CN114773441B - Application of Hot1p as positive control factor in improving protein expression in host cell - Google Patents
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Abstract
The invention relates to application of a transcription regulatory factor expressed by eukaryotic genes, in particular to application of a transcription regulatory factor Hot1p of a constitutive promoter Pgap. The invention discloses an application of a Hot1p as a positive regulatory factor in improving protein expression in a host cell, wherein the amino acid sequence of the Hot1p is encoded by a Hot1 gene with a nucleotide sequence of SEQ ID NO. 1; the application is to increase the expression of proteins in host cells by inserting the Hot1 gene after the promoter Pgap. The application of the invention can enhance the transcription of the Pichia pastoris constitutive promoter Pgap promoter, thereby enabling the subsequent exogenous gene to be efficiently expressed in Pichia pastoris, and avoiding the dilution effect generated by using the same promoter when multiple copies or multiple genes are expressed, so that the difference of the expression amounts of different genes is overlarge.
Description
Technical Field
The invention belongs to the fields of molecular biology and bioengineering, relates to application of a transcription regulatory factor of eukaryotic gene expression, and in particular relates to application of a transcription regulatory factor Hot1p of a constitutive promoter Pgap.
Background
Promoters are one of the most important elements regulating gene expression, P AOX1 Promoters (inducible) and Pgap promoters (constitutive) are the most representative promoters in pichia pastoris exogenous protein expression.
The methanol-inducible Pichia expression system is a common expression system currently used for expressing most heterologous proteins, however, not all exogenous proteins are suitable for methanol-inducible expression and methanol has great potential safety hazards in large-scale production. Compared with methanol induction, the constitutive pichia pastoris expression system does not use methanol induction when expressing heterologous proteins, but most of constitutive promoters have relatively weak strength, and cannot obtain higher protein yield, so that the application of the constitutive pichia pastoris expression system is limited.
Aiming at the defect of methanol induction, the optimization and transformation of the pichia pastoris expression system are in recent years becoming research hot spots. In promoter engineering studies, a new promoter library is currently constructed mainly by various methods. Among them, there are many studies by AOX1 Deletion or insertion of cis-acting elements, point mutation of 5' UTR or core promoter region, etc. to engineer P AOX1 Thereby leading to P AOX1 But these modifications do not seem to eliminate the effects of high levels of glucose and glycerolOil, and the like, are substituted for carbon sources and are far from reaching the level of industrial application. For construction of Pgap library, the only study was that Qin et al constructed GAP promoter library by random mutation through error-prone PCR, but the method was random and could not explain the regulation of Pgap. With the application of transcriptome data analysis, the development work of the promoter is greatly improved. With respect to regulation and enhancement of P AOX1 There have been many reports on the study on the expression intensity of methanol, which has been suggested to be able to control a plurality of trans-acting elements (mainly focused on P AOX1 Transcription regulatory factor of promoter or carbon source repression-related transcription factor, etc.), or subcellular localization, at the level of transcription, of P AOX1 Thereby affecting the expression of genes related to methanol metabolic pathways. Nevertheless, participate in P AOX1 The regulation mechanism is complex, and partial carbon source de-repression is not superior to traditional methanol induction in protein expression. At present, research on promoter regulation is mainly focused on PAOX1, while for research on exploring transcriptional regulation of Pgap, only Ozge Ata and the like are currently used, a high-expression rhGH strain of a promoter variant is constructed by deleting or copying a Transcription Factor Binding Site (TFBS) in a targeted manner, and reports on enhancing the yield of target proteins expressed by the Pgap promoter through improvement of transcriptional regulation of the Pgap promoter are almost blank in the published literature.
In Pichia pastoris Hot1p (SEQ ID NO: NC_012963.1, putative: PAS_chr1-1_1049) belongs to the Gcr 1_c conserved protein domain, a family capable of activating transcription of glycolytic enzymes. The gene is homologous to the Gcr1 gene in Saccharomyces cerevisiae. Gcr1p is a transcriptional regulator of glycerol biosynthetic genes in Saccharomyces cerevisiae, in response to high osmotic pressure. According to the literature report, the research on the gene is mainly on saccharomyces cerevisiae, and if the Gcr1p (Gcr 1 protein) can be combined with a TPI1 promoter, the gene participates in forward regulation of glycolytic genes, and the deletion of the gene can lead to the saccharomyces cerevisiae to show growth defects, myoinositol level defects, abnormal vacuole structure and autophagy reduction. Additional studies have shown that overexpression of Gcr1 can enhance cell growth and glucose uptake. However, there is no report on the transcription regulation of Pgap.
Disclosure of Invention
The invention mainly aims at solving the problems and defects of the expression of heterologous proteins in host cells, and provides a transcription factor Hot1 gene for regulating the expression of the heterologous proteins in the host cells, which can enhance the transcription of a Pichia pastoris constitutive promoter Pgap promoter, so that the subsequent exogenous genes are efficiently expressed in Pichia pastoris.
In a first aspect of the invention, there is provided the use of Hot1p as a positive regulator for increasing protein expression in a host cell, the amino acid sequence of said Hot1p being encoded by a Hot1 gene having the nucleotide sequence SEQ ID NO. 1; the application is to increase the expression of proteins in host cells by inserting the Hot1 gene after the promoter Pgap.
According to the use of the invention, the host cell is selected from the group consisting of: pichia pastoris, saccharomyces cerevisiae and candida glycerinogenes.
According to the prior art, pichia pastoris, saccharomyces cerevisiae, candida glycerogenes and the like can accept a yeast expression vector using Pgap as a promoter for expressing heterologous proteins, and thus can be used as a host cell of the present invention.
In a second aspect of the present invention, there is provided a gene expression cassette for regulating expression of a heterologous protein in a host cell, using Pgap as a promoter and inserting the Hot1 gene.
In a third aspect of the invention, there is provided a vector comprising the gene expression cassette of the invention.
In a fourth aspect of the invention, there is provided a host cell comprising a gene expression cassette according to the invention, or comprising a vector according to the invention.
In a fifth aspect of the invention, there is provided a method of enhancing protein expression in a host cell comprising: providing a host cell according to the invention, culturing the host cell under conditions suitable for expression of the heterologous protein, and isolating the expressed protein from the culture medium.
The invention discloses by experiments: the gene Hot1 plays a role in transcriptional activation in a heterologous protein synthesis path taking Pgap as a promoter, and a transcription factor Hot1p formed after translation of the sequence participates in forward regulation of the Pgap promoter, so that the expression efficiency of exogenous genes is improved, and dilution effect generated by using the same promoter during multicopy or multi-gene expression can be avoided, so that the expression quantity difference of different genes is overlarge.
The invention can construct a gene expression cassette taking Pgap as a promoter and inserting the Hot1 gene, and the Hot1p transcription activator can enhance the transcription of the Pichia pastoris constitutive promoter Pgap promoter, so that the exogenous gene in the gene expression cassette can be efficiently expressed in Pichia pastoris (or other yeasts which can accept the Pgap as the promoter), and the expression intensity of the exogenous gene can be obviously improved.
Drawings
FIG. 1 is a map of the ppic3.5k-Pgap-Hot1 plasmid.
FIG. 2 shows the growth rate of xylanase xynB expressed by a strain (Pichia pastoris), wherein the control group is a non-co-expressed strain; clone 1 to clone 5 are Hot1 gene co-expression strains.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods. The molecular cloning techniques used in the following examples are described in J.Sambucus et al, code "molecular cloning Experimental guidelines", or according to the manufacturer's recommendations.
In this context, hot1p means a protein expressed by Hot 1.
Example 1: construction of transcription control factor Hot1p expression cassette
1. Amplification of Pgap promoter
The invention takes the Pgap sequence of pichia pastoris as a reference, adopts software DNAMAN 8 to design and synthesize two oligonucleotide primers, and amplifies the Pgap target gene by a PCR method.
The two PCR primers were as follows:
1-F:ACTTACGAGCTCGAGATCTTTTTTGTAG(SEQ ID NO:4)
1-R:ACTTACGCGGATCCGCGATAGTTGTTCAATTG(SEQ ID NO:5)
the underlined bases are SacI and BamHI restriction enzyme sites respectively.
The PCR reaction system is shown in Table 1 below.
Table 1:
| component (A) | Volume (mu L) |
| 2×Q5 Master Mix | 12.5 |
| Primer F(10μM) | 1.25 |
| Primer R(10μM) | 1.25 |
| Template DNA | 1(100ng) |
| ddH 2 O | 9ul |
| Total volume of | 25ul |
The PCR program settings are shown in Table 2.
Table 2:
after electrophoresis of the PCR amplified product by 1% agarose gel, gel recovery was performed by using a DNA recovery kit to obtain a fragment of about 480 bp.
2. Construction of ppic3.5k-Pgap vector
The ppic3.5K plasmid and the Pgap promoter PCR product were digested with restriction enzymes SacI and BamHI at 37℃for 20min, respectively, under the conditions shown in Table 3 below.
Table 3:
the two target fragments were recovered after electrophoresis on a 1% agarose gel, and ligated with T4DNA ligase, with the ligation system shown in Table 4 below.
Table 4:
| component (A) | Volume (mu L) |
| 10×T4 ligase buffer | 1 |
| T4DNA ligase | 0.5 |
| Ligation vector insert | 1:3-1:10 (molar concentration ratio) |
| ddH 2 O | Supplement to 10 |
| Total volume of | 10 |
Ligation was performed at 16℃for 12h with ligase, DH5a competent cells transformed with the ligation product, plasmids were extracted with a plasmid extraction kit, and electrophoresis results after double digestion with BamHI and SacI showed two bands of 8.2kb and 483bp, indicating successful ligation, and the Pgap gene was determined by DNA sequencing.
3. Amplification and sequencing of the transcription control factor Hot1 Gene
The invention takes synonymous mutant genes with the sequence of SEQ ID NO:2 in pichia pastoris genome as reference, adopts software DNAMAN 8 to design and synthesize 4 oligonucleotide primers, and amplifies a transcription regulatory factor Hot1 gene (SEQ ID NO: 1) by an overlay PCR method.
2-F：ACTTACCGGAATTCCGGATGAATGCATCTAGTATCC(SEQ ID NO:6)
2-R:CCTGTAGGGGGCTCAATTCCACCGAATCCATTTGAC(SEQ ID NO:7)
3-F:CGGTGGAATTGAGCCCCCTACAGGGTATTCGATTGTGT(SEQ ID NO:8)
3-R:ACTTATTTGCGGCCGCTTTACTACAAGTCATAATT(SEQ ID NO:9)
The underlined bases are EcoRI and NotI restriction endonuclease cleavage sites respectively.
The Hot1 gene was amplified by dividing it into two parts using 2-F/R and 3-F/R, respectively, and then amplifying the two parts by overlapping extension using 2-F/3-R.
The PCR reaction system and the PCR procedure were set as described above.
The overlay PCR amplification reaction is shown in Table 5.
Table 5:
note that: segment 1: fragment 2 molar mass ratio 1:1
After electrophoresis of the PCR amplification product by 1% agarose gel, gel recovery was performed using a DNA recovery kit, to obtain a fragment of about 1.2kb (SEQ ID NO: 1).
4. Ligation of the ppic3.5k-Pgap-Hot1 expression cassette
The ppic3.5K-Pgap plasmid (SEQ ID NO: 3) and the transcription control factor Hot1 gene PCR product (SEQ ID NO: 1) are respectively digested with restriction enzymes EcoRI and NotI endonuclease, purified and recovered.
The PCR product connection method of the ppic3.5K-Pgap plasmid and the transcription control factor Hot1 gene is shown in the step 2, and the electrophoresis result shows that the two bands of 8.7kb and 1.2kb are obtained after double digestion by BamHI and SacI, which shows that the connection is successful, and the transcription control factor Hot1 gene is determined by DNA sequencing.
The transcription regulatory factor Hot1p expression cassette was thus successfully constructed (as shown in FIG. 1).
Example 2: hot1 Gene integration Pichia pastoris genome
In order to improve the integration efficiency of the single cooperite expression cassette on the pichia pastoris chromosome, the expression cassette was linearized with the restriction enzyme Sal I and recovered by purification with the kit. The recipient bacteria of this experiment were Pichia pastoris SMD1168 (recombinant bacteria containing xylanase xynB gene inserted after Pgap, EX 6), screened after electrotransformation using G418 plates containing 0.3mg/mL, and identified by genomic PCR.
The result of sequencing the PCR product shows that the screened strain is positive clone of the co-expression Hot1 p.
Example 3: determination of heterologous protein expression driven by Hot1p enhanced constitutive promoter Pgap
The positive clone 1 selected in example 1 and the positive clone 2 selected in example 2 were fermented at 28℃and 200rpm for 72 hours. Meanwhile, pichia pastoris (EX 6) containing the xynB gene is used as a control, supernatant is taken after culturing for 72 hours, SDS-PAGE electrophoresis detection is carried out, and xylanase xynB expression level is analyzed.
The expression level of the selected clone 1 is improved by 99% compared with that of a control group after the Hot1p transcription factor is over-expressed, the expression level of the clone 2 is improved by 101% compared with that of the control group, the expression level of the clone 3 is improved by 244% compared with that of the control group, the expression level of the clone 4 is improved by 129% compared with that of the control group, and the expression level of the clone 5 is improved by 180% compared with that of the control group.
SEQUENCE LISTING
<110> and university of south China
<120> use of Hot1p as a Positive regulatory factor for increasing protein expression in host cells
<130>
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 1209
<212> DNA
<213> Pichia pastoris
<400> 1
atgaatgcat ctagtatcca aagaaaaaac ccataccaaa gcaaacaaac agcctattta 60
aattggtgtc acaatgaggg gaagaaattc attccaggtc cgcccaaatt gcgtgagatc 120
gttacgccaa agagactctt cctgttcctg aaaactcata ttgaacccgc caaagtacga 180
gaaatccccc tctcgtttgc atccttcgat gcatatgtga gcgctatcgt agacttatat 240
cataggcaac gtactgagtt caaagttaga cctgtattag acgatccaaa cgagcctgat 300
gcggattggg tgacaccaag agacgacaga gttaaagagt atttgaagca gatcaagaaa 360
gagagacaga acgtacatcg cgtgactcaa tcgcatgaga tggcccaaga aagaacggtt 420
accccaacat caactaactc ttcaaatatg ctgataccca ctacgttcac aggaaacagc 480
gacccatccc tgctgtcaaa tgaattcggt ggaattgagc cccctacagg gtattcgatt 540
gtgtctagcc aaggattgag gggttcctcc agcagtacat cttcttctgg aaccgatatt 600
tcatatctca accagcaatt agctaaaaca aatgataagg ttaacagatt atccatggat 660
gttcaacagt taaaagaatt gctgactaat accaacaaca aggtgacaca gatcgtggaa 720
atgctcactt tagtgtctaa agcaggacaa gttggagctc accgaacacc ttcgcaatct 780
gagccagtct ctaattcaat ggaacctcca caacctgcaa acggtcaata tggcattgct 840
ccagtatctg ttccgttctc caaccccact ggagctcctc ctgctccaac aacaactgta 900
atacctgaaa ttattctcaa taacaaagca aaaacaatct ctgccatctg gcgggagtac 960
aagattggat acaaaggaca accttcttta gcagaaatgg aacggaaata tggtactagc 1020
tggcgtcgtg gacgtatcag gaaaactgtg cagagaagga gaaggatagc acaggctata 1080
gagacctata tcaaccaggg gcttacagaa gaagaagcgc tttttcggtt ggagaagtac 1140
cgacaagaaa ggtcaaagag ccttttttgg ctctattcca atgttccaga aaattcttat 1200
gacttgtag 1209
<210> 2
<211> 1209
<212> DNA
<213> Pichia pastoris
<400> 2
atgaatgcat ctagtatcca aagaaaaaac ccataccaaa gcaaacaaac agcctattta 60
aattggtgtc acaatgaggg gaagaaattc attccaggtc cgcccaaatt gcgtgagatc 120
gttacgccaa agagactctt cctgttcctg aaaactcata ttgaacccgc caaagtacga 180
gaaatccccc tctcgtttgc atccttcgat gcatatgtga gcgctatcgt agacttatat 240
cataggcaac gtactgagtt caaagttaga cctgtattag acgatccaaa cgagcctgat 300
gcggattggg tgacaccaag agacgacaga gttaaagagt atttgaagca gatcaagaaa 360
gagagacaga acgtacatcg cgtgactcaa tcgcatgaga tggcccaaga aagaacggtt 420
accccaacat caactaactc ttcaaatatg ctgataccca ctacgttcac aggaaacagc 480
gacccatccc tgctgtcaaa tggattcggt ggaattgagc cccctacagg gtattcgatt 540
gtgtctagcc aaggattgag gggttcctcc agcagtacat cttcttctgg aaccgatatt 600
tcatatctca accagcaatt agctaaaaca aatgataagg ttaacagatt atccatggat 660
gttcaacagt taaaagaatt gctgactaat accaacaaca aggtgacaca gatcgtggaa 720
atgctcactt tagtgtctaa agcaggacaa gttggagctc accgaacacc ttcgcaatct 780
gagccagtct ctaattcaat ggaacctcca caacctgcaa acggtcaata tggcattgct 840
ccagtatctg ttccgttctc caaccccact ggagctcctc ctgctccaac aacaactgta 900
atacctgaaa ttattctcaa taacaaagca aaaacaatct ctgccatctg gcgggagtac 960
aagattggat acaaaggaca accttcttta gcagaaatgg aacggaaata tggtactagc 1020
tggcgtcgtg gacgtatcag gaaaactgtg cagagaagga gaaggatagc acaggctata 1080
gagacctata tcaaccaggg gcttacagaa gaagaagcgc tttttcggtt ggagaagtac 1140
cgacaagaaa ggtcaaagag ccttttttgg ctctattcca atgttccaga aaattcttat 1200
gacttgtag 1209
<210> 3
<211> 8759
<212> DNA
<213> Synthesis
<400> 3
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctca gatctttttt gtagaaatgt cttggtgtcc 240
tcgtccaatc aggtagccat ctctgaaata tctggctccg ttgcaactcc gaacgacctg 300
ctggcaacgt aaaattctcc ggggtaaaac ttaaatgtgg agtaatggaa ccagaaacgt 360
ctcttccctt ctctctcctt ccaccgcccg ttaccgtccc taggaaattt tactctgctg 420
gagagcttct tctacggccc ccttgcagca atgctcttcc cagcattacg ttgcgggtaa 480
aacggaggtc gtgtacccga cctagcagcc cagggatgga aaagtcccgg ccgtcgctgg 540
caataatagc gggcggacgc atgtcatgag attattggaa accaccagaa tcgaatataa 600
aaggcgaaca cctttcccaa ttttggtttc tcctgaccca aagactttaa atttaattta 660
tttgtcccta tttcaatcaa ttgaacaact atggatccta cgtagaattc cctagggcgg 720
ccgcgaatta attcgcctta gacatgactg ttcctcagtt caagttgggc acttacgaga 780
agaccggtct tgctagattc taatcaagag gatgtcagaa tgccatttgc ctgagagatg 840
caggcttcat ttttgatact tttttatttg taacctatat agtataggat tttttttgtc 900
attttgtttc ttctcgtacg agcttgctcc tgatcagcct atctcgcagc tgatgaatat 960
cttgtggtag gggtttggga aaatcattcg agtttgatgt ttttcttggt atttcccact 1020
cctcttcaga gtacagaaga ttaagtgaga cgttcgtttg tgcaagctta tcgataagct 1080
ttaatgcggt agtttatcac agttaaattg ctaacgcagt caggcaccgt gtatgaaatc 1140
taacaatgcg ctcatcgtca tcctcggcac cgtcaccctg gatgctgtag gcataggctt 1200
ggttatgccg gtactgccgg gcctcttgcg ggatatcgtc cattccgaca gcatcgccag 1260
tcactatggc gtgctgctag cgctatatgc gttgatgcaa tttctatgcg cacccgttct 1320
cggagcactg tccgaccgct ttggccgccg cccagtcctg ctcgcttcgc tacttggagc 1380
cactatcgac tacgcgatca tggcgaccac acccgtcctg tggatctatc gaatctaaat 1440
gtaagttaaa atctctaaat aattaaataa gtcccagttt ctccatacga accttaacag 1500
cattgcggtg agcatctaga ccttcaacag cagccagatc catcactgct tggccaatat 1560
gtttcagtcc ctcaggagtt acgtcttgtg aagtgatgaa cttctggaag gttgcagtgt 1620
taactccgct gtattgacgg gcatatccgt acgttggcaa agtgtggttg gtaccggagg 1680
agtaatctcc acaactctct ggagagtagg caccaacaaa cacagatcca gcgtgttgta 1740
cttgatcaac ataagaagaa gcattctcga tttgcaggat caagtgttca ggagcgtact 1800
gattggacat ttccaaagcc tgctcgtagg ttgcaaccga tagggttgta gagtgtgcaa 1860
tacacttgcg tacaatttca acccttggca actgcacagc ttggttgtga acagcatctt 1920
caattctggc aagctccttg tctgtcatat cgacagccaa cagaatcacc tgggaatcaa 1980
taccatgttc agcttgagac agaaggtctg aggcaacgaa atctggatca gcgtatttat 2040
cagcaataac tagaacttca gaaggcccag caggcatgtc aatactacac agggctgatg 2100
tgtcattttg aaccatcatc ttggcagcag taacgaactg gtttcctgga ccaaatattt 2160
tgtcacactt aggaacagtt tctgttccgt aagccatagc agctactgcc tgggcgcctc 2220
ctgctagcac gatacactta gcaccaacct tgtgggcaac gtagatgact tctggggtaa 2280
gggtaccatc cttcttaggt ggagatgcaa aaacaatttc tttgcaacca gcaactttgg 2340
caggaacacc cagcatcagg gaagtggaag gcagaattgc ggttccacca ggaatataga 2400
ggccaacttt ctcaataggt cttgcaaaac gagagcagac tacaccaggg caagtctcaa 2460
cttgcaacgt ctccgttagt tgagcttcat ggaatttcct gacgttatct atagagagat 2520
caatggctct cttaacgtta tctggcaatt gcataagttc ctctgggaaa ggagcttcta 2580
acacaggtgt cttcaaagcg actccatcaa acttggcagt tagttctaaa agggctttgt 2640
caccattttg acgaacattg tcgacaattg gtttgactaa ttccataatc tgttccgttt 2700
tctggatagg acgacgaagg gcatcttcaa tttcttgtga ggaggcctta gaaacgtcaa 2760
ttttgcacaa ttcaatacga ccttcagaag ggacttcttt aggtttggat tcttctttag 2820
gttgttcctt ggtgtatcct ggcttggcat ctcctttcct tctagtgacc tttagggact 2880
tcatatccag gtttctctcc acctcgtcca acgtcacacc gtacttggca catctaacta 2940
atgcaaaata aaataagtca gcacattccc aggctatatc ttccttggat ttagcttctg 3000
caagttcatc agcttcctcc ctaattttag cgttcaacaa aacttcgtcg tcaaataacc 3060
gtttggtata agaaccttct ggagcattgc tcttacgatc ccacaaggtg gcttccatgg 3120
ctctaagacc ctttgattgg ccaaaacagg aagtgcgttc caagtgacag aaaccaacac 3180
ctgtttgttc aaccacaaat ttcaagcagt ctccatcaca atccaattcg atacccagca 3240
acttttgagt tgctccagat gtagcacctt tataccacaa accgtgacga cgagattggt 3300
agactccagt ttgtgtcctt atagcctccg gaatagactt tttggacgag tacaccaggc 3360
ccaacgagta attagaagag tcagccacca aagtagtgaa tagaccatcg gggcggtcag 3420
tagtcaaaga cgccaacaaa atttcactga cagggaactt tttgacatct tcagaaagtt 3480
cgtattcagt agtcaattgc cgagcatcaa taatggggat tataccagaa gcaacagtgg 3540
aagtcacatc taccaacttt gcggtctcag aaaaagcata aacagttcta ctaccgccat 3600
tagtgaaact tttcaaatcg cccagtggag aagaaaaagg cacagcgata ctagcattag 3660
cgggcaagga tgcaacttta tcaaccaggg tcctatagat aaccctagcg cctgggatca 3720
tcctttggac aactctttct gccaaatcta ggtccaaaat cacttcattg ataccattat 3780
tgtacaactt gagcaagttg tcgatcagct cctcaaattg gtcctctgta acggatgact 3840
caacttgcac attaacttga agctcagtcg attgagtgaa cttgatcagg ttgtgcagct 3900
ggtcagcagc atagggaaac acggcttttc ctaccaaact caaggaatta tcaaactctg 3960
caacacttgc gtatgcaggt agcaagggaa atgtcatact tgaagtcgga cagtgagtgt 4020
agtcttgaga aattctgaag ccgtattttt attatcagtg agtcagtcat caggagatcc 4080
tctacgccgg acgcatcgtg gccgacctgc aggggggggg ggggcgctga ggtctgcctc 4140
gtgaagaagg tgttgctgac tcataccagg cctgaatcgc cccatcatcc agccagaaag 4200
tgagggagcc acggttgatg agagctttgt tgtaggtgga ccagttggtg attttgaact 4260
tttgctttgc cacggaacgg tctgcgttgt cgggaagatg cgtgatctga tccttcaact 4320
cagcaaaagt tcgatttatt caacaaagcc gccgtcccgt caagtcagcg taatgctctg 4380
ccagtgttac aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa 4440
ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa 4500
tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc 4560
gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt 4620
atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg 4680
catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc 4740
atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct 4800
gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc 4860
atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc 4920
ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt 4980
cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt 5040
ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa 5100
tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa 5160
atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc gttgaatatg 5220
gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga 5280
tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttcccc 5340
cccccccctg caggtcggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc 5400
cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg 5460
cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc 5520
accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat 5580
gcaggagtcg cataagggag agcgtcgagt atctatgatt ggaagtatgg gaatggtgat 5640
acccgcattc ttcagtgtct tgaggtctcc tatcagatta tgcccaacta aagcaaccgg 5700
aggaggagat ttcatggtaa atttctctga cttttggtca tcagtagact cgaactgtga 5760
gactatctcg gttatgacag cagaaatgtc cttcttggag acagtaaatg aagtcccacc 5820
aataaagaaa tccttgttat caggaacaaa cttcttgttt cgaacttttt cggtgccttg 5880
aactataaaa tgtagagtgg atatgtcggg taggaatgga gcgggcaaat gcttaccttc 5940
tggaccttca agaggtatgt agggtttgta gatactgatg ccaacttcag tgacaacgtt 6000
gctatttcgt tcaaaccatt ccgaatccag agaaatcaaa gttgtttgtc tactattgat 6060
ccaagccagt gcggtcttga aactgacaat agtgtgctcg tgttttgagg tcatctttgt 6120
atgaataaat ctagtctttg atctaaataa tcttgacgag ccaaggcgat aaatacccaa 6180
atctaaaact cttttaaaac gttaaaagga caagtatgtc tgcctgtatt aaaccccaaa 6240
tcagctcgta gtctgatcct catcaacttg aggggcacta tcttgtttta gagaaatttg 6300
cggagatgcg atatcgagaa aaaggtacgc tgattttaaa cgtgaaattt atctcaagat 6360
ctctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 6420
acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 6480
gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg 6540
tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt 6600
gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct 6660
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6720
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6780
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6840
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6900
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6960
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 7020
ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 7080
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 7140
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 7200
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 7260
acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 7320
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 7380
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 7440
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 7500
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7560
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7620
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7680
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7740
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7800
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7860
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt 7920
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7980
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 8040
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 8100
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 8160
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 8220
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 8280
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 8340
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 8400
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 8460
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 8520
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8580
acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 8640
cctttcgtct tcaagaatta attctcatgt ttgacagctt atcatcgata agctgactca 8700
tgttggtatt gtgaaataga cgcagatcgg gaacactgaa aaataacagt tattattcg 8759
<210> 4
<211> 28
<212> DNA
<213> Synthesis
<400> 4
acttacgagc tcgagatctt ttttgtag 28
<210> 5
<211> 32
<212> DNA
<213> Synthesis
<400> 5
acttacgcgg atccgcgata gttgttcaat tg 32
<210> 6
<211> 36
<212> DNA
<213> Synthesis
<400> 6
acttaccgga attccggatg aatgcatcta gtatcc 36
<210> 7
<211> 36
<212> DNA
<213> Synthesis
<400> 7
cctgtagggg gctcaattcc accgaatcca tttgac 36
<210> 8
<211> 38
<212> DNA
<213> Synthesis
<400> 8
cggtggaatt gagcccccta cagggtattc gattgtgt 38
<210> 9
<211> 35
<212> DNA
<213> Synthesis
<400> 9
acttatttgc ggccgcttta ctacaagtca taatt 35
Claims (2)
1. Application of Hot1p as positive regulatory factor in improving expression quantity of heterologous protein in host cell, wherein the amino acid sequence of Hot1p is represented by SEQ ID NO. 1Hot1A gene encoding the same; the application is by inserting the promoter PgapHot1A gene that allows the host cell to overexpress Hot1p to enhance expression of a heterologous protein that uses Pgap as a promoter; the host cell is pichia pastoris.
2. A method for enhancing the expression of a heterologous protein in a host cell, comprising: insertion of the nucleotide sequence SEQ ID No. 1 after the promoter PgapHot1Genes that allow host cells to overexpress theHot1The Hot1p coded by the gene can enhance the expression of a heterologous protein taking Pgap as a promoter; culturing the host cell under conditions suitable for expression of the heterologous protein, and isolating the expressed heterologous protein from the culture medium; the host cell is pichia pastoris.
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| CN113564166A (en) * | 2021-07-26 | 2021-10-29 | 浙江大学 | Growth-dependent promoter from pichia pastoris and application thereof |
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| DK2900690T3 (en) * | 2012-09-25 | 2019-10-21 | Vib Vzw | MUTANT Yeast strain with REDUCED GLYCEROL PRODUCTION |
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