility of the target protein is enhanced and/or the folding of a spatial structure of the target protein in the prokaryotic system is improved. The signal peptide sequence of the specific protein or the mutant sequence of the signal peptide sequence or the chimeric sequence of the signal peptide sequence or the polypeptide sequence designed by artificial simulation cannot or cannot efficiently guide the transmembrane transport of the target protein to the outside of the cell. The methods provided by the invention can be used for expression of a variety of proteins of interest.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method capable of improving target protein expression in a prokaryotic system.
Background
The prokaryotic expression system is a very mature foreign protein expression system at present, and various commercial strains and plasmids (vectors) can be used. For example, pET expression systems based on the T7 promoter. The system fully utilizes the high efficiency of the T7 promoter and the adjustability of the lactose operon to realize the high efficiency induction expression of the exogenous protein in special Escherichia coli strains, such as BL21 (DE 3). In addition, other bacterial-based high-efficiency expression systems are also very mature, such as the bacillus subtilis (Bacillus subtilis) expression system, the Brevibacillus expression system, and the like.
The high efficiency of prokaryotic expression system can increase the yield of target protein, but the level of functional expression (Functional expression) varies with the difference of protein types, especially for some exogenous proteins with special or complex spatial structures, such as membrane proteins, etc. "functional expression" is defined herein as the ability of a protein of interest to be expressed in a soluble manner, thereby forming a reasonable three-dimensional structure and exhibiting normal function. Since the protein folding mechanism of the prokaryotic system itself mostly belongs to Post-translational folding (Post-translational folding mechanism), i.e. protein or polypeptide is synthesized first and spatial folding is performed after synthesis is completed. A large amount of nascent peptides (i.e., polypeptide chains that have just completed synthesis in a protein synthesis system) accumulate in large amounts to form insoluble inclusion bodies before forming the correct spatial structure, and thus cannot form the correct spatial structure. The aggregated polypeptide chain generally does not have the normal function of the protein, and needs further denaturation and renaturation to recover the space structure, thereby obtaining the corresponding function. However, the success rate of renaturation of proteins varies greatly depending on the kind of proteins, and the renaturation process requires more time periods, manpower, material resources, or financial resources.
In the use of prokaryotic systems for protein expression of interest, to increase protein solubility and prevent inclusion body formation, various soluble tags can be used for regulation, such as MBP (maltose-binding protein) (di Guanetal 1988; kapush and Waugh 1999) tag, GST (glutethione-S-transferase) (Smith and Johnson, 1988) tag, SUMO (small ubiquitin related modifier) (button et al 2005; marblestone et al 2006), nusA (N-utilization substance) (Davis et al 1999) tag and Trx (Thioredoxin) (Lavallie et al 1993) and the like. After the label is connected to the N end of the target protein, the solubility of the target protein can be increased, so that the target protein is easier to form a correct spatial structure. Some tags also increase the expression level of the target protein. Of course, the positive effect difference of different labels on the expression of different target proteins is larger, so that the development of the novel label has good application prospect. The present invention calls for and proposes a novel polypeptide tag that can be used to improve the expression of a protein of interest in a prokaryotic system.
Disclosure of Invention
The technical problem solved by the invention is to provide a method capable of improving the expression of a target protein in a prokaryotic system, and a specific guide sequence is used for guiding the expression of the target protein, so that the expression quantity of the target protein in the expression system is increased and/or the solubility of the target protein is enhanced and/or the folding of the spatial structure of the target protein in the prokaryotic system is improved. Specific leader sequences refer to naturally occurring signal peptide sequences of part of the protein or mutant sequences of signal peptide sequences or chimeric sequences of signal peptide sequences or polypeptide sequences designed by artificial simulation or multiple combinations of these polypeptide sequences or these sequences with other polypeptide sequences.
The technical scheme of the invention is as follows:
first, the method for improving the expression of a target protein in a prokaryotic system provided by the invention is to guide the expression of the target protein by using a signal peptide sequence of a specific protein or a mutant sequence of the signal peptide sequence or a chimeric sequence of the signal peptide sequence or a polypeptide sequence designed by artificial simulation, thereby improving the expression amount of the target protein in the expression system and/or enhancing the solubility of the target protein and/or improving the folding of the spatial structure of the target protein in the prokaryotic system.
Further, in the above scheme, the specific guide sequence refers to the signal peptide of the specific protein, specifically refers to the signal peptide sequence of a part of the naturally occurring protein or a mutant sequence of the signal peptide sequence or a chimeric sequence of the signal peptide sequence or a polypeptide sequence designed by artificial simulation, and the nucleotide sequence of the specific guide sequence is shown in SEQ ID NO. 1-7.
Further, in the above scheme, the specific guide sequence further comprises a mutation sequence of the signal peptide sequence of the specific protein or a sequence with homology higher than 30% between the chimeric sequence of the signal peptide sequence and the signal peptide sequence, as shown in SEQ ID NO. 8-18.
Further, in the above scheme, the specific guide sequence further comprises a mutant sequence of the specific protein signal peptide sequence or a chimeric sequence of the signal peptide sequence or a multiple combination sequence of polypeptide sequences designed by artificial simulation, as shown in SEQ ID NO. 19-22.
Further, in the above-described embodiment, the specific leader sequence further includes a signal peptide sequence of the aforementioned specific protein or a mutant sequence of the signal peptide sequence or a chimeric sequence of the signal peptide sequence or a multiple combination of polypeptide sequences designed by artificial simulation, or a multiple combination of these sequences with other polypeptide sequences. In the polypeptide sequence combination process, the mutant sequence of the signal peptide sequence of the specific protein or the chimeric sequence of the signal peptide sequence or the polypeptide sequence designed by artificial simulation can be appropriately modified. The polynucleotide sequence encoding a specific guide sequence which is capable of increasing the expression level of a target protein in an expression system and/or enhancing the solubility of the target protein and/or improving the folding of the spatial structure of the target protein in a prokaryotic system can be deduced from the genetic code. The preference of codon usage can also be adjusted according to the type of host cell. The polynucleotide sequence can be prepared by conventional molecular biological methods or chemical synthesis methods well known in the art.
Further, in the above scheme, the specific guide sequence and the target protein sequence are spliced to form a fusion protein, and the specific guide sequence is located at the N-terminal of the target protein; the specific guide sequence may be directly linked to the target protein sequence, or a spacer sequence may be present. The spacer sequence may be a non-coding sequence, or may be a coding sequence of another gene, such as an operon (opera) -like structure, or a 2A-like structure (Szymczak-Workman AL, vignali KM, vignali DAA,2012; ha S-H, et AL 2010).
Further, in the above-described scheme, the spacer sequence in the fusion protein may be an amino acid sequence encoded by a polynucleotide sequence of a restriction enzyme recognition site; other sources of polypeptide sequences are also possible.
Further, in the above-described scheme, the polynucleotide sequence encoding the specific guide sequence may be expressed under the control of regulatory sequences such as a promoter, an enhancer, a terminator, an operator, and a 2A polypeptide.
Further, in the above-described scheme, the polynucleotide sequence encoding the fusion protein formed by splicing the specific guide sequence with the target protein sequence may be placed in any DNA vector, expressed by a corresponding regulatory sequence, such as the 3' end of a promoter for promoting the expression of the target gene, or directly substituted for the existing gene sequence in the vector.
Further, the DNA vector is a natural or artificial plasmid vector, a natural or artificial phage genome, a natural or artificial microorganism genome, a natural or artificial eukaryotic genome, or other natural or artificial DNA fragments
Further, in the above-described embodiments, the target protein includes a protein or polypeptide that can be expressed in a prokaryotic system by biological techniques, including a protein or polypeptide that is native to the host system, and also includes a protein or polypeptide that is not native to the host system.
Further, in the above-described embodiments, the prokaryotic system includes a target protein expression system based on a living prokaryotic cell, and also includes an in vitro protein expression system constructed using all or a part of components required for protein synthesis in a prokaryotic cell.
Further, in the above-described embodiments, when the prokaryotic system is a living prokaryotic cell, the specific guide sequence may not or may not be capable of efficiently transporting the target protein to the periplasmic space or extracellular space.
The beneficial effects of the invention are as follows: the invention firstly provides a polypeptide sequence which can improve the expression effect of target protein in a prokaryotic system. Such polypeptide sequences include signal peptide sequences of specific proteins or mutated sequences of signal peptide sequences or chimeric sequences of signal peptide sequences or artificially designed polypeptide sequences as well as various combinations between such sequences or various combinations between such sequences and other polypeptides. Such polypeptides and combinations thereof can be used to improve the expression of a protein of interest in a prokaryotic system, including increasing the expression of the protein of interest in a prokaryotic system and/or enhancing the solubility of the protein of interest and/or promoting the correct formation of the spatial structure of the protein of interest. The polypeptide sequence can be used as a powerful supplement of the existing protein expression tag.
Drawings
FIG. 1 is a graph showing comparison of expression of IEGFP and EGFP in E.coli BL21 (DE 3) star strain in example 3; wherein, "E" represents EGFP; "IE" means IEGFP; "1-6" means 6 independent clones; "M" represents the standard protein molecular weight, "-" represents the negative control (prepared with BL21 (DE 3) star strain containing pET28a plasmid); the arrows indicate the bands of interest for EGFP and IEGFP, respectively.
FIG. 2 is a graph comparing the expression of three tag-directed EGFP in E.coli BL21 (DE 3) star strain in example 4; wherein, "IPTG-" means that IPTG is not added; "IPTG+" means induction of expression by addition of IPTG; "1-6" means 6 independent clones; "M" represents the standard protein molecular weight, "-" represents the negative control (prepared with BL21 (DE 3) star strain containing pET28a plasmid); the arrows indicate the destination strips, respectively.
FIG. 3 is a comparison of the expression of various tag-directed EGFP in E.coli BL21 (DE 3) star strain in example 8; wherein "S" represents a soluble moiety; "IS" means insoluble moieties; "M" represents the standard protein molecular weight, "-" represents the negative control (prepared with BL21 (DE 3) star strain containing pET28a plasmid); the arrows indicate the destination strips, respectively.
Detailed Description
The present invention will be described in detail by the following examples for better understanding of the technical aspects of the present invention, but the scope of the present invention is not limited thereto.
Example 1: construction of fusion protein IEGFP
This example exemplifies a fusion protein IEGFP in which the N-terminal signal peptide (SEQ ID NO: 1) of the naturally occurring protein Cry1Ia was spliced with the EGFP protein.
The IEGFP protein is characterized by: the N-terminal signal peptide sequence of Cry1Ia is positioned at the N-terminal of EGFP protein; no other redundant amino acids exist between the N-terminal signal peptide (SEQ ID NO: 1) of Cry1Ia and the amino acid sequence of EGFP protein. The polynucleotide sequence encoding the IEGFP protein can be obtained from at least three pathways: 1) The polynucleotide sequences of the N-terminal signal peptide encoding Cry1Ia and the EGFP protein amino acid sequence are respectively obtained by polymerase chain reaction (PCR reaction), and then the polynucleotide sequences are spliced together by a method of overlapping PCR (Horton, R.M. et al BioTechniques, 1990) to form a complete fusion gene; 2) The polynucleotide sequences of the N-terminal signal peptide and EGFP protein amino acid sequence of coding Cry1Ia are respectively obtained by polymerase chain reaction (PCR reaction), and then are spliced into a complete fusion gene by a recombinant cloning method; 3) The two polynucleotide sequences are directly spliced by artificial synthesis. All three methods are conventional molecular biological methods or conventional synthetic methods well known in the art.
Example 2: construction of prokaryotic cell expression vector p28aD-Iegfp of fusion protein IEGFP
This example illustrates a prokaryotic expression vector for the fusion protein described in example 1 herein. First, bamHI (5 ') and SacI (3') sites were added to both ends of the Iegfp gene, respectively. The gene was then inserted into the pET28aDel vector (Gao et al 2011) at the corresponding site to form a p28aD-Iegfp expression vector. As a control, bamHI (5 ') and SacI (3') sites were added to both ends of the egfp gene, respectively, to form a p28aD-egfp expression vector. Two plasmids were transformed into E.coli BL21 (DE 3) strain to obtain BLp28aD-Iegfp and BLp28aD-egfp strains, respectively.
Example 3: expression and comparison of IEGFP protein and EGFP protein
BLp28aD-Iegfp and BLp28aD-egfp strains were streaked, respectively, in solid LB medium (Luria-Bertani medium) containing 50. Mu.g/mL kanamycin, and cultured overnight at 37 ℃. 6 individual clones were selected for each strain, individually picked and inoculated into liquid LB medium containing 50. Mu.g/mL kanamycin, and shake cultured (37 ℃,200 rpm) to OD600 = 0.8. IPTG (isopropylβ -D-1-thiohinging) was added to the medium at a final concentration of 1 mmol/L. Expression was induced for 4 hours at 16 ℃. After the induction expression is finished, each tube of bacterial liquid is sucked into a new 2mL centrifuge tube, and the bacterial cells are collected after the bacterial liquid is centrifuged at 12000rpm for 5 min. 160. Mu.L of PBS buffer (137mM NaCl,2.7mM KCl,10mM Na) 2 HPO 4 ,and 2mM KH 2 PO 4 pH 7.4) and 40. Mu.L of 5 Xprotein loading buffer (250 mM Tris (pH 6.8), 10% (w/v) SDS,0.5% (w/v) BPB,50% (v/v) glycerol, 5% (v/v) beta-mercaptoethanol) were added and mixed. After boiling for 10min, centrifugation was carried out at 12000rpm for 5min, 10. Mu.L of the supernatant was spotted, and SDS-PAGE was performed to analyze the protein expression. As shown in FIG. 1, EGFP expression level is low, molecular weight is very close to host cell self protein, and IEGFP expression level is significantly higher than EGFP.
Example 4: differential tag directed EGFP expression comparison
The egfp gene was inserted into the 3' end of MBP (major binding protein) and NusA (N-utilization substance protein A) tag coding sequences, respectively, and the two obtained fusion genes were inserted into BamHI (5 ') and SacI (3 ') sites of pET28aDel vector, respectively, to form p28aD-MBP-egfp and p28aD-NusA-egfp expression vectors. The two plasmids were transformed into E.coli BL21 (DE 3) strain, respectively, to obtain BLp28aD-MBP-egfp and BLp28aD-NusA-egfp strains.
BLp28aD-Iegfp, BLp28aD-egfp, BLp28aD-MBP-egfp and BLp28aD-NusA-egfp strains were streaked, respectively, in solid LB medium (Luria-Bertani medium) containing 50. Mu.g/mL kanamycin, and cultured overnight at 37 ℃. 6 individual clones were selected for each strain, individually picked and inoculated into liquid LB medium containing 50. Mu.g/mL kanamycin, and shake cultured (37 ℃,200 rpm) to OD600 = 0.8. IPTG (isopropylβ -D-1-thiohinging) was added to the medium at a final concentration of 1 mmol/L. Expression was induced for 4 hours at 16 ℃. After the induction expression is finished, each tube of bacterial liquid is sucked into a new 2mL centrifuge tube, and the bacterial cells are collected after the bacterial liquid is centrifuged at 12000rpm for 5 min. 160. Mu.L of PBS buffer (137mM NaCl,2.7mM KCl,10mM Na) 2 HPO 4 ,and 2mM KH 2 PO 4 pH 7.4) and 40. Mu.L of 5 Xprotein loading buffer (250 mM Tris (pH 6.8), 10% (w/v) SDS,0.5% (w/v) BPB,50% (v/v) glycerol, 5% (v/v) beta-mercaptoethanol) were added and mixed. After boiling for 10min, centrifugation was carried out at 12000rpm for 5min, 10. Mu.L of the supernatant was spotted, and SDS-PAGE was performed to analyze the protein expression. The results show that all three tags can enhance the expression level of EGFP protein. As a result, as shown in FIG. 2, the expression level of IEGFP was inferior to that of NusA-EGFP.
Example 5: construction of fusion protein HI-EGFP
This example illustrates the complex expression tag (with the amino acid sequence SEQ ID NO: 19) formed by a naturally occurring N-terminal signal peptide (SEQ ID NO: 1) of the Cry1Ia protein and a histidine tag (6 histidines) that directs the expression of the EGFP protein to form the fusion protein HI-EGFP.
The HI-EGFP protein is characterized in that: the three polypeptides are formed by the following steps: histidine tag (6 histidines), cry1Ia protein N-terminal signal peptide, EGFP sequence. The encoding polynucleotide sequence of the HI-EGFP protein can be obtained from at least three pathways: 1) The polynucleotide sequences of the N-terminal signal peptide and EGFP protein of the coding Cry1Ia are respectively obtained by polymerase chain reaction (PCR reaction), wherein when the coding sequence of the N-terminal signal peptide of the Cry1Ia is amplified, a coding sequence of a histidine tag is added into a 5' primer of the coding sequence, and then the coding sequence and the coding sequence are spliced together by an overlapping PCR method (Horton, R.M. et al BioTechniques, 1990) to form a complete fusion gene; 2) The polynucleotide sequences of the N-terminal signal peptide and the EGFP protein of the coding Cry1Ia are respectively obtained by polymerase chain reaction (PCR reaction), wherein when the coding sequence of the N-terminal signal peptide of the Cry1Ia is amplified, a coding sequence of a histidine tag is added into a 5' primer, and then the coding sequence is spliced into a complete fusion gene by a recombinant cloning method; 3) The three polynucleotide sequences are directly spliced by artificial synthesis. All three methods are conventional molecular biological methods or conventional synthetic methods well known in the art.
Example 6: cry1Ia protein N-terminal signal peptide or mutant thereof participates in different composite tags
This example exemplifies the different combinations of the N-terminal signal peptide (SEQ ID NO: 1) or mutants thereof of a naturally occurring protein Cry1Ia, which was then spliced with EGFP to form a fusion protein. Three complex expression tags are exemplified, namely IIHI (with the amino acid sequence of SEQ ID NO: 20), I4HI (with the amino acid sequence of SEQ ID NO: 21) and I8HI (with the amino acid sequence of SEQ ID NO: 22). Fusion proteins IIHI-EGFP, I4HI-EGFP and I8HI-EGFP spliced with EGFP proteins respectively from the three composite tags.
The IIHI tag is characterized in that: the three Cry1Ia N-terminal signal peptide sequences (SEQ ID NO: 1) are concatenated with one histidine tag (containing 6 histidines) inserted at the C-terminus of the second signal peptide. The I4HI tag is characterized in that: the sequences of SEQ ID NO. 18 and SEQ ID NO.1 are connected in series, and 6 histidine is contained between the two sequences. The I8HI tag is characterized in that: the sequences of SEQ ID NO. 17 and SEQ ID NO.1 are connected in series, and 6 histidine is contained between the two sequences.
The coding sequence of the above composite expression tag can be obtained from at least two approaches: 1) Firstly, obtaining an independent label in each composite expression label through polymerase chain reaction (PCR reaction) (the coding sequence of a histidine label is directly designed in a primer without being amplified separately), and then respectively splicing by a method of overlapping PCR (Horton, R.M. et al BioTechniques, 1990) to form a complete composite label; 2) The corresponding composite expression label is directly obtained by artificial synthesis. After the coding sequence of the composite expression tag is obtained, the coding sequence is spliced with the coding polynucleotide sequence of EGFP protein respectively through overlapping PCR reaction. The coding poly-and glutamate sequences of the three fusion proteins IIHI-EGFP, I4HI-EGFP and I8HI-EGFP can also be synthesized directly. In summary, the polynucleotide sequences encoding the fusion proteins described above are obtained in a variety of ways, but are well known in the art as conventional molecular biology methods or conventional synthetic methods.
Example 7: construction of HI-EGFP, IIHI-EGFP, I4HI-EGFP and I8HI-EGFP protein expression vectors
Polynucleotide sequences encoding HI-EGFP, IIHI-EGFP, I4HI-EGFP and I8HI-EGFP were inserted into the BamHI (5 ') and SacI (3') sites of the pET28aDel vector, respectively, to form p28aD-HI-EGFP, p28aD-IIHI-EGFP, p28aD-I4HI-EGFP and p28aD-I8HI-EGFP expression vectors. The four plasmids were transformed into E.coli BL21 (DE 3) strain, respectively, to obtain BLp28aD-HI-egfp, BLp28aD-IIHI-egfp, BLp28aD-I4HI-egfp and BLp28aD-I8HI-egfp strains.
Example 8: expression and comparison of multiple fusion proteins
BLp28aD-Iegfp, BLp28aD-egfp, BLp28aD-HI-egfp, BLp28aD-IIHI-egfp, BLp28aD-I4HI-egfp and BLp28aD-I8HI-egfp strains were streaked, respectively, in solid LB medium (Luria-Bertani medium) containing 50 μg/mL kanamycin, and incubated overnight at 37 ℃. Each strain was picked up as a single clone and inoculated into liquid LB medium containing 50. Mu.g/mL kanamycin, and cultured with shaking (37 ℃ C., 200 rpm) overnight. Transfer to 50mL of liquid LB medium containing 50 μg/mL kanamycin, and continue culturing until od600=0.8. IPTG (isopropyless-D-1-thiogalactopyranoside) was added at a final concentration of 1mmol/L and induced for 4 hours (16 ℃ C.).
Each bottle of cells was collected separately and washed repeatedly 3 times with PBS buffer. Finally20mL of pre-chilled PBS buffer (137mM NaCl,2.7mM KCl,10mM Na) 2 HPO4,and 2mM KH 2 PO 4 pH 7.4) resuspended cells. After the cells were sonicated, centrifuged at 12000rpm for 15min, the supernatant and pellet were separated. The pellet was fully resuspended with an equal volume of PBS buffer. mu.L of each supernatant or pellet was pipetted into each tube and 40. Mu.L of 5 Xprotein loading buffer (250 mM Tris (pH 6.8), 10% (w/v) SDS,0.5% (w/v) BPB,50% (v/v) glycerol, 5% (v/v) beta-mercaptoethanol) was added and mixed. After boiling for 10min, centrifugation was performed at 12000rpm for 5min, 10. Mu.L of the supernatant was spotted, and SDS-PAGE analysis was performed.
As shown in FIG. 3, the results showed that EGFP and HI-EGFP alone were expressed very low, while the other four fusion proteins (IEGFP, IIHI-EGFP, I4HI-EGFP and I8 HI-EGFP) were expressed higher, and SDS-PAGE was easily resolved. In addition, all four fusion proteins are expressed in soluble form.
The specific sequences of SEQ ID NO.1-SEQ ID NO.22 are shown below:
SEQ ID NO.1：MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIELQNINHEDCLKM
SEQ ID NO.2：MKPNNQNNHQTLLSNVTVDKPFADALKNETTMELKNSNHEDCLKM
SEQ ID NO.3：MKLKNPDKHQSLSSNAKVDKIATDSLKNETDIELKNMNNEDYLRM
SEQ ID NO.4：MKLKNPDKHQTLSSNAKVDKIATDSLKNETDIELKNMNNEDYLRM
SEQ ID NO.5：MKSKNQNMYRSFSSNATVDKSFTDPLEHNTNMELQNSNHEDCLKM
SEQ ID NO.6：MKLKNPDKHQSLSSNAKVDKIATDSLKNETDIELKNINHEDFLRM
SEQ ID NO.7：MKSKNQNMHQSLSNNATVDKNFTGSLENNTNTELQNFNHEG
SEQ ID NO.8：MNLNNQDNHQSFSSNAKVDKISTDSLKNETDIELQNINHEDCLKM
SEQ ID NO.9：MNLNNQDNHQSFSSNAKVDKISTDSLKNETDIELQNINHEDSLCI
SEQ ID NO.10：MNLNNQDNHQSFSSNANVDNISTDSLNNETDIELQNINHEDCLKM
SEQ ID NO.11：MNLNNQDNHQSFSSNANVDNISTDSLNNETDIELQNINHEDSLCI
SEQ ID NO.12：MNLKNQDKHQSFSSNAKVDNISTDSLNNETDIELQNINHEDCLKM
SEQ ID NO.13：MNLKNQDKHQSFSSNAKVDNISTDSLNNETDIELQNINHEDSLCI
SEQ ID NO.14：MNLKNQDKHQSFSSNAKVDNISTDSLQNETDIELQNINHEDSLCV
SEQ ID NO.15：MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIELQNINHEDSLCV
SEQ ID NO.16：MNLKNQDKHQSFSSNAKVDNISTDSLQNETDIELQNINHEDCLKM
SEQ ID NO.17：MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIELQNIRIEDSLCI
SEQ ID NO.18：MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIELQNINHEDSLCI
SEQ ID NO.19：MHHHHHHKLKNQDKHQSFSSNAKVDKISTDSLKNETDIEFQNINHEDCLKM
SEQ ID NO.20：
MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIELQNINHEDCLKMMKLKNQ
DKHQSFSSNAKVDKISTDSLKNETDIELQNINHEDCLKMHHHHHHMKLKNQ
DKHQSFSSNAKVDKISTDSLKNETDIEFQNINHEDCLKM
SEQ ID NO.21：
MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIELQNINHEDSLCIHHHHHH
MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIEFQNINHEDCLKM
SEQ ID NO.22：
MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIELQNIRIEDSLCIHHHHHH
MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIEFQNINHEDCLKM
finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> Shanxi university of agriculture
<120> a method for enhancing protein expression in a prokaryotic system
<130> none of
<170> PatentIn version 3.5
<210> 1
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence
<400> 1
Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met
35 40 45
<210> 2
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence
<400> 2
Met Lys Pro Asn Asn Gln Asn Asn His Gln Thr Leu Leu Ser Asn Val
1 5 10 15
Thr Val Asp Lys Pro Phe Ala Asp Ala Leu Lys Asn Glu Thr Thr Met
20 25 30
Glu Leu Lys Asn Ser Asn His Glu Asp Cys Leu Lys Met
35 40 45
<210> 3
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence
<400> 3
Met Lys Leu Lys Asn Pro Asp Lys His Gln Ser Leu Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ala Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Lys Asn Met Asn Asn Glu Asp Tyr Leu Arg Met
35 40 45
<210> 4
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence
<400> 4
Met Lys Leu Lys Asn Pro Asp Lys His Gln Thr Leu Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ala Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Lys Asn Met Asn Asn Glu Asp Tyr Leu Arg Met
35 40 45
<210> 5
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence
<400> 5
Met Lys Ser Lys Asn Gln Asn Met Tyr Arg Ser Phe Ser Ser Asn Ala
1 5 10 15
Thr Val Asp Lys Ser Phe Thr Asp Pro Leu Glu His Asn Thr Asn Met
20 25 30
Glu Leu Gln Asn Ser Asn His Glu Asp Cys Leu Lys Met
35 40 45
<210> 6
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence
<400> 6
Met Lys Leu Lys Asn Pro Asp Lys His Gln Ser Leu Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ala Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Lys Asn Ile Asn His Glu Asp Phe Leu Arg Met
35 40 45
<210> 7
<211> 41
<212> PRT
<213> A
<221> LIPID
<222> (1)..(41)
<223> protein sequence
<400> 7
Met Lys Ser Lys Asn Gln Asn Met His Gln Ser Leu Ser Asn Asn Ala
1 5 10 15
Thr Val Asp Lys Asn Phe Thr Gly Ser Leu Glu Asn Asn Thr Asn Thr
20 25 30
Glu Leu Gln Asn Phe Asn His Glu Gly
35 40
<210> 8
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 8
Met Asn Leu Asn Asn Gln Asp Asn His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met
35 40 45
<210> 9
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 9
Met Asn Leu Asn Asn Gln Asp Asn His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Ser Leu Cys Ile
35 40 45
<210> 10
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 10
Met Asn Leu Asn Asn Gln Asp Asn His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Asn Val Asp Asn Ile Ser Thr Asp Ser Leu Asn Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met
35 40 45
<210> 11
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 11
Met Asn Leu Asn Asn Gln Asp Asn His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Asn Val Asp Asn Ile Ser Thr Asp Ser Leu Asn Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Ser Leu Cys Ile
35 40 45
<210> 12
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 12
Met Asn Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Asn Ile Ser Thr Asp Ser Leu Asn Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met
35 40 45
<210> 13
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 13
Met Asn Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Asn Ile Ser Thr Asp Ser Leu Asn Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Ser Leu Cys Ile
35 40 45
<210> 14
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 14
Met Asn Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Asn Ile Ser Thr Asp Ser Leu Gln Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Ser Leu Cys Val
35 40 45
<210> 15
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 15
Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Ser Leu Cys Val
35 40 45
<210> 16
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 16
Met Asn Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Asn Ile Ser Thr Asp Ser Leu Gln Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met
35 40 45
<210> 17
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 17
Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Arg Ile Glu Asp Ser Leu Cys Ile
35 40 45
<210> 18
<211> 45
<212> PRT
<213> A
<221> LIPID
<222> (1)..(45)
<223> protein sequence mutation
<400> 18
Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Ser Leu Cys Ile
35 40 45
<210> 19
<211> 51
<212> PRT
<213> A
<221> LIPID
<222> (1)..(51)
<223> protein sequence complexing
<400> 19
Met His His His His His His Lys Leu Lys Asn Gln Asp Lys His Gln
1 5 10 15
Ser Phe Ser Ser Asn Ala Lys Val Asp Lys Ile Ser Thr Asp Ser Leu
20 25 30
Lys Asn Glu Thr Asp Ile Glu Phe Gln Asn Ile Asn His Glu Asp Cys
35 40 45
Leu Lys Met
50
<210> 20
<211> 141
<212> PRT
<213> A
<221> LIPID
<222> (1)..(141)
<223> protein sequence complexing
<400> 20
Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met Met Lys Leu
35 40 45
Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala Lys Val Asp
50 55 60
Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile Glu Leu Gln
65 70 75 80
Asn Ile Asn His Glu Asp Cys Leu Lys Met His His His His His His
85 90 95
Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
100 105 110
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
115 120 125
Glu Phe Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met
130 135 140
<210> 21
<211> 96
<212> PRT
<213> A
<221> LIPID
<222> (1)..(96)
<223> protein sequence complexing
<400> 21
Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Glu Asp Ser Leu Cys Ile His His His
35 40 45
His His His Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser
50 55 60
Ser Asn Ala Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu
65 70 75 80
Thr Asp Ile Glu Phe Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met
85 90 95
<210> 22
<211> 96
<212> PRT
<213> A
<221> LIPID
<222> (1)..(96)
<223> protein sequence complexing
<400> 22
Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Arg Ile Glu Asp Ser Leu Cys Ile His His His
35 40 45
His His His Met Lys Leu Lys Asn Gln Asp Lys His Gln Ser Phe Ser
50 55 60
Ser Asn Ala Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu
65 70 75 80
Thr Asp Ile Glu Phe Gln Asn Ile Asn His Glu Asp Cys Leu Lys Met
85 90 95
Claims (3)
1. A method for improving the expression of a protein of interest in a prokaryotic system, characterized in that the expression of the protein of interest is guided by a specific guide sequence, and the expression of the protein of interest in the prokaryotic system is improved;
the amino acid sequence of the specific guide sequence is selected from the group consisting of:
an amino acid sequence shown as SEQ ID NO. 1;
the amino acid sequence shown in SEQ ID NO. 20-22.
2. A method according to claim 1, wherein the protein of interest comprises a protein or polypeptide that can be expressed in a prokaryotic system by a related technique, including a protein or polypeptide that is native to the host system or a protein or polypeptide that is not native to the host system.
3. A method according to claim 1, wherein the prokaryotic system comprises a protein expression system of interest in a host of living prokaryotic cells and further comprises an in vitro protein expression system constructed using all or part of the components required for protein synthesis in the prokaryotic cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910234754.6A CN109971777B (en) | 2019-03-26 | 2019-03-26 | Method for enhancing protein expression in prokaryotic system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910234754.6A CN109971777B (en) | 2019-03-26 | 2019-03-26 | Method for enhancing protein expression in prokaryotic system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109971777A CN109971777A (en) | 2019-07-05 |
| CN109971777B true CN109971777B (en) | 2023-04-25 |
Family
ID=67080852
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910234754.6A Active CN109971777B (en) | 2019-03-26 | 2019-03-26 | Method for enhancing protein expression in prokaryotic system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109971777B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480460B (en) * | 2022-01-28 | 2023-06-16 | 山西农业大学 | Method for simultaneously enhancing expression quantity and solubility of target protein in prokaryotic system |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6103511A (en) * | 1996-10-04 | 2000-08-15 | University Of Georgia Research Foundation, Inc. | Lichenase and coding sequences |
| CN103228670A (en) * | 2010-12-13 | 2013-07-31 | 先正达参股股份有限公司 | Cry1I proteins and genes for insect control |
| CN108409867A (en) * | 2018-03-02 | 2018-08-17 | 山西农业大学 | A method of it can be used in improving fluorescent protein fluorescence signal strength |
-
2019
- 2019-03-26 CN CN201910234754.6A patent/CN109971777B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6103511A (en) * | 1996-10-04 | 2000-08-15 | University Of Georgia Research Foundation, Inc. | Lichenase and coding sequences |
| CN103228670A (en) * | 2010-12-13 | 2013-07-31 | 先正达参股股份有限公司 | Cry1I proteins and genes for insect control |
| CN108409867A (en) * | 2018-03-02 | 2018-08-17 | 山西农业大学 | A method of it can be used in improving fluorescent protein fluorescence signal strength |
Non-Patent Citations (1)
| Title |
|---|
| 信号肽及其在蛋白质表达中的应用;韦雪芳等;《生物技术通报》;20061226(第06期);第38-42页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109971777A (en) | 2019-07-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3362050B2 (en) | Method for mass production of antimicrobial peptides | |
| Korepanova et al. | Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein | |
| EP1220933B1 (en) | Purification of recombinant proteins fused to multiple epitopes | |
| CN113195521A (en) | Mtu Delta I-CM intein variants and uses thereof | |
| US8304210B2 (en) | Method for the secretory production of heterologous protein in Escherichia coli | |
| US20160168226A1 (en) | Process for production of insulin and insulin analogues | |
| CN109971777B (en) | Method for enhancing protein expression in prokaryotic system | |
| US20220411764A1 (en) | Thioredoxin mutant, preparation method thereof, and application thereof in production of recombinant fusion protein | |
| US6740507B2 (en) | Engineering of living cells for the expression of holo-phycobiliprotein-based constructs | |
| CN114480460B (en) | Method for simultaneously enhancing expression quantity and solubility of target protein in prokaryotic system | |
| JP7016552B2 (en) | How to increase the secretion of recombinant proteins | |
| US11267863B2 (en) | N-terminal fusion partner for producing recombinant polypeptide, and method for producing recombinant polypeptide using same | |
| JPWO2020045530A1 (en) | Soluble expression of proteins using peptide tags | |
| EP1981978B1 (en) | Affinity polypeptide for purification of recombinant proteins | |
| US20110287484A1 (en) | Expression vector containing the major envelope protein p9 of cystovirus phi6 as a fusion partner, and process for producing a membrane protein using the same | |
| CN107629112B (en) | High-affinity LC3 protein targeting peptide and application thereof | |
| US20160009779A1 (en) | Novel fusion tags and expression vector system for the expression of human parathyroid hormone (rhpth) | |
| Zhang et al. | Molecular cloning and expression analysis of a new bilin lyase: The cpcT gene encoding a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the β-subunit of phycocyanin in Arthrospira platensis FACHB314 | |
| KR101505697B1 (en) | Membrane protein expression vector comprising major envelope protein p9 of systovirus phi12 as a fusion partner and method for producing membrane protein using the same | |
| CN114807101B (en) | Fusion protein containing bovine enterokinase light chain protein, expression vector and recombinant engineering bacteria thereof | |
| KR102009709B1 (en) | Method of preparing human parathyroid hormone 1-84 using fusion polypeptide | |
| KR102084054B1 (en) | method of increasing signal sequence-mediated secretion of recombinant proteins | |
| CA2529282C (en) | Recombinant igf expression systems | |
| KR20230120361A (en) | Peptide tags increaing the solubility of proteins | |
| Oh et al. | Use of carboxypeptidase Y propeptide as a fusion partner for expression of small polypeptides in Escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |