CN115807022A - Intein-mediated polyprotein co-expression method - Google Patents
Intein-mediated polyprotein co-expression method Download PDFInfo
- Publication number
- CN115807022A CN115807022A CN202211003936.0A CN202211003936A CN115807022A CN 115807022 A CN115807022 A CN 115807022A CN 202211003936 A CN202211003936 A CN 202211003936A CN 115807022 A CN115807022 A CN 115807022A
- Authority
- CN
- China
- Prior art keywords
- intein
- expression
- protein
- promoter
- polyprotein
- 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.)
- Pending
Links
- 230000017730 intein-mediated protein splicing Effects 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 32
- 230000004186 co-expression Effects 0.000 title claims abstract description 15
- 108010076039 Polyproteins Proteins 0.000 title claims abstract description 14
- 230000001404 mediated effect Effects 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 97
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 71
- 230000014509 gene expression Effects 0.000 claims abstract description 64
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 17
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 17
- 239000013612 plasmid Substances 0.000 claims description 32
- 210000004027 cell Anatomy 0.000 claims description 12
- 238000003776 cleavage reaction Methods 0.000 claims description 7
- 239000013600 plasmid vector Substances 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 230000007017 scission Effects 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 4
- 210000004899 c-terminal region Anatomy 0.000 claims description 3
- 239000013613 expression plasmid Substances 0.000 claims description 3
- 241000203069 Archaea Species 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241000233866 Fungi Species 0.000 claims description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 claims description 2
- 210000004102 animal cell Anatomy 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000010276 construction Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 17
- 230000037361 pathway Effects 0.000 abstract description 4
- 238000007036 catalytic synthesis reaction Methods 0.000 abstract description 2
- 238000000855 fermentation Methods 0.000 abstract description 2
- 230000004151 fermentation Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract 1
- 201000001718 Roberts syndrome Diseases 0.000 description 19
- 208000012474 Roberts-SC phocomelia syndrome Diseases 0.000 description 19
- 238000010586 diagram Methods 0.000 description 17
- 239000013604 expression vector Substances 0.000 description 9
- 238000005001 rutherford backscattering spectroscopy Methods 0.000 description 8
- OTPDWCMLUKMQNO-UHFFFAOYSA-N 1,2,3,4-tetrahydropyrimidine Chemical compound C1NCC=CN1 OTPDWCMLUKMQNO-UHFFFAOYSA-N 0.000 description 7
- 230000004927 fusion Effects 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 108010024026 Nitrile hydratase Proteins 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000009465 prokaryotic expression Effects 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- CKLJMWTZIZZHCS-UWTATZPHSA-N D-aspartic acid Chemical compound OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229960003966 nicotinamide Drugs 0.000 description 2
- 235000005152 nicotinamide Nutrition 0.000 description 2
- 239000011570 nicotinamide Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- GZPHSAQLYPIAIN-UHFFFAOYSA-N 3-pyridinecarbonitrile Chemical compound N#CC1=CC=CN=C1 GZPHSAQLYPIAIN-UHFFFAOYSA-N 0.000 description 1
- 101100002068 Bacillus subtilis (strain 168) araR gene Proteins 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 101800001646 Protein n Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 1
- 101150044616 araC gene Proteins 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 101150092716 ectB gene Proteins 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an intein-mediated multi-protein co-expression method, wherein one or more inteins with one inactivated end are connected among a plurality of proteins to finally form a fusion protein for expression. During protein expression, the self-cleaving activity of the intein is used to release the individual protein from the fusion protein. The expression of the polyprotein by the method has three advantages: 1) The expression of each protein is high-efficient and stable, and the difference of the expression quantity is small; 2) The protein is convenient to assemble; 3) And the steric hindrance does not exist after the assembly, and the influence on the activity of the protein is small. The method is suitable for equivalent expression of polyprotein or joint expression of multiple genes in a synthetic pathway, and has important application value in the fields of substance fermentation and catalytic synthesis.
Description
Technical Field
The invention belongs to the technical field of protein engineering, and particularly relates to an intein-mediated multi-protein co-expression method.
Background
There are four traditional ways of co-expression of multiple proteins:
method 1, multiple vectors: the expression of the polyprotein was performed using the resistance of different selection markers, as shown in FIGS. 1-2. For example, the protein 1 is expressed by using an ampicillin-resistant pET28a prokaryotic expression vector, the protein 2 is expressed by using a kanamycin-resistant pET32a prokaryotic expression vector, the protein 3 is expressed by using a chloramphenicol-resistant prokaryotic expression vector, and the protein 4 is expressed by using a tetracycline-resistant expression vector. The method has the advantages that each protein can be independently expressed, and has the defects that the expression quantity of the proteins is generally different, the plasmid vectors have replication or transcription competition, and the copy number and the expression protein fluctuation of the plasmid vectors are large in the subsequent culture process. In addition, the proteins expressed by the method have larger spatial difference, which is not beneficial to the assembly of multi-subunit proteins or the common form function of the same metabolic pathway protein.
Method 3 polycistronic expression, as shown in FIGS. 5-6. The expression mode uses polycistrons in a prokaryotic expression system, namely, a plurality of proteins are expressed in one mRNA, and the gene arrangement structure of promoter-RBS-protein 1-RBS-protein 2-RBS-protein 3 and the like-terminator is utilized. In the method, all proteins share one promoter and are constructed in one expression vector, so that the method has wider applicability to the expression vector and the promoter and the plasmid vector is stable. However, this method has problems such as a large difference in expression (in particular, the later gene RBS is hardly recognized and translated by ribosomes) and a poor protein assembly effect.
Method 4, fusion expression, as shown in FIGS. 7-8. This expression mode is to fuse a plurality of proteins into one large protein, and to use the gene arrangement structure of "promoter-RBS-protein 1-protein 2-protein 3 etc. -terminator" for common functions. The fusion expression has the advantages that different proteins can be ensured to be approximately equal in molecular quantity, and are constructed on a plasmid expression vector and are relatively stable in the culture process. In addition, fusion expression allows different proteins to be spatially closer, making assembly of the proteins simpler. However, due to the intramolecular steric hindrance effect, the formed fusion protein may cause that the assembled protein complex cannot form the correct spatial conformation, and the function of the protein complex is affected.
Disclosure of Invention
The invention aims to provide an intein-mediated polyprotein co-expression method, which can obviously improve the yield of polyprotein synthesis.
In order to achieve the above object, the technical solutions adopted are as follows:
a method for intein-mediated co-expression of multiple proteins, comprising the steps of:
(1) Constructing an expression plasmid vector, wherein the plasmid vector contains a gene of a promoter-RBS-protein 1-intein-protein 2 … … with one end inactivated and an intein-protein N-terminator with one end inactivated, wherein the 'intein-protein with one end inactivated' is a repeating unit, or the plasmid vector contains a promoter-RBS-protein 1-intein-connecting peptide with C end inactivated-N end-intein-protein 2 … … -intein-connecting peptide with C end inactivated-N end-intein-protein N-terminator, wherein the 'intein-connecting peptide with C end inactivated-N end-intein-protein' is a repeating unit, and N is 2-5;
(2) Transferring the plasmid into host cells, and carrying out induced expression to display the fusion protein;
(3) The cleavage function of the intein was activated, the bacteria were removed by centrifugation and the assembled protein in the supernatant was collected.
Wherein, the protein 1 to the protein n are all protein subunits of the same protease, and the subunits are assembled into complete protein with enzyme activity after fusion expression.
Preferably, the intein is a full-length intein or a mini-intein.
Preferably, the promoter is a T7 promoter, a Lac promoter, a Tac promoter, a CMV promoter or an H1 promoter.
Preferably, the plasmid vector is pET28a (+), pET22b (+), or pBAD33.
Preferably, the host cell is a bacterium, archaea, fungus, plant cell or animal cell.
Preferably, the host cell is escherichia coli containing a T7 RNA polymerase gene, and the promoter is a T7 promoter.
The invention discloses an equimolecular protein expression technology based on self-cutting intein, which is characterized in that one or more intein with one inactivated end are connected among a plurality of proteins to finally form a fusion protein for expression. During protein expression, the self-cleaving activity of the intein is used to release the individual protein from the fusion protein. The expression of the polyprotein by the method has three advantages: 1) The expression of each protein is high-efficient and stable, and the difference of the expression quantity is small; 2) The protein is convenient to assemble; 3) And the steric hindrance does not exist after the assembly, and the influence on the activity of the protein is small. The method is suitable for equivalent expression of polyprotein or co-expression of multiple genes in a synthetic pathway, and has important application value in the fields of material fermentation and catalytic synthesis.
Drawings
FIG. 1 is a schematic diagram of a plasmid expressing various proteins in a multi-plasmid manner 1.
FIG. 2 is a schematic diagram of the principle of multi-plasmid expression of various proteins in mode 1.
FIG. 3 is a schematic diagram of a plasmid in which the same plasmid proteins are independently transcribed in mode 2.
FIG. 4 is a schematic diagram of the principle of independent transcription of the same plasmid protein in mode 2.
FIG. 5 is a schematic diagram of a plasmid in which proteins of the same plasmid are independently translated in mode 3.
FIG. 6 is a schematic diagram of the principle of independent translation of the same plasmid protein in mode 3.
FIG. 7 is a plasmid schematic of the same plasmid fusion protein in mode 4.
FIG. 8 is a schematic diagram of the same plasmid fusion protein in mode 4.
FIG. 9 is a schematic diagram of a plasmid isolated by cleavage of intein in the same plasmid fusion protein in mode 5.
FIG. 10 is a schematic diagram of the cleavage and separation of intein in the same plasmid fusion protein in the mode 5.
FIG. 11 is a schematic diagram of a plasmid with cleavage and separation of intein from the same plasmid fusion protein in mode 6.
FIG. 12 is a schematic diagram of the manner 6 for cleavage and separation of intein in the same plasmid fusion protein.
FIG. 13 is a Western blotting test for comparing the expression effects of 6 expression modes on three proteins of nitrile hydratase.
FIG. 14 is a comparison of Western blotting for detecting the expression effects of 6 expression modes on three proteins of tetrahydropyrimidine synthase.
FIG. 15 is a comparison of HPLC detection of the effects of 6 ways of nitrile hydratase expression on the synthesis of nicotinamide.
FIG. 16 is a comparison of HPLC detection results of three proteins expressing tetrahydropyrimidine synthase in synthesizing tetrahydropyrimidine products in 6 expression modes.
Detailed Description
The present invention is further illustrated by the construction and validation of an expression modification system for L593, which should not be construed as in any way limiting the scope of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
In this example, we designed six three protein expression patterns. The first mode is shown in a schematic diagram in figure 1, the expression schematic diagram is shown in figure 2, three protein genes respectively use expression plasmid vectors of three different resistance markers, the plasmids are respectively pET28a (+), pET22b (+), and pBAD33, the resistance is kanamycin, ampicillin or chloramphenicol carried by the vectors, each gene is provided with an independent promoter, RBS and terminator, the promoter is a T7 promoter, and the RBS is araC. RBS was designed website with https:// sbi. Post. Ac. Kr/utr _ library/. The three transcribed RNAs are independently translated into corresponding proteins, and finally, the proteins are respectively used for performing functions or assembled into a functional compound for performing the functions. The schematic diagram of the second mode is shown in figure 3, the schematic diagram of expression is shown in figure 4, three protein genes are positioned on the same plasmid expression vector pET28a (+), respective T7 promoters, RBSs (RBS) designed by websites and terminators are respectively used, the three RNAs after transcription are independently translated into corresponding proteins, and finally, the proteins are respectively used for performing functions or are assembled into a functional compound for performing functions. The third mode is shown in figure 5, the expression scheme is shown in figure 6, three protein genes are positioned on the same plasmid expression vector, the plasmid is pET28a, a promoter and a terminator are jointly used, but each protein gene has an independent RBS, the protein genes are transcribed into a fusion RNA, the corresponding protein is translated according to the independent RBS, and finally, the three protein genes respectively perform functions or are assembled adjacent to each other to form a functional compound to perform functions. The scheme of the fourth mode is shown in figure 7, the expression scheme is shown in figure 8, three proteins are fused into a large fusion protein, and the large fusion protein functions through intramolecular assembly after expression, but serious steric hindrance phenomenon can exist. Scheme five is schematically shown in figure 9, expression is schematically shown in figure 10, three proteins are fused into a large fusion protein, but adjacent proteins are connected by using an intein ssp DnaB (with inactivated N-terminal or inactivated C-terminal) with one end, and after a large protein assembly (with steric hindrance) is achieved, intein is translated and self-cleaved to release independent proteins (with residual intein at the end) and further assembled into a correct functional complex (with steric hindrance eliminated). The schematic diagram of the sixth mode is shown in figure 11, the expression schematic diagram is shown in figure 12, three proteins are fused into a large fusion protein, but adjacent proteins are connected by using C-terminal inactive intein-linker peptide-N-terminal inactive intein, and after translation, a large protein assembly (steric hindrance exists), the intein is released by self-cleavage to release independent proteins (the tail end does not have residual intein), and further assembled into a correct functional complex (the steric hindrance is eliminated).
Example 2 comparison of the expression ability of the engineered bacteria to express the proteins
In this example, we examined the difference comparison of the expression levels of the respective proteins by using Western blot for 6 expression patterns. We used nitrile hydratase complex NhhhA (SEQ ID No: 1)/NhB (SEQ ID No: 2)/NhG (SEQ ID No: 3) and tetrahydropyrimidine synthesis pathway gene ectoA (SEQ ID No: 4)/ectB (SEQ ID No: 5)/ectC (SEQ ID No: 6) as two complex enzymes and a multienzyme pathway to test, and constructed a plasmid (SEQ ID No: 7) having an expression gene of an intein-NhG-terminator with a promoter-RBS-NhhhhhhhhA-inactivated at one end, a plasmid (SEQ ID No: 8) having an expression gene of an intein-terminator with a promoter-RBS-NhhhhhA-inactivated at-C-inactivated intein-linker-N-inactivated intein-hhhB-C-inactivated at one end, an intein-inactivated at-N-end-inactivating peptide linker-N-inactivating intein-NhG-terminator, a plasmid (SEQ ID No: 8) having an expression gene of an intein-N-inactivated intein-NhG-terminator, a-C-linked gene linked to an expression gene of an intein-N-C-inactivated intein-terminator, and a-linked plasmid (SEQ ID No: 9-inactivated at one end, respectively. The sequence of the above expressed gene was designed by https:// sbi. After the constructed strains are induced and expressed, the His tag antibody is used for carrying out western blotting detection on each sample. The results are shown in FIGS. 13 and 14, respectively, and the ratio of the expression levels of each protein was more balanced (nearly equimolar expression) using the intein-mediated multi-protein expression pattern (pattern 5 and pattern 6) and the fusion expression pattern (pattern 4) than the conventional multi-protein expression pattern (patterns 1-3).
Example 3 comparison of Activity of Each protein expressed by engineering bacteria in 6 expression modes
In this example, we compared the effect of 6 expression patterns on the activity of each enzyme. The contents of nicotinamide, which is a nitrile hydratase product, and tetrahydropyrimidine, which is a tetrahydropyrimidine synthase product, are respectively detected by HPLC, and the change of enzyme activity is determined according to the yield.
Nitrile hydratase activity assay: the expression-inducing strain was resuspended in 10mM KPB (pH 7.4) solution, 10. Mu.L to 1.5mL of centrifuge tube and placed on a 25 ℃ metal bath. Adding 450 mu L of substrate (200 mM nicotinonitrile solution) into a centrifuge tube, fully and uniformly mixing by vortex, reacting for 10min at 25 ℃, centrifuging to remove thalli, and detecting the content of nicotinamide in the supernatant by HPLC.
Determination of tetrahydropyrimidine synthetase Activity: weighing 1g of the cells, suspending the cells in 20mL of reaction solution (50 mM PBS buffer, pH 7.5, 150mM L-Aspartic acid,100mM Glycerol), performing shaking culture at 40 ℃ and 200rmp for 3h, centrifuging the cells to remove the cells, and detecting the content of tetrahydropyrimidine in the supernatant by HPLC (high performance liquid chromatography).
As a result, as shown in fig. 15 and 16, the ability to synthesize a multi-protein product can be significantly improved using intein-mediated multi-protein expression patterns (patterns 5 and 6) compared to other protein expression patterns.
Claims (6)
1. A method for intein-mediated co-expression of multiple proteins, comprising the steps of:
(1) Construction of an expression plasmid vector containing the promoter-RBS-protein 1 Intein-proteins inactivated at one end 2 … … -intein-protein with inactivated end n Gene of terminator, or promoter-RBS-protein 1 C-terminal inactivated intein-linker peptide- -N-terminal inactivated intein-protein 2 … … -C terminal inactivated intein-linker-N terminal inactivated intein-protein n -a terminator, wherein n is 2-5;
(2) Transferring the plasmid into host cells, and carrying out induced expression to display the fusion protein;
(3) The cleavage function of the intein was activated, the bacteria were removed by centrifugation, and the assembled protein in the supernatant was collected.
2. The method of intein-mediated polyprotein co-expression of claim 1, wherein: the intein is a full-length intein or a mini-intein.
3. The method of intein-mediated polyprotein co-expression of claim 2, wherein: the promoter is a T7 promoter, a Lac promoter, a Tac promoter, a CMV promoter or an H1 promoter.
4. The method of intein-mediated polyprotein co-expression of claim 1, wherein: the plasmid vector is pET28a (+), pET22b (+), or pBAD33.
5. The method of intein-mediated polyprotein co-expression of claim 1, wherein: the host cell is a bacterium, archaea, fungus, plant cell or animal cell.
6. The intein-mediated polyprotein co-expression method of claim 5, wherein: the host cell is escherichia coli containing a T7 RNA polymerase gene, and the promoter is a T7 promoter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211003936.0A CN115807022A (en) | 2022-08-19 | 2022-08-19 | Intein-mediated polyprotein co-expression method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211003936.0A CN115807022A (en) | 2022-08-19 | 2022-08-19 | Intein-mediated polyprotein co-expression method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115807022A true CN115807022A (en) | 2023-03-17 |
Family
ID=85482507
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211003936.0A Pending CN115807022A (en) | 2022-08-19 | 2022-08-19 | Intein-mediated polyprotein co-expression method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115807022A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101595228A (en) * | 2005-07-21 | 2009-12-02 | 艾博特公司 | The method that comprises multi-gene expression and use polyprotein, precursor protein and the proteolysis of SORF construct |
| CN102676534A (en) * | 2012-04-12 | 2012-09-19 | 上海育臣生物工程技术有限公司 | Method for preparing thymosin polypeptide by using interin |
| CN107267539A (en) * | 2016-04-06 | 2017-10-20 | 沈阳药科大学 | A kind of efficient EHEC solubility expression carrier for obtaining recombinant protein |
| US20180155763A1 (en) * | 2005-09-15 | 2018-06-07 | Duke University | Non-fouling polymeric surface modification and signal amplification method for biomolecular detection |
| CN115261398A (en) * | 2022-08-02 | 2022-11-01 | 态创生物科技(广州)有限公司 | Oligopeptide expression and purification method based on phage display and intein cleavage |
-
2022
- 2022-08-19 CN CN202211003936.0A patent/CN115807022A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101595228A (en) * | 2005-07-21 | 2009-12-02 | 艾博特公司 | The method that comprises multi-gene expression and use polyprotein, precursor protein and the proteolysis of SORF construct |
| US20180155763A1 (en) * | 2005-09-15 | 2018-06-07 | Duke University | Non-fouling polymeric surface modification and signal amplification method for biomolecular detection |
| CN102676534A (en) * | 2012-04-12 | 2012-09-19 | 上海育臣生物工程技术有限公司 | Method for preparing thymosin polypeptide by using interin |
| CN107267539A (en) * | 2016-04-06 | 2017-10-20 | 沈阳药科大学 | A kind of efficient EHEC solubility expression carrier for obtaining recombinant protein |
| CN115261398A (en) * | 2022-08-02 | 2022-11-01 | 态创生物科技(广州)有限公司 | Oligopeptide expression and purification method based on phage display and intein cleavage |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Adachi et al. | Post-transcriptional pseudouridylation in mRNA as well as in some major types of noncoding RNAs | |
| Albayrak et al. | Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation | |
| EP2880171B1 (en) | Methods and compositions for controlling gene expression by rna processing | |
| Huang et al. | Direct inhibition of tombusvirus plus-strand RNA synthesis by a dominant negative mutant of a host metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, in yeast and plants | |
| EP2995683B1 (en) | Method for producing peptide library, peptide library, and screening method | |
| Salim et al. | Requirement of upstream Hfq-binding (ARN) x elements in glmS and the Hfq C-terminal region for GlmS upregulation by sRNAs GlmZ and GlmY | |
| CN107012121A (en) | Carry the structure of the stable cell lines of orthogonal tRNA/ aminoacyl tRNA synthetases | |
| EP2217702A2 (en) | Directed evolution using proteins comprising unnatural amino acids | |
| Mendez-Perez et al. | A translation-coupling DNA cassette for monitoring protein translation in Escherichia coli | |
| US20230056404A1 (en) | Systems and methods for protein expression | |
| KR102755545B1 (en) | Promoter | |
| Liu et al. | Bicistronic expression strategy for high‐level expression of recombinant proteins in Corynebacterium glutamicum | |
| Zhang et al. | Synthetic sRNA‐based engineering of Escherichia coli for enhanced production of full‐length immunoglobulin G | |
| Yu et al. | Development of a novel platform for recombinant protein production in Corynebacterium glutamicum on ethanol | |
| Harris et al. | Determination and control of low‐level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system | |
| JP7028986B2 (en) | Tandem DNA element that can increase protein synthesis efficiency | |
| CN115807022A (en) | Intein-mediated polyprotein co-expression method | |
| Yumerefendi et al. | Library-based methods for identification of soluble expression constructs | |
| Zhang et al. | Construction of an expression vector that uses the aph promoter for protein expression in Corynebacterium glutamicum | |
| CN101928719A (en) | T vector for screening protein soluble expression and its construction method and application | |
| Ke et al. | Activation of the promoter of the fengycin synthetase operon by the UP element | |
| CN115340996A (en) | Co-expression method of multi-subunit protein by using specific enzyme cutting site | |
| Hohnholz et al. | Recombinant multicopy plasmids in yeast–interactions with the endogenous 2 μm | |
| Fages-Lartaud et al. | Standard Intein Gene Expression Ramps (SIGER) for protein-independent expression control | |
| CN116286926B (en) | Construction of double-fluorescence reporter plasmid and application of double-fluorescence reporter plasmid in detection of intracellular c-di-GMP level of 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 |