CN117126875B - 4-isopropylbenzoic acid induced expression system and induced expression method suitable for pichia pastoris - Google Patents
4-isopropylbenzoic acid induced expression system and induced expression method suitable for pichia pastoris Download PDFInfo
- Publication number
- CN117126875B CN117126875B CN202311362429.0A CN202311362429A CN117126875B CN 117126875 B CN117126875 B CN 117126875B CN 202311362429 A CN202311362429 A CN 202311362429A CN 117126875 B CN117126875 B CN 117126875B
- Authority
- CN
- China
- Prior art keywords
- cymr
- pichia pastoris
- promoter
- expression
- gcwcuo1
- 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
- 230000014509 gene expression Effects 0.000 title claims abstract description 89
- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 46
- CKMXAIVXVKGGFM-UHFFFAOYSA-N p-cumic acid Chemical compound CC(C)C1=CC=C(C(O)=O)C=C1 CKMXAIVXVKGGFM-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 15
- 108010034634 Repressor Proteins Proteins 0.000 claims abstract description 30
- 102000009661 Repressor Proteins Human genes 0.000 claims abstract description 30
- 101150045505 cymR gene Proteins 0.000 claims abstract description 29
- 230000001105 regulatory effect Effects 0.000 claims abstract description 20
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 18
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 18
- 230000006698 induction Effects 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 35
- 210000004027 cell Anatomy 0.000 claims description 18
- 230000001939 inductive effect Effects 0.000 claims description 17
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 210000000349 chromosome Anatomy 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 239000013613 expression plasmid Substances 0.000 claims description 3
- 239000000411 inducer Substances 0.000 abstract description 16
- 230000000694 effects Effects 0.000 abstract description 9
- 230000001276 controlling effect Effects 0.000 abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 5
- 229910052799 carbon Inorganic materials 0.000 abstract description 5
- 239000008103 glucose Substances 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 3
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000005090 green fluorescent protein Substances 0.000 description 9
- 230000033228 biological regulation Effects 0.000 description 8
- 230000035897 transcription Effects 0.000 description 8
- 238000013518 transcription Methods 0.000 description 8
- 230000002103 transcriptional effect Effects 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000010276 construction Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 5
- 241000235648 Pichia Species 0.000 description 5
- 230000010354 integration Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 4
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 3
- 102000004533 Endonucleases Human genes 0.000 description 3
- 108010042407 Endonucleases Proteins 0.000 description 3
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 3
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000007222 ypd medium Substances 0.000 description 3
- 102100036826 Aldehyde oxidase Human genes 0.000 description 2
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000009830 intercalation Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 101710118538 Protease Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241001041887 Pseudomonas putida F1 Species 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108700026226 TATA Box Proteins 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000002687 intercalation Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000000754 repressing effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- 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
- C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of agricultural biology, in particular to a 4-isopropylbenzoic acid induced expression system and an induced expression method suitable for pichia pastoris. The invention selects the strong constitutive promoter P GCW14 And a promoter P containing the operator CuO and having a promoter activity was selected GCWCuO1 The method comprises the steps of carrying out a first treatment on the surface of the At the same time by truncating the strong promoter P GAP Screening expression element P capable of regulating and controlling expression of repressor protein CymR moderately GAP200 cymR. P-based GCWCuO1 And P GAP200 /cymRAnd (5) constructing a novel Pichia pastoris stringency induction expression system. The pichia pastoris induction expression system according to the present application uses inexpensive glucose as a carbon source and low-concentration inexpensive nontoxic 4-isopropylbenzoic acid as a carbon source when expressing recombinant proteinsThe inducer can realize the safe and low-cost production of the recombinant protein, especially the recombinant protein toxic to pichia pastoris cells.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to a 4-isopropylbenzoic acid induced expression system and an induced expression method suitable for pichia pastoris.
Background
Pichia pastoris is one of the most widely used recombinant protein expression hosts at present, and more than 4000 recombinant proteins are expressed; with the development of synthetic biology, pichia pastoris has also been used as a chassis cell for efficient synthesis of active substances. Inducible promoters allow for the expression of recombinant proteins in a time-sequential manner, and are commonly used for the expression of recombinant proteins, particularly those that are toxic to the host cell, and for the regulated expression of specific genes in synthetic biology. Pichia methanol-inducible promoter P AOX1 The advantages of high-strength transcription and strict induction by methanol are widely applied, but methanol as an inducer is inflammable, explosive and toxic and has great potential safety hazard in production.
In view of the above drawbacks, many researchers have developed P AOX1 But not satisfactory results. Other inducible promoters reported in many documents have not been used in general because of the disadvantages of low transcription strength and low stringency. Rhamnose inducible promoter P LRA3 And P LRA4 Is strictly induced by rhamnose, wherein P LRA3 Has higher transcription strength, can be used for time sequence expression of target genes, but rhamnose as a carbon source and an inducer limits the wide application of the promoter due to higher price. Therefore, the development of low-cost inducible promoters which are not induced by methanol and are not inhibited by glucose has important application value in the safe and efficient production and synthesis biology of recombinant proteins in pichia pastoris.
From Pseudomonas putida f1%pseudomonas putidaf1 CymR) can bind to the operator CuO on the chromosome, resulting in the inability of genes downstream of the operator CuO to be transcribed, the inability of the protein to be transcribedCan express; after 4-isopropylbenzoic acid (Cumate) binds to CymR, cymR dissociates from CuO, allowing transcription and expression of genes downstream of the operator CuO. The construction of induction systems based on Cumate and CymR/CuO is generally to embed CuO in specific positions of a constitutive promoter sequence, so as to construct a promoter which contains CuO and has biological functions; elements for moderate expression of the repressor protein CymR are constructed on plasmids or chromosomes from constitutive promoters. The induction expression system constructed according to the elements shows strict induction in escherichia coli, corynebacterium glutamicum and mammalian cells to be applied, but has not been reported in yeast cells with important application value.
Disclosure of Invention
It is an object of the present application to provide a 4-isopropylbenzoic acid inducible expression system suitable for pichia pastoris.
It is another object of the present application to provide a method for inducing expression of recombinant proteins in pichia pastoris.
The 4-isopropylbenzoic acid induced expression system suitable for pichia pastoris comprises a promoter P with CuO as an operator and promoter activity GCWCuO1 And regulatory expression element P for realizing moderate expression of repressor protein CymR GAP200 cymR, wherein the promoter P GCWCuO1 The nucleotide sequence of (2) is shown as SEQ ID No. 12, and the repressor protein CymR regulates and expresses the element P GAP200 The nucleotide sequence of/cymR is shown as SEQ ID No. 21.
The method for inducing expression of recombinant protein in pichia pastoris according to the present invention comprises the steps of:
promoter P containing operator CuO and having promoter Activity GCWCuO1 Ligating the coding gene of the target protein to the promoter P GCWCuO1 Regulating the expression of the gene encoding the target protein, wherein the promoter P GCWCuO1 The nucleotide sequence of (2) is shown as SEQ ID No. 12;
regulating expression element P of repressor protein CymR GAP200 cymR, the promoter linked to the gene encoding the target proteinP GCWCuO1 Integration into the Pichia chromosome, wherein the repressor CymR regulates the expression element P GAP200 The nucleotide sequence of/cymR is shown as SEQ ID No. 21;
and adding 4-isopropylbenzoic acid into the culture medium of the recombinant pichia pastoris cells to induce and express the recombinant target protein.
According to the technical scheme, the invention relates to two functional elements, namely a promoter for regulating and controlling the moderate expression of a repressor protein CymR and a promoter containing an operator CuO and having transcriptional activity for regulating and controlling the expression of a target gene, and the four-isopropylbenzoic acid induction expression system can be realized when the two elements exist on a pichia pastoris chromosome at the same time. In the invention, the two elements are constructed on an integrated plasmid, and when the plasmid is introduced into a pichia pastoris cell, the two elements are integrated at the same site (gas 1 site) of a chromosome; other measures can be taken to integrate the two fragments into the pichia chromosome in succession.
According to a specific embodiment of the present invention, the method for inducing expression of recombinant proteins in pichia pastoris of the present application comprises the steps of:
promoter P containing CuO of operator and having promoter activity GCWCuO1 Is connected with plasmid containing coding gene of target protein and then connected with repressor protein CymR regulatory expression element P GAP200 The cymR connection, 4-isopropylbenzoic acid induced expression plasmid of pichia pastoris is obtained, wherein the promoter P GCWCuO1 The nucleotide sequence of (2) is shown as SEQ ID No. 12, and the repressor protein CymR regulates and expresses the element P GAP200 The nucleotide sequence of/cymR is shown as SEQ ID No. 21;
introducing the 4-isopropylbenzoic acid induced expression of the pichia pastoris into pichia pastoris cells to obtain recombinant pichia pastoris cells;
and adding 4-isopropylbenzoic acid to 5-150 mug/mL in the culture medium of the recombinant pichia pastoris cell, and inducing to express the recombinant target protein.
According to the technical scheme of the invention, the strong constitutive promoter P is selected GCW14 And is combined withPromoter P containing operator CuO and having promoter Activity was selected GCWCuO1 The method comprises the steps of carrying out a first treatment on the surface of the At the same time by truncating the strong promoter P GAP Screening expression element P capable of regulating and controlling expression of repressor protein CymR moderately GAP200 cymR. P-based GCWCuO1 And P GAP200 /cymRAnd (5) constructing a novel Pichia pastoris stringency induction expression system. When the pichia pastoris induction expression system expresses recombinant proteins, the recombinant proteins, especially the recombinant proteins toxic to pichia pastoris cells, can be safely produced at low cost by taking the low-cost glucose as a carbon source and the low-concentration low-cost nontoxic 4-isopropylbenzoic acid as an inducer.
The high-efficiency expression of the recombinant protein is realized by using a strong constitutive promoter, and the invention uses a strong constitutive promoter P GCW14 Based on (a) design an inducible promoter P GCWCuO1 . P in the presence of the uninhibited protein CymR GCWCuO1 The high-efficiency expression of the downstream gene can be realized; in the presence of CymR, cymR binds to P GCWCuO1 The upper operator CuO blocks transcription of the RNA polymerase, thereby repressing downstream gene expression. In an inducible expression system, the level of repressor protein expression largely determines the effect of inducible expression; repressor expression levels are too low and they do not bind efficiently to the operator, resulting in induction of expression systems exhibiting higher leaky expression; when the expression level of the repressor is too high, part of the repressor is still combined with the operator after the inducer molecule is added, so that the expression of the target gene is repressed, and the induction expression intensity is not high. The repressor CymR regulatory expression element P in the present invention GAP200 cymR, which can achieve moderate expression of the repressor protein cymR, is fully combined with the operator CuO in the absence of an inducer, and has lower leakage expression of the expression system; after the inducer is added, the inducer can be recombined with the CymR to dissociate from the operator CuO, thereby realizing the high-efficiency expression of the target gene.
Drawings
FIG. 1 shows the operator CuO in promoter P GCW14 Upper embedding location, wherein a: promoter P GCWCuO1 And B: promoter P GCWCuO2 ；
FIG. 2 shows the phenotype of different strains grown in YPD medium, wherein A: pichia pastoris GS115, B: chromosomal integration P GCWCuO1 Regulation and controlgfpExpressed pichia pastoris, C: chromosomal integration P GCWCuO2 Regulation and controlgfpExpressed pichia pastoris;
FIG. 3 shows the fluorescence intensity of different strains in liquid MD medium containing Cumate at different concentrations, at different culture times, wherein 50 bp, 200 bp and 480 bp are regulatory repressor genes, respectivelycymRExpressed promoter P GAP Cumate concentrations in the medium were 0. Mu.g/ml and 25. Mu.g/ml, respectively, and induction times were 16 h and 36h, respectively.
Detailed Description
The strains and media used in the following examples: pichia pastoris GS115 @Pichia pastorisGS 115); YPD Medium (g/L): yeast powder 10, peptone 20 and glucose 20; MD Medium reference (Current Genetics,2019, 65:785-798).
Example 1 design of CuO-containing PGCW14
P GCW14 Is a strong constitutive promoter, and is transformed into an inducible promoter, so that an operator CuO which can be combined by a repressor CymR is embedded in a specific position; on the other hand, the promoter may lose the gene transcription initiation activity after intercalation into CuO. Therefore, there is a need to optimize the obtaining of CuO-intercalated and transcriptionally active P GCW14 。
Eukaryotic promoters are more complex in structure than prokaryotic promoters, and comprise mostly core promoter elements and upstream regulatory elements. Wherein the core promoter element comprises a TATA box and a transcription initiation point, and ensures the combination of RNA polymerase and the initiation of DNA transcription. Embedding foreign DNA sequences on the original promoter affects RNA polymerase binding and transcription efficiency. The invention constructs a promoter which can be induced by 4-isopropylbenzoic acid and needs to be positioned on a strong promoter P GCW14 The upper intercalating repressor CymR-bondable operator CuO, and CuO insertion site selection and P-pair thereof GCW14 The influence of transcriptional activity is of paramount importance. At P GCW14 Is embedded with an operator CuO after the TATA sequence to construct P GCWCuO1 The method comprises the steps of carrying out a first treatment on the surface of the At P GCW14 Embedding an operator CuO before the TATA sequence to construct P GCWCuO2 。
1.1 promoter P GCWCuO1 Construction
1.1.1 obtaining DNA fragment P GCWL
The Pichia pastoris GS115 chromosome is used as a template, and a primer P is used GCWL -F1(SEQ ID No:1)、P GCWL Amplification of P by R1 (SEQ ID No: 2) GCW14 DNA fragment P of 1-727 bp GCWL 。
1.1.2 splicing to obtain a CuO-containing DNA fragment P GCWR1
With P GCWR1 -F1(SEQ ID No:3)、P GCWR1 -F2(SEQ ID No:4)、P GCWR1 -F3(SEQ ID No:5)、P GCWR1 -R1(SEQ ID No:6)、P GCWR1 -R2(SEQ ID No:7)、P GCWR1 PCR is carried out using R3 (SEQ ID No: 8) as template and primer, where the primer serves as template at the same time, and the complete DNA sequence is spliced by the primer to form the CuO-containing DNA fragment P GCWR1 。
1.1.3 fusion to obtain a CuO-containing promoter P GCWCuO1
P obtained in 1.1.1 step GCWL And 1.1.2 step P GCWR1 As a template, primer P GCWL -F1、P GCWR1 R3 performs Overlap-PCR, P GCWL And P GCWR1 Fused to full length P containing CuO GCW14 It is named P GCWCuO1 ，CuO。
1.2 promoter P GCWCuO2 Construction
1.2.1 construction of DNA fragment P GCWR2
The Pichia pastoris GS115 chromosome is used as a template, and a primer P is used GCWR2 -F1(SEQ ID No:9)、P GCWR2 -F2(SEQ ID No:10)、P GCWR2 R (SEQ ID No: 11) amplifying a CuO-containing DNA fragment P GCWR2 。
1.2.2 fusion construction of full-Length promoter P containing CuO GCWCuO2
P obtained in 1.1.1 step GCWL And 1.2.1 step P obtained GCWR2 As a template, primer P GCWL -F1、P GCWR2 R is subjected to Overlap-PCR, P GCWL And P GCWR2 Fused to full length P containing CuO GCW14 It is named P GCWCuO2 。
1.3 promoter P GCWCuO1 And P GCWCuO2 Transcriptional Activity verification
To detect P GCWCuO1 (SEQ ID No: 12) and P GCWCuO2 (SEQ ID No: 13) transcriptional Activity with Green fluorescent protein GenegfpThe expression intensity of the reporter gene is characterized. P (P) GCWCuO1 And P GCWCuO2 Respectively inscribe inAscI andEcor I digested and ligated with the plasmid pGHGAP/gfp treated by the same endonuclease (T4 ligase), thereby constructing promoters P, respectively GCWCuO1 And P GCWCuO2 Regulation and controlgfpThe expressed plasmids pGCWCuO1/gfp and pGHGCWCuO2/gfp. After the plasmid was treated with the endonuclease SwaI, pichia conversion was performed according to the conversion protocol in the Pichia expression Manual of Invitrogen corporation, and transformants were selected on MD medium. Positive transformants verified by PCR were streaked on YPD medium and the green fluorescence intensity in the cells was observed. As shown in FIG. 2, the integration on the chromosome is represented by P GCWCuO1 Regulation and controlgfpThe expressed strain GS115-pGCWCuO1/gfp was green (B in FIG. 2), while the control strain (Pichia pastoris GS 115) (A in FIG. 2) and chromosomal integration consisted of P GCWCuO2 Regulation and controlgfpThe expressed strain GS115-pGCWCuO1/gfp was not green fluorescent (C in FIG. 2). The results show that P GCWCuO1 Still has good transcriptional activity and can be used for constructing a Cumate induction system.
Example 2 design of the expression intensity control Unit of the repressor protein CymR
Repressor protein CymR binds to promoter P GCWCuO1 The CuO sequence of (2) represses the transcription of the downstream gene, and the dissociation degree and dissociation degree of the CymR are regulated by adding the time and the dosage of an inducer Cumate, so that the expression intensity of the downstream gene is regulated. If the expression level of the repressor protein is too high, the concentration of the inducer which is required to be dissociated from the operator is high; if the expression level of the repressor protein is too low, there will be a weak repression effect and leakage expression will occur in the absence of the inducer. Thus, controlling the expression intensity of the repressor protein is also one of the key factors in constructing an inducible expression system.
P GAP Is a commonly used strong constitutive promoter, which is selected in the present invention to regulate expression of the repressor protein CymR. To achieve expression of different intensities of CymR, promoter P containing 50, bp, 200, 480, bp upstream of the start codon was constructed by promoter truncation GAP50 、P GAP200 、P GAP480 . With the decrease of the strength of the promoter, the transcriptional activity of the promoter is gradually reduced, so that the expression of different strengths of CymR is realized.
The pichia pastoris chromosome is used as a template, and the forward primer P is respectively used GAP480 -F(SEQ ID No:14)、P GAP200 -F(SEQ ID No:15)、P GAP50 -F (SEQ ID No: 16) and reverse primer P GAP R (SEQ ID No: 17) amplification of promoters P of different lengths GAP480 、P GAP200 、P GAP50 . The 3' -end is added with the coding gene cymR-nls of the repressor protein cymR of the protein nuclear localization signal.
The Saccharomyces cerevisiae chromosome is used as a template, and the terminator Tact is amplified by using primers Tact-F (SEQ ID No: 18) and Tact-R (SEQ ID No: 19). With P GAP480 CymR-nls and Tact as templates, primer P was used GAP480 Performing overlay-PCR on F and Tact-R to construct CymR expression unit P GAP480 cymR. Similarly, cymR expression unit P was constructed GAP200 cymR and P GAP50 /cymR。
Example 3, cumate Induction System construction and application Effect
Plasmid pGCWCuO1/gfp is treated by endoprotease AscI and then regulated and expressed by element P with repressor CymR GAP480 /cymR（P GAP480 /cymR ，SEQ ID No:20）、P GAP200 /cymR（P GAP200 /cymR，SEQ ID No:21）、P GAP50 /cymR（P GAP50 Seamless cloning is carried out on/cymR and SEQ ID No. 22), and plasmids pGAPGCW480/gfp-cymR, pGAPGCW200/gfp-cymR and pGAPGCW50/gfp-cymR are respectively constructed. After the plasmid is treated by endonuclease SwaI, pichia pastoris GS115 is transformed, and transformants are screened by MD culture medium. The transformant was verified by PCR (primer P GAP50 After F and the universal primer 3-AOX 1), positive transformants were selected. Selecting pGAPGCW480/gfp-cymR, pGAPGCW200/gfp-cymR and pGAPGCW50/gfp-cymR respectivelygfpAndcymRpositive transformant with expression unit integrated in Pichia pastoris GS115 chromosome is inoculated in liquid YPD culture medium and cultured in shaking at 30 deg.C until OD 600 5. Respectively inoculating 100 μl of the bacterial solutions into liquid MD culture medium, shake culturing at 30deg.C to OD 600 1, after Cumate was added to the medium to 0. Mu.g/ml and 25. Mu.g/ml, OD of the bacterial liquid was measured for 16 h and 36 hours 600 And green fluorescence value GFP, and GFP/OD 600 The magnitude of the values characterizes the intensity of green fluorescent protein expressed by the cells.
As can be seen from FIG. 3, from P GAP50 When cymR expression is regulated, in the presence or absence of inducer Cumate, the thallus has strong green fluorescence value, which indicates that the repressor protein CymR can not be effectively expressed due to too short length of the promoter, and the repressor protein CymR is expressed by P GCWCuO1 Regulation and controlgfpAnd expressed. From P GAP200 When cymR expression is regulated, GFP/OD is obtained after 16 h and 36h are cultured without addition of inducer 600 The lower value indicates that the green fluorescent protein is weakly expressed, and also indicates that the repressor protein CymR is efficiently expressed, and the repressor protein CymR is combined with the promoter P GCWCuO1 CuO represses the expression of green fluorescent protein; GFP/OD after addition of inducer 600 The numerical value is obviously increased, cumate is combined with CymR to cause the Cumate to be dissociated from CuO, and P is used for preparing the CuO GCWCuO1 Regulation and controlgfpAnd expressed. From P GAP480 GFP/OD with or without the addition of an inducer when controlling cymR expression 600 The values are low, and the phenomenon is derived from P GAP480 Has strong transcriptional activity, and can over-express the repressor protein, even if 25 mug/ml Cumate is added, the repressor protein can not be completely dissociated from CuO, resulting in P GCWCuO1 Regulation and controlgfpAnd cannot be expressed effectively.
When the liquid MD medium contains 25 mug/ml Cumate, the growth of the bacterial cells is inhibited; further increasing the Cumate concentration may promote dissociation of the repressor protein CymR from CuO, thereby allowinggfpExpression, however, cell growth is significantly inhibited, and thus is not suitable for practical production. Notably, the Cumate induction concentration is closely related to the culture medium used for the growth characteristics of pichia pastoris, and when the pichia pastoris is cultured by a nutrient-rich culture medium (such as YPD), the growth of the pichia pastoris is not obviously inhibited when the Cumate concentration is increased to 200 ug/ml.
In general, a Pichia pastoris expression system for 4-isopropylbenzoic acid was constructed by screening for a promoter for the expression strength of the repressor protein CymR and constructing a promoter containing the operator CuO and having transcriptional activity. The induction expression system uses cheap glucose as a carbon source and 4-isopropylbenzoic acid with low safety and low concentration as an inducer, so that the low-cost and safe production of the recombinant protein can be realized, and the induction expression system can also be used for the time sequence expression of key proteins in the synthetic biology.
The above embodiments are only used for explaining the technical solution of the present application, and do not limit the protection scope of the present application.
Claims (4)
1. A4-isopropylbenzoic acid induced expression system suitable for pichia pastoris is characterized by comprising a promoter P containing an operator CuO and having a promoter activityGCWCuO1 And a repressor CymR regulatory expression element PGAP200cymR, wherein the promoter PGCWCuO1 The nucleotide sequence of (2) is shown as SEQ ID No. 12, and the repressor protein CymR regulates and expresses the element PGAP200The nucleotide sequence of/cymR is shown as SEQ ID No. 21.
2. A method for inducing expression of a recombinant protein in pichia pastoris, said method comprising the steps of:
promoter P containing operator CuO and having promoter ActivityGCWCuO1 Ligating the coding gene of the target protein to the promoter PGCWCuO1Regulating the expression of the gene encoding the target protein, wherein the promoter PGCWCuO1 The nucleotide sequence of (2) is shown as SEQ ID No. 12;
regulating expression element P of repressor protein CymRGAP200cymR, the promoter P linked to the gene encoding the target proteinGCWCuO1Integrating into chromosome of Pichia pastoris cell to obtain recombinant Pichia pastoris cell, wherein the repressor protein CymR regulates expression element PGAP200The nucleotide sequence of/cymR is shown as SEQ ID No. 21;
and adding 4-isopropylbenzoic acid into the culture medium of the recombinant pichia pastoris cells to induce and express the recombinant target protein.
3. The method of inducing expression of recombinant proteins in pichia pastoris according to claim 2, wherein the method comprises the steps of:
promoter P containing operator CuO and having promoter ActivityGCWCuO1 Is connected with plasmid containing coding gene of target protein and then connected with repressor protein CymR regulatory expression element PGAP200The cymR connection is carried out, and 4-isopropylbenzoic acid induction expression plasmid of pichia pastoris is constructed and obtained;
introducing the 4-isopropylbenzoic acid induced expression plasmid of the pichia pastoris into pichia pastoris cells to obtain recombinant pichia pastoris cells;
and adding 4-isopropylbenzoic acid into the culture medium of the recombinant pichia pastoris cells to induce and express the recombinant target protein.
4. The method for inducing expression of recombinant proteins in pichia pastoris according to claim 2 or 3, wherein 4-isopropylbenzoic acid is added to 5 μg/mL to 150 μg/mL in the culture medium of the recombinant pichia pastoris cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311362429.0A CN117126875B (en) | 2023-10-20 | 2023-10-20 | 4-isopropylbenzoic acid induced expression system and induced expression method suitable for pichia pastoris |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311362429.0A CN117126875B (en) | 2023-10-20 | 2023-10-20 | 4-isopropylbenzoic acid induced expression system and induced expression method suitable for pichia pastoris |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117126875A CN117126875A (en) | 2023-11-28 |
| CN117126875B true CN117126875B (en) | 2024-02-06 |
Family
ID=88856683
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311362429.0A Active CN117126875B (en) | 2023-10-20 | 2023-10-20 | 4-isopropylbenzoic acid induced expression system and induced expression method suitable for pichia pastoris |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117126875B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734061A (en) * | 2016-03-25 | 2016-07-06 | 中国农业科学院生物技术研究所 | Rhamnose induced promoter separated from pichia pastoris and application thereof |
| KR20170017415A (en) * | 2015-08-06 | 2017-02-15 | 경희대학교 산학협력단 | Cassette for gene expression regulated by Cumate Operon |
| WO2017087731A1 (en) * | 2015-11-18 | 2017-05-26 | Industrial Microbes, Inc. | Functional expression of monooxygenases and methods of use |
| CN109136119A (en) * | 2017-06-19 | 2019-01-04 | 武汉生物技术研究院 | Microorganisms and uses thereof |
| CN112877352A (en) * | 2021-02-08 | 2021-06-01 | 华南理工大学 | Cumate induction system suitable for corynebacterium glutamicum, plasmid vector constructed by induction system and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2619989C (en) * | 2005-08-23 | 2014-06-17 | National Research Council Of Canada | Regulation of heterologous recombinant protein expression in methylotrophic and methanotrophic bacteria |
| JP2021511834A (en) * | 2018-01-23 | 2021-05-13 | サザン・アイ・エクイップメント | Expression vector and method |
-
2023
- 2023-10-20 CN CN202311362429.0A patent/CN117126875B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170017415A (en) * | 2015-08-06 | 2017-02-15 | 경희대학교 산학협력단 | Cassette for gene expression regulated by Cumate Operon |
| WO2017087731A1 (en) * | 2015-11-18 | 2017-05-26 | Industrial Microbes, Inc. | Functional expression of monooxygenases and methods of use |
| CN105734061A (en) * | 2016-03-25 | 2016-07-06 | 中国农业科学院生物技术研究所 | Rhamnose induced promoter separated from pichia pastoris and application thereof |
| CN109136119A (en) * | 2017-06-19 | 2019-01-04 | 武汉生物技术研究院 | Microorganisms and uses thereof |
| CN112877352A (en) * | 2021-02-08 | 2021-06-01 | 华南理工大学 | Cumate induction system suitable for corynebacterium glutamicum, plasmid vector constructed by induction system and application |
Non-Patent Citations (7)
| Title |
|---|
| Guo,Y..Komagataella pastoris isolate EAMORG09 chromosome 1.《Genbank》.2023,FEATURES、ORIGIN. * |
| How to Choose the Right Inducible Gene Expression System for Mammalian Studies?;Kallunki T等;《Cells》;第8卷(第8期);doi: 10.3390/cells8080796 * |
| Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning;Broussau S等;《Mol Ther Methods Clin Dev》;第40卷;第40-57页 * |
| The cumate gene-switch: a system for regulated expression in mammalian cells;Mullick A等;《BMC Biotechnol》;第6卷;doi: 10.1186/1472-6750-6-43 * |
| Yeast (S.cerevisiae) actin gene, complete cds.;Gallwitz D.等;《EMBL》;SEQUENCE * |
| 低温下不同饱和度冻结砂岩动态力学行为试验研究;许军策等;《煤炭科学技术》;第51卷(第9期);第88-99页 * |
| 芽胞杆菌中重组蛋白分泌表达的优化策略及研究进展;曹雅蒙等;《微生物学通报》;第48卷(第9期);第3095-3105页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117126875A (en) | 2023-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Trassaert et al. | New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica | |
| AU2016262494B2 (en) | Expression constructs and methods of genetically engineering methylotrophic yeast | |
| Leuker et al. | Sequence and promoter regulation of the PCK1 gene encoding phosphoenolpyruvate carboxykinase of the fungal pathogen Candida albicans | |
| Remaut et al. | Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication | |
| EP2390334B1 (en) | Expression system | |
| FI127283B (en) | Expression system for eukaryotic microorganisms | |
| JP6910358B2 (en) | Yeast cells | |
| Liu et al. | Engineering an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species | |
| JP2011083296A5 (en) | ||
| EP2106447A1 (en) | Method for methanol independent induction from methanol inducible promoters in pichia | |
| WO2019128837A1 (en) | Improved promoter and carrier composed of same and application thereof | |
| JP5010598B2 (en) | Tetrahymena heat-inducible promoter and use thereof | |
| Li et al. | A novel vector for construction of markerless multicopy overexpression transformants in Pichia pastoris | |
| Gregor et al. | Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea | |
| AU2005293516A1 (en) | Homologous amdS genes as selectable marker | |
| WO2023011659A1 (en) | Transcription regulation system based on crispri and crispra, and establishment method therefor and use thereof | |
| Liu et al. | Use of transcription activator-like effector for efficient gene modification and transcription in the filamentous fungus Trichoderma reesei | |
| CN117126875B (en) | 4-isopropylbenzoic acid induced expression system and induced expression method suitable for pichia pastoris | |
| EP0317254A1 (en) | Yeast expression system | |
| Bass et al. | Application of the E. coli trp promoter | |
| CN109913490B (en) | Enhanced expression vector suitable for corynebacterium glutamicum | |
| Tamayo‐Ramos et al. | Enhanced production of Aspergillus niger laccase‐like multicopper oxidases through mRNA optimization of the glucoamylase expression system | |
| CN113481231B (en) | Construction method of strain for producing recombinant protein containing unnatural amino acid and strain obtained by construction method | |
| CN116064627A (en) | Engineering bacterium for specifically removing arsenic | |
| JP6640226B2 (en) | Expression element, expression cassette, and vector containing them |
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 |