CN113122565A - Novel practical pichia pastoris secretory expression vector, construction method and kit - Google Patents
Novel practical pichia pastoris secretory expression vector, construction method and kit Download PDFInfo
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- 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
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a novel practical pichia pastoris secretory expression vector, a construction method and a kit, wherein a vector framework is derived from pPICZaA, and the final vector framework is only about 4K, so that the subsequent molecular cloning operation experiment efficiency is facilitated. The resistance of the prokaryotic cells is kanamycin, which is different from the resistance of bleomycin of a pPICZaA vector, the bleomycin is expensive and colonies with uneven growth, commonly called large and small spots, often appear in the transformation and screening process, thereby bringing great difficulty to the cloning and screening work. The mutant AOX promoter which is proved to be capable of obviously improving the protein fermentation expression amount is selected as the carrier, and the protein expression amount can be obviously improved particularly in large-scale fermentation. The vector of the invention selects MF4I signal peptide, can efficiently secrete and express target protein, enables the subsequent separation and purification work to be simple and rapid, and further saves the purification cost.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a novel and practical pichia pastoris secretory expression vector, a construction method and a kit.
Background
At present, Pichia pastoris is the most common protein expression system second only to escherichia coli at present, is widely applied to aspects of laboratory-scale protein preparation, characterization, structure analysis and the like, and thousands of proteins are successfully expressed in the Pichia pastoris system. Through the development of the years, pichia pastoris expression vectors have been widely applied to the fields of food, medicine and industrial enzyme preparations, and are a very important recombinant protein expression system. The advantages of the pichia pastoris expression system mainly include: 1. multiple post-translational modifications applicable to a clear genetic background; 2. can grow rapidly in a medium with known components; 3. is easy to use and is cheaper than mammalian cells; 4. easy to perform high-density culture and fermentation, and high expression level.
There is no natural plasmid in pichia pastoris body, so the recombinant carrying exogenous gene must be integrated in chromosome to realize the expression of exogenous gene, can maintain the stability of gene and can produce gene with various copy number. However, the recombinant strain with single copy number is often expressed in a low amount, and the expression level of the target protein is generally increased by increasing the copy number of the exogenous gene in pichia pastoris, and the following 3 methods are used: 1. inserting end-to-end multi-copy reading frames into the expression vector; 2. inserting kan gene into the vector, wherein the G418 resistance level and the gene copy number are in a linear relation; 3. shble gene (bleomycin) is inserted into the vector, the gene can express and play a biological function in large intestine and yeast, and the resistance level and the gene copy number are in linear relation in yeast.
The commonly used multicopy plasmids are pPIC9k and pPICZa, wherein the pPIC9k vector carries kan gene, can be screened by G418, but is larger and has more than 9000bp, and the molecular biological operation for in vitro multicopy construction is more complicated and inconvenient; the pPICZaA vector is small and only has the size of 3600bp, but the vector uses bleomycin which is expensive and often has colonies with uneven growth in the transformation screening process, commonly called large and small spots, and brings great difficulty to the cloning screening work.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) in the prior art, the carrier skeleton is inconvenient for subsequent molecular cloning operation experiment efficiency.
(2) In the prior art, bleomycin is expensive, and colonies with uneven growth, commonly called large and small spots, often appear in the transformation screening process, which brings great difficulty to the cloning screening work.
(3) In the prior art, the protein expression quantity cannot be obviously improved in large-scale fermentation.
(4) In the prior art, the carrier can not ensure that the subsequent separation and purification work is simple and rapid, and the purification cost is high.
The difficulty in solving the above problems and defects is:
(1) searching a proper means to modify the yeast vector into a proper size;
(2) effective replacement of part of the original part of the vector is carried out, so that the screening and transformation of yeast recombinants are more facilitated.
The significance of solving the problems and the defects is as follows:
(1) the yeast carrier is miniaturized, so that the molecular biological operation is facilitated;
(2) bleomycin is not used, so that the cost is reduced, and the problem of large and small spots is solved;
(3) the strength of the promoter is changed, and the protein expression quantity is improved;
(4) the construction efficiency and success rate of the multi-copy carrier are improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a novel and practical pichia pastoris secretory expression vector, a construction method and a kit.
The invention is realized in this way, a novel and practical pichia pastoris secretory expression vector, the sequence of the DNA of the novel and practical pichia pastoris secretory expression vector is SEQ ID NO: 1.
the nucleotide sequence of SEQ ID NO: 1 is derived from pPICZaA and has a length of less than 4K.
Another object of the present invention is to provide a kanamycin, wherein the sequence of the DNA of the kanamycin is SEQ ID NO: 2; the kanamycin is SEQ ID NO: screening in 1.
The nucleotide sequence of SEQ ID NO: the N end of the 2 sequence is provided with BamHI and SpeI enzyme cutting sites and a 20bp sequence which is homologous with the vector framework; the C end of the sequence is provided with MluI enzyme cutting sites and a 20bp sequence which is homologous with the vector framework; the sequence of the restriction enzyme site DNA is SEQ ID NO: 3;
the sequence of the homologous arm DNA is SEQ ID NO: 4.
another object of the present invention is to provide a BDM promoter, wherein the sequence of the DNA of the BDM promoter is SEQ ID NO: 5.
another objective of the present invention is to provide an MF4I signal peptide, wherein the DNA sequence of MF4I signal peptide is SEQ ID NO: 6.
another object of the present invention is to provide a multiple cloning site region, wherein the sequence of the DNA of the multiple cloning site region is SEQ ID NO: 7.
another object of the present invention is to provide a method for constructing a novel and useful pichia pastoris secretory expression vector, which comprises:
(1) the bleomycin antibody of the pPICZaA vector is changed into Kanamycin resistance;
(2) fusion of the Kana ORF sequence with a backbone vector;
(3) the promoter and the signal peptide of the pPICK vector are replaced by a BDM promoter and an MF4I signal peptide;
(4) fusion of the Kana ORF sequence with the backbone vector.
The step (1) comprises the following steps:
(1.1) carrying out double digestion on the pPICZaA vector by using BamHI and MluI to obtain a vector framework with the size of 2495bp, and the sequence is shown as SEQ ID NO: 1;
(1.2) synthesizing a Kana ORF sequence by the whole gene, wherein the N end of the sequence is provided with BamHI and SpeI enzyme cutting sites and a 20bp sequence which is homologous with a vector framework; the C end of the sequence is provided with MluI enzyme cutting site and 20bp sequence which is homologous with the vector framework. The complete sequence of the synthetic Kana ORF is shown as SEQ ID NO: 2;
the fusion of the fragment and the vector in the step (2) utilizes a homologous recombination method, and the specific method comprises the following steps: 100ng of recovered vector framework, 300ng of recovered Kana ORF fragment, 5ul of NEB Gibson Assembly Master Mix, 10ul of total reaction system, immediately converting escherichia coli TOP10 competent cells after the reaction system is placed at 50 ℃ for reaction for 1 hour, identifying transformants, sending the transformants for testing, and storing the vector with correct sequencing for later use;
in the step (3), the pPICK carrier is subjected to double enzyme digestion by BglII and SalI to be used as a carrier skeleton, so that the carrier skeleton with the size of 2810bp is obtained; comprises the amino acid sequence of SEQ ID NO: 3; the sequence of the homologous arm DNA is SEQ ID NO: 4;
the step (4) comprises the following steps:
(4.1) carrying out full-gene synthesis on BDM starting, MF4I signal peptide and MCS region complete sequence, wherein a 20bp sequence which is homologous with a pPICK carrier skeleton is arranged on a sequence C end band, and a 20bp sequence which is homologous with the other end of the pPICK carrier is arranged on a sequence N end same band; the method comprises the following steps: SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7;
(4.2) the fusion of Kana ORF sequence and skeleton carrier, the fusion of fragment and carrier uses homologous recombination method, the concrete method is: 150ng of recovered pPICK vector skeleton, 450ng of recovered BDM-MF4I-MCS fragment, 10ul of NEB Gibson Assembly Master Mix 5ul of total reaction system, immediately converting escherichia coli TOP10 competent cells after the reaction system is placed at 50 ℃ for reaction for 1 hour, identifying and sending transformants, and obtaining the vector with correct sequencing, namely pPICK-BDM.
It is another object of the invention to provide a kit comprising the sequence SEQ ID NO: 1 to SEQ ID NO: 7.
by combining all the technical schemes, the invention has the advantages and positive effects that:
the vector skeleton is derived from pPICZaA, and the final vector skeleton is only about 4K, so that the subsequent molecular cloning operation experiment efficiency is facilitated.
The resistance of the prokaryotic cells is kanamycin, which is different from the resistance of bleomycin of a pPICZaA vector, the bleomycin is expensive and colonies with uneven growth, commonly called large and small spots, often appear in the transformation and screening process, thereby bringing great difficulty to the cloning and screening work.
The mutant AOX promoter which is proved to be capable of obviously improving the protein fermentation expression amount is selected as the carrier, and the protein expression amount can be obviously improved particularly in large-scale fermentation.
The vector selects MF4I signal peptide, can efficiently secrete and express target protein, so that the subsequent separation and purification work is simple and rapid, and the purification cost is further saved.
The carrier can efficiently construct a target protein multi-copy expression carrier in vitro by utilizing the principle of biological bricks, obviously improves the protein expression quantity and has great advantages in the production process.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flow chart of vector construction provided by an embodiment of the present invention.
FIG. 2 is an SDS-PAGE examination of shake flask induction supernatants of recombinant strains of MPH-R yeast provided in the examples of the invention.
FIG. 3 is a diagram showing the expression of L-amino acid oxidase (LAAO) in Pichia pastoris, according to an embodiment of the present invention.
FIG. 4 is a diagram showing the expression of L-glutamate oxidase (LGOX) in Pichia pastoris, according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a novel and practical pichia pastoris secretory expression vector, and the invention is described in detail below with reference to the attached drawings.
The present invention will be further described with reference to the following examples.
Examples
The bleomycin antibody of the pPICZaA vector is changed into Kanamycin resistance
(1) The vector backbone of 2495bp is obtained by double digestion of pPICZaA vector with BamHI and MluI, and the sequence is as follows SEQ ID NO: 1:
CGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATCAGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACGATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTGAATTCACGTGGCCCAGCCGGCCGTCTCGGATCGGTACCTCGAGCCGCGGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTGTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCG
(2) synthesizing Kana ORF sequence by whole gene, wherein the N end of the sequence is provided with BamHI and SpeI enzyme cutting sites and 20bp sequence homologous with vector skeleton; the C end of the sequence is provided with MluI enzyme cutting site and 20bp sequence which is homologous with the vector framework. The synthetic Kana ORF complete sequence is as follows SEQ ID NO: 2: (the gray highlighting markers are cleavage sites (SEQ ID NO: 3ACTAG, GGATCC, ACGCGT) and the lower case sequences are homology arms (SEQ ID NO: 4 aagtgagaccttcgtttgtg and gtacgcatgtaacattatac))
aagtgagaccttcgtttgtgACTAGTTCGACCCAATGGATCCCGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGACCTGCAGGGGGGGGGGGGAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCCCCCCCCCCCCCTGCAGGTCGGCCACGATGCGTCCGGCGTAGAGGATCTCCTGATGACTGACTCACTGATAATAAAAATACGGCTTCAGAATTTCTCAAGACTACACTCACTGTCCGACTTCAAGTACGCGTgtacgcatgtaacattatac
In the present invention, the primers used for the whole gene synthesis are (all primers are 5 '-3'):
Kana ORF-1
AAGTGAGACCTTCGTTTGTGACTAGTTCGACCCAATGGATCCCGCTCTCCCTTATGCGA
Kana ORF-2
GCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCG
Kana ORF-3
CAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAG
Kana ORF-4
GGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCC
Kana ORF-5
CCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTG
Kana ORF-6
ATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAG
Kana ORF-7
GGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCG
Kana ORF-8
TTTGAGACACAACGTGGCTTTCCCCCCCCCCCCTGCAGGTCGGCATCACCGGCGCCA
Kana ORF-9
CCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCAT
Kana ORF-10
CCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTA
Kana ORF-11
AAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGC
Kana ORF-12
TATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTT
Kana ORF-13
TGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCT
Kana ORF-14
TCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGAT
Kana ORF-15
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGA
Kana ORF-16
TCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCA
Kana ORF-17
ATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTAC
Kana ORF-18
CTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCA
Kana ORF-19
AGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGG
Kana ORF-20
AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATA
Kana ORF-21
GATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGC
Kana ORF-22
CAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGA
Kana ORF-23
TGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAG
Kana ORF-24
TGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCA
Kana ORF-25
ACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGG
Kana ORF-26
CGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGG
Kana ORF-27
TGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCG
Kana ORF-28
ATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGG
Kana ORF-29
ACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATT
Kana ORF-30
ACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTA
Kana ORF-31
AGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCG
Kana ORF-32
TGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTC
Kana ORF-33
GCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAA
Kana ORF-34
GCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTC
Kana ORF-35
TCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGG
Kana ORF-36
CGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAA
Kana ORF-37
GCAACACCTTCTTCACGAGGCAGACCTCAGCGCCCCCCCCCCCCCTGCAGGTCGGCCAC
Kana ORF-38
CAGTGAGTCAGTCATCAGGAGATCCTCTACGCCGGACGCATCGTGGCCGACCTGCAGGG
Kana ORF-39
TGATGACTGACTCACTGATAATAAAAATACGGCTTCAGAATTTCTCAAGACTACACTCA
Kana ORF-40
GTATAATGTTACATGCGTACACGCGTACTTGAAGTCGGACAGTGAGTGTAGTCTTGAGA
in the present invention, the Kana ORF sequence is fused to a backbone vector.
The fusion of the fragment and the vector in the experiment utilizes a homologous recombination method, and the specific method comprises the following steps: 100ng of recovered vector framework, 300ng of recovered Kana ORF fragment, 5ul of NEB Gibson Assembly Master Mix, 10ul of total reaction system, immediately transforming Escherichia coli TOP10 competent cells after the reaction system is placed at 50 ℃ for reaction for 1 hour, identifying transformants, sending the transformants, and storing the vector (marked as pPICK in the text) with correct sequencing for later use.
The invention is further described below in connection with the exchange of the promoter and signal peptide of the pPICK vector for the BDM promoter and MF4I signal peptide.
In the present invention, the promoter and signal peptide of the pPICK vector were replaced with BDM promoter and MF4I signal peptide.
The pPICK vector uses BglII and SalI double enzyme digestion as a vector skeleton to obtain a vector skeleton with the size of 2810bp, and the specific sequence is as follows (is a sequence vector) SEQ ID NO: 8:
CATCATCATCATCATCATTGAGTTTGTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGACTAGTTCGACCCAATGGATCCCGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGACCTGCAGGGGGGGGGGGGAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCCCCCCCCCCCCCTGCAGGTCGGCCACGATGCGTCCGGCGTAGAGGATCTCCTGATGACTGACTCACTGATAATAAAAATACGGCTTCAGAATTTCTCAAGACTACACTCACTGTCCGACTTCAAGTACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATCTCTAGA
(1) the BDM start, MF4I signal peptide and MCS region complete sequence are synthesized by whole gene, 20bp sequence homologous with pPICK carrier skeleton is carried on the C end of the sequence, and 20bp sequence homologous with the other end of the pPICK carrier is carried on the same band of the N end of the sequence. The synthetic full sequence is as follows: (the light grey marker BDM promoter
SEQ ID NO: 5(AACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGCCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGTTGTTGATCTTGACTACTTTTAACGATAACTTGAGAAGATCTAAGAACAACTAACTGTTTGAAACTATGGCTATTCCAAGATTT), dark grey labelled MF4I signal peptide sequence SEQ ID NO: 6(CCATCTATCTTTATAGCTGTCTTGTTTGCTGCATCTTCTGCTTTGGCTGCTCCAGTTAACACTACTACTGAAGATGAAACTGCTCAAATTCCAGCTGAGGCTGTTATTGGTTACTCTGATTTGGAAGGTGATTTTGATGTTGCTGTTTTGCCATTTTCTAACTCTACTAACAACGGTTTGTTAGAGGAAGCCGAGGCTGAAGCTGAACCAAAGTTCATTAATACTACTATTGCCTCTATTGCTGCTAAGGAAGAAGGTGTTTCTTTGGAA), light gray marked by the multiple cloning site region (SEQ ID NO: 7GAATTCATTGGGGTACCTCGAGCCGCGGCCGCCCAATGTCGAC) and lower case sequences are homology arms (ttggtcatgagatctctaga and catcatcatcatcatcattga)
ttggtcatgagatctctagaAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGCCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGTTGTTGATCTTGACTACTTTTAACGATAACTTGAGAAGATCTAAGAACAACTAACTGTTTGAAACTATGGCTATTCCAAGATTTCCATCTATCTTTATAGCTGTCTTGTTTGCTGCATCTTCTGCTTTGGCTGCTCCAGTTAACACTACTACTGAAGATGAAACTGCTCAAATTCCAGCTGAGGCTGTTATTGGTTACTCTGATTTGGAAGGTGATTTTGATGTTGCTGTTTTGCCATTTTCTAACTCTACTAACAACGGTTTGTTAGAGGAAGCCGAGGCTGAAGCTGAACCAAAGTTCATTAATACTACTATTGCCTCTATTGCTGCTAAGGAAGAAGGTGTTTCTTTGGAAGAATTCATTGGGGTACCTCGAGCCGCGGCCGCCCAATGTCGACcatcatcatcatcatcattga
In the present invention, the primers used for the whole gene synthesis are (all primers are 5 '-3'):
BDM-MF4I-MCS-1
TTGGTCATGAGATCTCTAGAAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCC
BDM-MF4I-MCS-2GGCACTTATGTGTGAGAATGGACCTGTGGATGTCGGATGGCAAAAAGGTTTCATTBDM-MF4I-MCS-3
TCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTT
BDM-MF4I-MCS-4
GGTGTTGAGGAGAAGAGGAGTGGAGGTCCTGCGTTTGCAACGGTCTGCTGCTAGT
BDM-MF4I-MCS-5CTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCT
BDM-MF4I-MCS-6GCCTAATAGAAGGAATTGGAATGAGCGAGCTCCAATCAAGCCCAATAACTGGGCTBDM-MF4I-MCS-7AATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGC
BDM-MF4I-MCS-8
GCATTCGGAAATAAACAAACATGAACCTCGCCAGGGGGGCCAGGATAGACAGGCT
BDM-MF4I-MCS-9TGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATG
BDM-MF4I-MCS-10GAACATGAAACTATTTGACCCCACACTCAGAAAGCCCTCATCTGGAGTGATGTTCBDM-MF4I-MCS-11CAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTT
BDM-MF4I-MCS-12ATCTTGGATGAGATCACGCTTTTGTCATATTAGGTTCCAAGACAGCGTTTAAACT
BDM-MF4I-MCS-13GTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTT
BDM-MF4I-MCS-14CAAACGGTATGGCGACTTTTGGAAGTTTCTTTTTGACCAACTGGCCGTTAGCATT
BDM-MF4I-MCS-15
AGTCGCCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCT
BDM-MF4I-MCS-16
CGGGGTTCAGAAGCGATAGAGAGACTGCGCTAAGCATTAATGAGATTATTTTTGAGCAT
BDM-MF4I-MCS-17
ATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTT
BDM-MF4I-MCS-18
ATCTTGGAAGCATACAATGTGGAGACAATGCATAATCATCCAAAAAGCGGGTGTTTCCC
BDM-MF4I-MCS-19
TTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAA
BDM-MF4I-MCS-20
TGTTTATATATTGCTGTCAAGTAGGGGTTAGAACAGTTAAATTTTGATCATGAACGTTA
BDM-MF4I-MCS-21
ACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCAT
BDM-MF4I-MCS-22
TTGTCAATTGGAACCAGTCGCAATTATGAAAGTAAGCTAATAATGATGATAAAAAAAAA
BDM-MF4I-MCS-23
CTGGTTCCAATTGACAAGTTGTTGATCTTGACTACTTTTAACGATAACTTGAGAAGATC
BDM-MF4I-MCS-24
AATCTTGGAATAGCCATAGTTTCAAACAGTTAGTTGTTCTTAGATCTTCTCAAGTTATC
BDM-MF4I-MCS-25
ATGGCTATTCCAAGATTTCCATCTATCTTTATAGCTGTCTTGTTTGCTGCATCTTCTGC
BDM-MF4I-MCS-26
GCAGTTTCATCTTCAGTAGTAGTGTTAACTGGAGCAGCCAAAGCAGAAGATGCAGCAAA
BDM-MF4I-MCS-27
ACTGAAGATGAAACTGCTCAAATTCCAGCTGAGGCTGTTATTGGTTACTCTGATTTGGA
BDM-MF4I-MCS-28
GTAGAGTTAGAAAATGGCAAAACAGCAACATCAAAATCACCTTCCAAATCAGAGTAACC
BDM-MF4I-MCS-29
CCATTTTCTAACTCTACTAACAACGGTTTGTTAGAGGAAGCCGAGGCTGAAGCTGAACC
BDM-MF4I-MCS-30
TCCTTAGCAGCAATAGAGGCAATAGTAGTATTAATGAACTTTGGTTCAGCTTCAGCCTC
BDM-MF4I-MCS-31
TCTATTGCTGCTAAGGAAGAAGGTGTTTCTTTGGAAGAATTCATTGGGGTACCTCGAGC
BDM-MF4I-MCS-32TCAATGATGATGATGATGATGGTCGACATTGGGCGGCCGCGGCTCGAGGTACCCCAAT
(2) fusion of the Kana ORF sequence with the backbone vector.
The vector construction flow chart 1 (the fusion of the fragment and the vector in the experiment utilizes a homologous recombination method), and the specific method comprises the following steps:
s101, recovering 150ng of pPICK vector skeleton;
s102, recovering 450ng of BDM-MF4I-MCS fragment, 5ul of NEB Gibson Assembly Master Mix and 10ul of total reaction system, and immediately transforming escherichia coli TOP10 competent cells after the reaction system is placed at 50 ℃ for reaction for 1 hour;
s103, identifying and sequencing the transformant, wherein the vector with correct sequencing is pPICK-BDM.
The technical solution of the present invention will be further described with reference to the following specific examples.
Example 1
This example is for the methyl Parathion hydrolase MPH (methyl Parathion Hydrolase) from the genus Plesiomonas, the Genbank of which is AAK 14390.1. The gene sequence of MPH was searched in NCBI, and the original signal peptide sequence in MPH gene was deleted. After being translated into an amino acid sequence, the gene is synthesized in Wuhan Jinkeri bioengineering GmbH according to the codon preference of pichia pastoris, the total length of the gene sequence mph gene is 912bp, and 304 amino acids are coded in total. And cloning the synthesized gene to a pPICK-BDM expression vector to obtain pPICK-BDM-mph. After linearization of the vector, Pichia pastoris GS115 competent cells were transformed electrically. Screening out positive bacterial colonies, inoculating the positive bacterial colonies in a BMGY culture medium, and fermenting in a shake flask. After 48 hours of methanol induction, the expression level of MPH-R in a 500mL shake flask is increased to 1.9U/mL, and the specific enzyme activity is 15.8U/mg. And the expression amount is increased to 18.4U/mL in 5L of basic salt high-density fermentation culture medium, and the expression amount is increased by 9.7 times again. FIG. 2 is an SDS-PAGE examination of shake flask induction supernatants of the MPH-R yeast recombinant strains.
Example 2
In the implementation, an L-amino acid oxidase gene laao (GenBank: WP _051653666.1) of a northern spore fungus (Kitasatospora cheerialensis) is synthesized through the preference of a heterologous table codon of the pichia pastoris, the gene is integrated on a pichia pastoris expression vector pPICK-BDM, and a recombinant is selected. According to a pichia pastoris inducible expression method, the L-amino acid oxidase (LAAO) is induced and expressed, and the result of enzyme activity property analysis of the LAAO in the fermentation liquid shows that the enzyme activity of the LAAO heterologously expressed by the pichia pastoris is 0.87U/mL, the optimal reaction temperature is 20 ℃, and the optimal reaction pH is 6.
The expression of the multi-copy plasmid and the strain of the LAAO gene is realized by using the pPICK-BDM vector, the gene has certain gene dose effect through the gene dose effect research of the enzyme, the kc-LAAO with 2, 3 and 4 copies is compared with the single-copy LAAO, and the enzyme activity is improved by 1.89, 2.47 and 3.06 times through comparing the enzyme activity through the shake flask fermentation. The wet weight of the thalli and the enzyme activity of LAAO are improved by 5.15 times and 45.24 times through fed-batch fermentation. The high density fermentation strategy improves the production efficiency of LAAO. FIG. 3 shows the expression of L-amino acid oxidase (LAAO) in Pichia pastoris.
The embodiment synthesizes L-glutamic acid oxidase gene (GenBank: EFE71695.1) derived from Streptomyces ghanaensis ATCC 14672, realizes the construction of multi-copy number plasmid of the gene in vitro by utilizing pPICK-BDM carrier and electrically converts pichia pastoris, and the shake flask experiment result shows that the enzyme activity of the multi-copy strain is greatly improved relative to single copy, and the enzyme activity levels of LGOX in the fermentation supernatant of multi-copy expression strains P-LGOX2, P-LGOX3 and P-LGOX4 are respectively 1.75, 2.41 and 2.75 times of that of the single-copy expression strain P-LGOX 1; and then, performing a high-density fermentation experiment by adopting 4 copies of recombinant strains, respectively trying 2 feed-batch fermentation processes of DO-stat and exponential feed-batch, and adopting the DO-stat feed-batch process, wherein the wet weight and the enzyme activity of the thalli at the end of fermentation respectively reach 386g/L and 150.1U/mL at the highest, and adopting the glycerol and methanol exponential feed-batch process, and the total enzyme activity and the unit production efficiency finally reach 247.8U/mL and 2581U/L/h at the end of fermentation.
Example 4 expression of L-glutamate oxidase (LGOX) in Pichia pastoris, as shown in FIG. 4.
In the implementation, a pPICK-BDM vector is used for displaying a LAAO gene and an LGOX gene on the surface of pichia pastoris, multiple copies of surface display vectors are constructed, namely PGLOD (1-3) -AG alpha 1 and PAAO (1-3) -AG alpha 1, and pichia pastoris cells are transformed by the vectors to obtain recombinant strains GLOD (1-3) -AG alpha 1 and AAO (1-3) -AG alpha 1. Subsequent experiments showed that the yield of GLOD of the high copy recombinant strain was 688.5U/g (dry weight) and the yield of LAAO was 626.7U/g (dry weight), which was greatly improved when compared to the conventional method.
Example 5
According to the embodiment, the expression of the chitosan enzyme gene CCHA from Aspergillus oryzae NKY2017 in Pichia pastoris is realized, and a multi-copy expression vector of CCHA is constructed by using pPICK-BDM, so that a corresponding multi-copy expression strain CCHA1/2/3/4 is obtained. Wherein the CCHA4 strain has the highest expression level, and the chitosanase total activity obtained by CCHA4 expression strain can reach 22,500U/mL when the high-density fermentation is finished.
In the description of the present invention, "a plurality" means two or more unless otherwise specified; the terms "upper", "lower", "left", "right", "inner", "outer", "front", "rear", "head", "tail", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are only for convenience in describing and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, should not be construed as limiting the invention. Furthermore, the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
<110> research center of biological pesticide engineering in Hubei province
<120> novel practical pichia pastoris secretory expression vector, construction method and kit
<160>8
<210>1
<211>2495
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
CGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATCAGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACGATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTGAATTCACGTGGCCCAGCCGGCCGTCTCGGATCGGTACCTCGAGCCGCGGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTGTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCG
<210>2
<211>1675
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
aagtgagaccttcgtttgtgACTAGTTCGACCCAATGGATCCCGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGACCTGCAGGGGGGGGGGGGAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCCCCCCCCCCCCCTGCAGGTCGGCCACGATGCGTCCGGCGTAGAGGATCTCCTGATGACTGACTCACTGATAATAAAAATACGGCTTCAGAATTTCTCAAGACTACACTCACTGTCCGACTTCAAGTACGCGTgtacgcatgtaacattatac
<210>3
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
ACTAG
GGATCC
ACGCGT
<210>4
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
aagtgagaccttcgtttgtg
gtacgcatgtaacattatac
<210>5
<211>952
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
AACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGCCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGTTGTTGATCTTGACTACTTTTAACGATAACTTGAGAAGATCTAAGAACAACTAACTGTTTGAAACTATGGCTATTCCAAGATTT
<210>6
<211>270
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
CCATCTATCTTTATAGCTGTCTTGTTTGCTGCATCTTCTGCTTTGGCTGCTCCAGTTAACACTACTACTGAAGATGAAACTGCTCAAATTCCAGCTGAGGCTGTTATTGGTTACTCTGATTTGGAAGGTGATTTTGATGTTGCTGTTTTGCCATTTTCTAACTCTACTAACAACGGTTTGTTAGAGGAAGCCGAGGCTGAAGCTGAACCAAAGTTCATTAATACTACTATTGCCTCTATTGCTGCTAAGGAAGAAGGTGTTTCTTTGGAA
<210>7
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
GAATTCATTGGGGTACCTCGAGCCGCGGCCGCCCAATGTCGAC
<210>8
<211>2810
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
CATCATCATCATCATCATTGAGTTTGTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGACTAGTTCGACCCAATGGATCCCGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGACCTGCAGGGGGGGGGGGGAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCCCCCCCCCCCCCTGCAGGTCGGCCACGATGCGTCCGGCGTAGAGGATCTCCTGATGACTGACTCACTGATAATAAAAATACGGCTTCAGAATTTCTCAAGACTACACTCACTGTCCGACTTCAAGTACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATCTCTAGA
Claims (10)
1. The utility model provides a novel practical pichia pastoris secretory expression vector, which is characterized in that, the sequence of the DNA of the novel practical pichia pastoris secretory expression vector is SEQ ID NO: 1.
2. the novel and useful pichia secretory expression vector of claim 1, wherein said SEQ ID NO: 1 is derived from pPICZaA and has a length of less than 4K.
3. A kanamycin characterized by the fact that the DNA sequence of kanamycin has the sequence shown in SEQ ID NO: 2; the kanamycin is SEQ ID NO: screening in 1.
4. The kanamycin according to claim 3 wherein,
the nucleotide sequence of SEQ ID NO: the N end of the 2 sequence is provided with BamHI and SpeI enzyme cutting sites and a 20bp sequence which is homologous with the vector framework; the C end of the sequence is provided with MluI enzyme cutting sites and a 20bp sequence which is homologous with the vector framework; the sequence of the restriction enzyme site DNA is SEQ ID NO: 3;
the sequence of the homologous arm DNA is SEQ ID NO: 4.
5. a BDM promoter, wherein the DNA sequence of the BDM promoter is SEQ ID NO: 5.
6. an MF4I signal peptide, wherein the DNA sequence of the MF4I signal peptide is SEQ ID NO: 6.
7. a multiple cloning site region, wherein the DNA of said multiple cloning site region has the sequence of SEQ ID NO: 7.
8. the utility model provides a method of construction of novel practical pichia pastoris secretory expression vector, which is characterized in that the method of construction of novel practical pichia pastoris secretory expression vector comprises:
(1) the bleomycin antibody of the pPICZaA vector is changed into Kanamycin resistance;
(2) fusion of the Kana ORF sequence with a backbone vector;
(3) the promoter and the signal peptide of the pPICK vector are replaced by a BDM promoter and an MF4I signal peptide;
(4) fusion of the Kana ORF sequence with the backbone vector.
9. The method for constructing a novel and useful secretory expression vector of Pichia pastoris according to claim 8, wherein step (1) comprises:
(1.1) carrying out double digestion on the pPICZaA vector by using BamHI and MluI to obtain a vector framework with the size of 2495bp, and the sequence is shown as SEQ ID NO: 1;
(1.2) synthesizing a Kana ORF sequence by the whole gene, wherein the N end of the sequence is provided with BamHI and SpeI enzyme cutting sites and a 20bp sequence which is homologous with a vector framework; the C end of the sequence is provided with MluI enzyme cutting site and 20bp sequence which is homologous with the vector framework. The complete sequence of the synthetic Kana ORF is shown as SEQ ID NO: 2;
the fusion of the fragment and the vector in the step (2) utilizes a homologous recombination method, and the specific method comprises the following steps: 100ng of recovered vector framework, 300ng of recovered Kana ORF fragment, 5ul of NEB Gibson Assembly Master Mix, 10ul of total reaction system, immediately converting escherichia coli TOP10 competent cells after the reaction system is placed at 50 ℃ for reaction for 1 hour, identifying transformants, sending the transformants for testing, and storing the vector with correct sequencing for later use;
in the step (3), the pPICK carrier is subjected to double enzyme digestion by BglII and SalI to be used as a carrier skeleton, so that the carrier skeleton with the size of 2810bp is obtained; comprises the amino acid sequence of SEQ ID NO: 3; the sequence of the homologous arm DNA is SEQ ID NO: 4;
the step (4) comprises the following steps:
(4.1) carrying out full-gene synthesis on BDM starting, MF4I signal peptide and MCS region complete sequence, wherein a 20bp sequence which is homologous with a pPICK carrier skeleton is arranged on a sequence C end band, and a 20bp sequence which is homologous with the other end of the pPICK carrier is arranged on a sequence N end same band; the method comprises the following steps: SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7;
(4.2) the fusion of Kana ORF sequence and skeleton carrier, the fusion of fragment and carrier uses homologous recombination method, the concrete method is: 150ng of recovered pPICK vector skeleton, 450ng of recovered BDM-MF4I-MCS fragment, 10ul of NEB Gibson Assembly Master Mix 5ul of total reaction system, immediately converting escherichia coli TOP10 competent cells after the reaction system is placed at 50 ℃ for reaction for 1 hour, identifying and sending transformants, and obtaining the vector with correct sequencing, namely pPICK-BDM.
10. A kit comprising the sequence of SEQ ID NO: 1 to SEQ ID NO: 7.
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Application publication date: 20210716 |