WO2022022197A1 - Procédé à base de séquence de codage n-terminale pour modifier l'expression de protéines régulatrices - Google Patents

Procédé à base de séquence de codage n-terminale pour modifier l'expression de protéines régulatrices Download PDF

Info

Publication number
WO2022022197A1
WO2022022197A1 PCT/CN2021/102986 CN2021102986W WO2022022197A1 WO 2022022197 A1 WO2022022197 A1 WO 2022022197A1 CN 2021102986 W CN2021102986 W CN 2021102986W WO 2022022197 A1 WO2022022197 A1 WO 2022022197A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
pullulanase
value
coding region
sfgfp
Prior art date
Application number
PCT/CN2021/102986
Other languages
English (en)
Chinese (zh)
Inventor
刘松
徐奎栋
李江华
陈坚
周景文
Original Assignee
江南大学
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2022022197A1 publication Critical patent/WO2022022197A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the invention relates to a method for regulating protein expression based on the modification of an N-terminal coding sequence, belonging to the technical field of genetic engineering and enzyme engineering
  • Mutation of genes is of great significance for changing the properties of proteins. Usually, mutation sequences with better properties can be found through mutation, thereby improving the application value of proteins. Synonymous mutation of genes is a commonly used mutation method, and the expression levels of synonymous mutations of genes can vary greatly.
  • the current commonly used method is to construct a synonymous mutation library and combine it with a high-throughput screening strategy to find the best mutant.
  • this method is time-consuming, labor-intensive, and specific, and cannot be used to guide the design of other genes.
  • synthesizing a series of short peptides is beneficial to widely improve gene expression, this method will have an impact on enzyme activity, because these expression-promoting short peptides occupy the position of the signal peptide, so that the Suitable for extracellular proteins that require the addition of a signal peptide.
  • NCS N-terminal coding region
  • the method of the present invention is established based on the bioinformatics analysis of representative samples, and by this method, the nucleotide sequence of the first 30 bases of the N-terminal of any gene can be de novo designed, and synonymous mutation can be performed on it.
  • the NCS nucleotide sequence of any gene is changed to the target nucleotide sequence by mutating the primers.
  • the present invention can be used to guide the design of any gene without adding additional amino acid sequence, and the properties of the protein are minimized. It can greatly improve the expression level of the target gene.
  • the present invention provides a method for screening nucleotide sequences encoding proteins with different expression levels, measuring the values of GC3 and ⁇ G, and then calculating the relative expression level of the protein by using the following equation, that is, the PsfGFP value, and screening according to the PsfGFP value The corresponding nucleotide sequence is obtained; the P sfGFP value is positively correlated with the actual expression of the protein:
  • PsfGFP 274497.657-108717.401 ⁇ GC3+4886.529 ⁇ G.
  • the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region.
  • the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 96 bp region of the N-terminal coding region.
  • the protein is any protein that can be expressed in Bacillus subtilis.
  • the protein includes, but is not limited to, pullulanase.
  • amino acid sequence of the pullulanase is shown in SEQ ID NO.19.
  • the PsfGFP value is positively correlated with the actual expression level of the protein.
  • the corresponding nucleotide sequences are screened according to the PsfGFP value.
  • the invention provides a method for regulating the protein expression of genetically engineered bacteria.
  • GC3 and ⁇ G parameters of the gene calculate the relative expression level of each nucleotide sequence according to the equation, select the nucleotide sequence with the required expression level, mutate the N-terminal coding region of the target protein accordingly, and transform it into in host cells;
  • the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region.
  • the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 96 bp region of the N-terminal coding region.
  • the nucleotide sequences whose P sfGFP value in the mutation library is in the top 10% are selected; when the protein expression needs to be down-regulated, the P sfGFP value in the mutation library is selected. sfGFP values are in the bottom 10% of nucleotide sequences.
  • the genetically engineered bacteria use Bacillus subtilis as a host.
  • the protein is any protein that can be expressed in Bacillus subtilis.
  • the protein includes, but is not limited to, pullulanase.
  • amino acid sequence of the pullulanase is shown in SEQ ID NO.19.
  • the invention provides a method for regulating the expression level of pullulanase.
  • the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region.
  • the recombinant plasmid is introduced into Bacillus subtilis, and the Bacillus subtilis is used to produce the protein.
  • amino acid sequence of the pullulanase is shown in SEQ ID NO.19.
  • the present invention also protects the application of the method for screening nucleotide sequences encoding high-expression proteins, or the method for regulating the protein expression of genetically engineered bacteria in regulating the expression of a target protein.
  • the present invention also protects the application of the method for regulating the expression of pullulanase in regulating pullulanase.
  • the calculated PsfGFP value is positively correlated with the actual expression level of the protein.
  • to calculate the PsfGFP value is to select the corresponding synonymous mutation sequence as needed. It was applied to transform the N-terminus of pullulanase fused to the nucleotide sequence of the Bgls signal peptide, and the selected synonymous mutation sequence could increase the extracellular enzyme activity by 2.67 times and decrease it by 48%.
  • Figure 1 is the map of the sfGFP expression plasmid P43-NMK-sfGFP.
  • Figure 2 is a graph showing the relative fluorescence intensity of the NCS library of sfGFP.
  • Figure 3 shows the nucleotide sequence indices and fluorescence values of the 1st to 60th samples among the 172 samples.
  • Figure 4 shows the nucleotide sequence indices and fluorescence values of the 61st to 120th samples among the 172 samples.
  • Figure 5 shows the nucleotide sequence indexes and fluorescence values of the 121st to 172nd samples in the 172 samples.
  • Figure 6 is the distribution of relative fluorescence values before and after transformation.
  • Figure 7 is the map of pullulanase expression plasmid P43-NMK-Bgls fused with BglS signal peptide.
  • Figure 8 is a protein gel image of the 5 NCS variants of the BglS signal peptide.
  • Figure 9 is a graph showing the correlation between the predicted expression value of pullulanase with the addition of five Bgls signal peptide sequences and the measured value of enzyme activity.
  • Seed medium (g/L): peptone 10, yeast extract 5, sodium chloride 5;
  • Fermentation medium (g/L): The following components were dissolved in 0.9L water: peptone 12g, yeast extract 24g, glycerol 4mL.
  • Seed culture Pick a single colony of engineering bacteria and insert it into the seed medium, the culture temperature is 37°C, the shaking speed is 200r/min, and the culture is 24h;
  • Fermentation culture The seed culture liquid is inserted into the fermentation medium according to the inoculum amount of 4%, the culture temperature is 37 °C, and the fermentation is carried out for 24 hours.
  • One-step cloning kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.
  • Glue concentration of 10% SDS-PAGE gel was used to analyze the protein expression level.
  • MES or MOPS buffer was used as the running buffer, and the loading volume was 10 ⁇ L.
  • the electrophoresis voltage was 150V.
  • Specific sample preparation and electrophoresis operations were performed according to the kit instructions.
  • the molecular weights (kDa) of the standard protein were: 188, 98, 62, 49, 38, 28, 17, 14, 6 and 3; and when electrophoresed in MOPS buffer, the molecular weight of the standard protein was Molecular weights (kDa) are: 191, 97, 64, 51, 39, 28, 19, 14
  • the PLytr promoter (nucleotide sequence shown in SEQ ID NO.1) was used with primers Lytr-F/Lytr-R (nucleotide sequence shown in SEQ ID NO.2 and 3) and Lytr-F-plasmid/ Lytr-R-plasmid (nucleotide sequence shown in SEQ ID NO. 4 and 5) was connected to the P43NMK plasmid by a one-step cloning kit to construct the plasmid P43NMK-Lytr;
  • the sfGFP fluorescent protein reporter gene (nucleotide sequence shown in SEQ ID NO.6) was used primers sfGFP-F/sfGFP-R (nucleotide sequence shown in SEQ ID NO.7 and 8) and sfGFP-F-plasmid/sfGFP-R-plasmid (nucleotide sequences shown in SEQ ID NO. 9 and 10) were fused to the downstream of PLytr by a one-step cloning kit to obtain the construction of P43NMK-Lytr_sfGFP, as shown in Figure 1. Show;
  • the recombinant plasmids with synonymous mutations constructed in Example 1 were transformed into the expression host Bacillus subtilis WB600, respectively, and the transformed single clones were inoculated into 96 shallow-well plates containing 200 ⁇ L of LB seed medium, and cultured for 8 hours;
  • a total of 8598 monoclonal host cells were characterized in Example 2, and the fluorescence value/OD was defined as the relative fluorescence intensity RFI. According to the level of the RFI value, the monoclonal cells were sorted from high to low, and every 50 cells were selected for sequencing identification (that is, the first One of the 1 to 50 strains was selected, one of the 51 to 100 strains was selected, and so on), and a total of 172 single clones were identified by sequencing.
  • GC3 The third base of the synonymous codon is the content of GC;
  • T3s, C3s, A3s, G3s After a synonymous mutation occurs at the first 30 bases of the N-terminal of the gene, the third synonymous codon is the frequency of T, C, A, and G, respectively;
  • CAI codon preference
  • Fop frequency of optimal codons (both above calculated ranges are 30 nucleotide sequences for NCS mutations).
  • ⁇ G the minimum free energy
  • the calculated range includes the region from the transcription start site to the downstream of NCS, in this example, 25 bases upstream of ATG (the transcription start site of the PLytr promoter) to 96 bases downstream of ATG were selected base;
  • TIR translation initiation rate
  • the range is the same as the calculation ⁇ G.
  • Example 3 Substitute the sequence of 172 samples in Example 3 into the regression prediction equation, calculate the predicted value, and compare it with the actual fluorescence value measured in Example 3, and perform correlation analysis. As shown in Figure 6, the sequence of The Pearson coefficient between the predicted value and the measured fluorescence value can reach 0.675, and the correlation is very strong, indicating that the regression prediction equation can be used to predict the protein fluorescence value.
  • Example 5 Using a prediction equation to guide NCS engineering of the signal peptide BglS gene
  • the BglS signal peptide (nucleotide sequence shown in SEQ ID NO. 13) was fused to the N-terminal of the pullulanase encoding gene (nucleotide sequence shown in SEQ ID NO. 14) to achieve pullulanase of extracellular expression.
  • the specific method is to clone the BglS signal peptide into the downstream of PLytr in P43NMK-Lytr by using the same one-step cloning method in the example to construct P43NMK-Lytr-BglS, as shown in FIG. 7 .
  • NCS region of BglS close to ATG was optimized: all the synonymous mutation combinations of the first ten amino acids of BglS were exhausted, and there were 131,072 possibilities; according to the examples 4 equations were used to calculate the GC3 and ⁇ G of each of the 131072 sequences and the theoretical value of PsfGFP, and according to the predicted value, 5 Bgls variants including wild type were selected: NCS+, NCS+', NCS-wt, NCS -', NCS-.
  • NCS+ represents the P sfGFP maximum variant
  • NCS+' represents the intermediate variant between the maximum and wild type of P sfGFP
  • NCS-wt represents the wild type
  • NCS- represents the P sfGFP minimum variant
  • NCS-' represents the intermediate variant Between the minimum of PsfGFP and the intermediate value variant of wild type, it has continuously decreasing predicted expression intensity.
  • step (1) signal peptide Bgls variants NCS+ (nucleotide sequence shown in SEQ ID NO. 15), NCS+' (nucleotide sequence shown in SEQ ID NO. 16), NCS- ' (the nucleotide sequence is shown in SEQ ID NO. 17), NCS- (the nucleotide sequence is shown in SEQ ID NO.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention se rapporte au domaine du génie génétique. Un procédé basé sur une séquence de codage N-terminale pour modifier une expression de protéine régulatrice est divulguée. Dans la présente invention, Bacillus subtilis sert d'hôte d'expression ; au moyen d'un modèle prédictif, la séquence nucléotidique la plus favorable pour favoriser l'expression génique est évaluée dans une mutation synonyme de région de codage N-terminale. Avec la combinaison de bibliothèques de mutations synonymes des dix meilleurs acides aminés du NCS d'une protéine fluorescente verte Superfolder (sfGFP, « superfolder green fluorescent protein »), l'intensité fluorescente des protéines dans les bibliothèques est mesurée, 172 échantillons représentatifs sont sélectionnés, séquencés puis identifiés et un procédé statistique est utilisé pour établir un modèle prédictif. La pullulanase d'un peptide signal BlgS est intégrée de manière optimale par l'intermédiaire du modèle, l'activité enzymatique extracellulaire de la pullulanase peut être augmentée à 2,67 fois de celle avant transformation et être réduite de 48 %, fournissant ainsi une direction pour une transformation rationnelle pour concevoir un gène N-terminal à partir de rien et favorisant une expression facilement régulée du gène.
PCT/CN2021/102986 2020-07-29 2021-06-29 Procédé à base de séquence de codage n-terminale pour modifier l'expression de protéines régulatrices WO2022022197A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010742774.7A CN111850096B (zh) 2020-07-29 2020-07-29 一种基于n端编码序列改造调控蛋白质表达的方法
CN202010742774.7 2020-07-29

Publications (1)

Publication Number Publication Date
WO2022022197A1 true WO2022022197A1 (fr) 2022-02-03

Family

ID=72945069

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/102986 WO2022022197A1 (fr) 2020-07-29 2021-06-29 Procédé à base de séquence de codage n-terminale pour modifier l'expression de protéines régulatrices

Country Status (2)

Country Link
CN (1) CN111850096B (fr)
WO (1) WO2022022197A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116072231A (zh) * 2022-10-17 2023-05-05 中国医学科学院病原生物学研究所 基于氨基酸序列的密码子优化在mRNA疫苗研发中的应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111850096B (zh) * 2020-07-29 2022-02-01 江南大学 一种基于n端编码序列改造调控蛋白质表达的方法
CN113201052B (zh) * 2021-04-21 2023-06-27 华东理工大学 HarpinEa的高效可溶性表达及生产方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102676480A (zh) * 2012-06-08 2012-09-19 江南大学 一种应用自诱导培养基和双温度调控策略生产胞外普鲁兰酶的方法
CN102791854A (zh) * 2009-12-22 2012-11-21 诺维信公司 普鲁兰酶变体及其用途
CN106190934A (zh) * 2016-07-05 2016-12-07 江南大学 一种生产普鲁兰酶的重组枯草芽孢杆菌及其构建
CN111850096A (zh) * 2020-07-29 2020-10-30 江南大学 一种基于n端编码序列改造调控蛋白质表达的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8679790B2 (en) * 2010-01-05 2014-03-25 The Trustees Of The University Of Pennsylvania Leader sequence to boost gene expression
US20180010136A1 (en) * 2014-05-30 2018-01-11 John Francis Hunt, III Methods for Altering Polypeptide Expression
CN104694452B (zh) * 2015-03-30 2019-03-01 中国科学院上海高等研究院 一种高产普鲁兰酶的重组枯草芽孢杆菌及其构建方法
CN106754833B (zh) * 2017-01-16 2020-06-09 广东溢多利生物科技股份有限公司 在枯草芽孢杆菌中高效表达普鲁兰酶的方法及重组枯草芽孢杆菌

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102791854A (zh) * 2009-12-22 2012-11-21 诺维信公司 普鲁兰酶变体及其用途
CN102676480A (zh) * 2012-06-08 2012-09-19 江南大学 一种应用自诱导培养基和双温度调控策略生产胞外普鲁兰酶的方法
CN106190934A (zh) * 2016-07-05 2016-12-07 江南大学 一种生产普鲁兰酶的重组枯草芽孢杆菌及其构建
CN111850096A (zh) * 2020-07-29 2020-10-30 江南大学 一种基于n端编码序列改造调控蛋白质表达的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 23 March 2016 (2016-03-23), ANONYMOUS : "pullulanase [synthetic construct]", XP055890670, retrieved from NCBI Database accession no. AMQ67157 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116072231A (zh) * 2022-10-17 2023-05-05 中国医学科学院病原生物学研究所 基于氨基酸序列的密码子优化在mRNA疫苗研发中的应用
CN116072231B (zh) * 2022-10-17 2024-02-13 中国医学科学院病原生物学研究所 基于氨基酸序列的密码子优化设计mRNA疫苗的方法

Also Published As

Publication number Publication date
CN111850096A (zh) 2020-10-30
CN111850096B (zh) 2022-02-01

Similar Documents

Publication Publication Date Title
WO2022022197A1 (fr) Procédé à base de séquence de codage n-terminale pour modifier l'expression de protéines régulatrices
Arai et al. Expression of the nir and nor genes for denitrification of Pseudomonas aeruginosa requires a novel CRP/FNR-related transcriptional regulator, DNR, in addition to ANR
JP5211559B2 (ja) 熱安定性を有する変異型ビリルビンオキシダーゼ
CN107012130B (zh) 一种葡萄糖氧化酶突变体及其编码基因和应用
CN111500597B (zh) 一种橡胶树白粉病抗性相关基因HbHSP90.8及其应用
CN111826377B (zh) 促进普鲁兰酶胞外表达的信号肽
CN110628738B (zh) 提高葡萄糖氧化酶活性的方法、突变体及其应用
CN1974768A (zh) 人肺癌细胞ngal基因启动子区转录调控元件
CN111850008B (zh) 促进蛋白胞外表达的信号肽
CN113106103B (zh) Ldlr基因突变体及其应用
CN110592084B (zh) 一种rhtA基因启动子改造的重组菌株及其构建方法与应用
CN107236758B (zh) 一种共表达热激蛋白提高外源蛋白表达量的方法
CN112391396A (zh) 构建于大肠杆菌中的粪肠球菌群体感应基因开关系统及其表达载体、工程菌和应用
CN114410496B (zh) 一种提高毕赤酵母外源蛋白产量的方法
CN111808177B (zh) 提高蛋白表达量的信号肽及其应用
CN111254143B (zh) 具有优良胁迫耐受性的简单节杆菌工程菌株的构建方法、菌株及其应用
CN109060744B (zh) 一种毕赤酵母重组蛋白分泌表达量的荧光分析方法
CN107083394B (zh) 一种共表达核糖体蛋白提高外源蛋白表达量的方法
JP7403590B2 (ja) エールビール及びその製造方法
CN113699174B (zh) 一种自诱导表达系统及其在促进基因表达中的应用
CN113755474B (zh) 一种羧肽酶及其编码基因和应用
CN115261363B (zh) Apobec3a的rna脱氨酶活性测定方法及rna高活性的apobec3a变体
CN116949042A (zh) 一种提高法夫驹形氏酵母外源蛋白表达量的醇氧化酶1启动子（paox1）及其方法
JP2024018971A (ja) イネ科植物リポキシゲナーゼ３の製造方法、及び組換え酵母
CN1305901C (zh) 肿瘤转移相关蛋白及其编码基因

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21849697

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21849697

Country of ref document: EP

Kind code of ref document: A1