WO2021252391A1 - Procédés et vecteurs permettant d'améliorer l'expression et/ou d'inhiber la dégradation d'une protéine - Google Patents

Procédés et vecteurs permettant d'améliorer l'expression et/ou d'inhiber la dégradation d'une protéine Download PDF

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WO2021252391A1
WO2021252391A1 PCT/US2021/036247 US2021036247W WO2021252391A1 WO 2021252391 A1 WO2021252391 A1 WO 2021252391A1 US 2021036247 W US2021036247 W US 2021036247W WO 2021252391 A1 WO2021252391 A1 WO 2021252391A1
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rad51
host cell
idr
vector
ntd
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Ting-Fang Wang
Chi-Ning CHUANG
Tai-Ting WOO
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Academia Sinica
Shih, Ming-Che
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/12—Dual-specificity kinases (2.7.12)
    • C12Y207/12001—Dual-specificity kinase (2.7.12.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

Definitions

  • the present invention relates to a field of recombinant protein expression.
  • the present disclosure pertains to an expression vector comprising an intrinsically disordered region (IDR) with high S/T and/or Q content or an S/T-Q cluster domain (SCD) and a method of enhancing protein expression using the expression vector.
  • IDR intrinsically disordered region
  • SCD S/T-Q cluster domain
  • the recombinant fusion protein approach has been widely applied in biological research and therapeutics. This approach typically involves joining two or more cDNA sequences in frame through ligation or overlapping polymerization chain reaction (PCR). The resulting DNA sequence then encodes a single polypeptide with a fusion carrier or protein tag, including an expression carrier, solubility carrier, fluorescent tag, targeting tag, reporter tag, chromatography (or affinity) tag and epitope tag, etc.
  • a fusion carrier or protein tag including an expression carrier, solubility carrier, fluorescent tag, targeting tag, reporter tag, chromatography (or affinity) tag and epitope tag, etc.
  • US 20160009779 provides for fusion tags of 20 to 50 amino acids and an expression vector system comprising a fusion tag of the invention.
  • US 20200385774 discloses a peptide sequence of a guide protein derived from the SH3 domain of the protein spectrin for the production of peptides of interest in the form of a fusion protein.
  • the present disclosure provides a method for enhancing expression and/or inhibiting degradation of an interested polypeptide in a host cell, comprising the steps of constructing an expression vector comprising a polynucleotide coding an intrinsically disordered region (IDR), and a polynucleotide coding the interested polypeptide; transforming the expression vector to the host cell and culturing the host cell under conditions that allow for expression of the interested polypeptide; wherein the overall percentage of the content of S, T and Q in the total amino acid content of the IDR is higher than 10%.
  • IDR intrinsically disordered region
  • the IDR comprises one or more S/T-Q cluster domains (SCDs).
  • SCDs S/T-Q cluster domains
  • one or more S, T or Q residues in the IDR are replaced with an amino acid with a hydrophobic side chain.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 residues in the IDR are replaced with an amino acid with a hydrophobic side chain.
  • one or more S, T or Q residues in the SCD are replaced with an amino acid with a negative-charged side chain.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 residues in the IDR are replaced with an amino acid with a negative-charged side chain.
  • one or more S, T or Q residues in the IDR are replaced with an another amino acid of S, T and Q.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 residues in the IDR are replaced with another amino acid of S, T and Q.
  • the present disclosure provides a vector comprising an intrinsically disordered region operably linked to a polypeptide coding an interested polypeptide, wherein the overall percentage of the content of S, T and Q in the total amino acid content of the IDR is higher than 10%.
  • the present disclosure provides a host cell comprising the vector of the present disclosure.
  • the SCDs or IDR are fused to the N-terminus of the interested polypeptide.
  • the IDR or SCDs are used as fusion tags for enhancing expression and/or inhibiting degradation of the interested polypeptide.
  • the interested polypeptide is a heterologous protein.
  • the SCD or IDR is phosphorylated.
  • the overall percentage of the content of S, T and Q in the total amino acid content of the IDR or SCD is higher than 15%. In some embodiments, the overall percentage of the content of S, T and Q in the total amino acid content of the IDR or SCD is higher than 15%, 20%, 25% or 30%.
  • the overall percentage of the content of S, T and Q in the total amino acid content of the IDR or SCD ranges from about 30% to about 40%.
  • the SCD is an IDR with high S/T and Q content (Traven A. and
  • the IDR or SCD is as listed in Table 1. Table 1. Amino acid sequences of SCDs or IDRs used in this disclosure
  • the host cell is a prokaryotic cell.
  • the host cell is a mammalian cell, a yeast cell, a fungal cell, an insect cell, a plant cell or a protozoan cell.
  • the host cell is Saccharomyces cerevisiae.
  • Figures 1 A to D show that NTD, a typical SCD, is essential for promoting high steady-state protein levels of S. cerevisiae Rad51 in vivo.
  • Figure 1 A shows that total cell lysates were prepared from vegetative cells under methyl methanesulfonate (MMS) treatment at indicated time-points and subjected to immunoblotting. Hsp104 was used as a loading control. Immunoblotting using goat anti-Rad51 antibody (yN-19) was purchased from Santa Cruz Biotechnology (CA, USA). This antibody was generated using a peptide (sc-8936) mapping at the N-terminus of yeast Rad51.
  • MMS methyl methanesulfonate
  • Figure 1 B shows that immunoblotting using guinea pig anti- Rad51 antibody was performed.
  • the predicted molecular weight of Rad51 - ⁇ N was 36,270 daltons.
  • Figure 1 C shows that total cell lysates from wild-type (WT) mitotic cells under MMS treatment were diluted with those of rad51- ⁇ N or empty sample buffer at indicated titers to estimate the relative steady-state protein levels of WT Rad51 and Rad51- ⁇ N
  • Figure 1 D shows that NTD is dispensable for DNA damage tolerance in vegetative growth. Spot assay showed five-fold serial dilutions of indicated haploid or diploid strains grown on YPD plates and YPD plates containing MMS at the indicated concentrations. Ploidy of different strains is indicated on the right.
  • Figure 1 E shows that spore viability was analyzed after 3 days on sporulation media at 30 °C. To score for spore viability, only tetrads (but not dyads or triads) were dissected on YPD.
  • Figures 2 A to C show that phosphorylation of Rad51-NTD not only promotes protein expression but also enhances protein stability.
  • the wild-type SK1 haploid strain was transformed with an indicated yeast expression vector, i.e., P RAD51 -lacZ-V5 (mock), P RAD51 -NTD-lacZ-V5, P RAD51 -NTD 3A -lacZ-V5 or P RAD51 -NTD 3D -lacZ-V5.
  • FIG. 1 shows that NTD-LacZ-V5 fusion proteins ( ⁇ 128 kilodaltons) were marked by white arrow heads. Endogenous Rad51 ( ⁇ 43 kilodaltons) was indicated by black arrows. Numbers below the anti-Rad51 immunoblot indicate fold-changes of NTD-LacZ-V5 fusion protein levels normalized to endogenous Rad51 protein levels in the same blot.
  • Figure 2 B shows that immunoblotting using anti-V5 antiserum was performed. Presumptive degraded products were indicated by black arrow heads.
  • Hsp104 was used as a loading control.
  • Figure 2 C shows that quantitative yeast LacZ assays were performed as described previously (Chuang C.N., Cheng Y.H., Wang T.F. Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis. Nucleic Acids Res. 2012; 40:11416–11427). Error bars indicate standard deviation between experiments. Asterisks indicate significant differences with p values calculated using a two-tailed t-test (*, p value ⁇ 0.05 and ***, p value ⁇ 0.001). [0024]
  • Figures 3 A and B show that the SCDs of three different S.
  • the wild-type haploid strain was transformed with an indicated yeast expression vector with the endogenous RAD51 promoter, i.e., P RAD51 -lacZ-V5- His 6 (mock), P RAD51 -Rad51-SCD-lacZ-V5-His 6 , P RAD51 -Rad53-SCD1-lacZ-V5-His 6 or P RAD51 - Hop1-SCD-lacZ-V5-His 6 .
  • Rad51-SCD and Rad53-SCD1 contain N-terminal 1-66 and 1-29 amino acid residues of Rad51 and Rad53, respectively.
  • Hop1-SCD contains a middle region (258-324 amino acid residues) of Hop1.
  • Total cell lysates were prepared from mitotic cells in exponential growth for visualization by immunoblotting with the corresponding antisera and quantitative yeast LacZ assays.
  • Figure 3 A shows that immunoblotting using anti-V5 antiserum was performed. Hsp104 was used as a loading control.
  • Figure 3 B shows that quantitative yeast LacZ ( ⁇ -galactosidase) assays were performed as described previously. Error bars indicate standard deviation between experiments. Asterisks indicate significant differences with p values calculated using a two-tailed t-test (*, p value ⁇ 0.05 and ***, p value ⁇ 0.001).
  • FIG. 4 shows that Rad53-SCD1 expresses four different target protein in S. cerevisiae on a large scale.
  • the wild-type haploid strain was transformed with an indicated yeast expression vector, i.e., P RAD51 -lacZ-V5-His 6 (mock), P RAD51 -Rad53-SCD1-lacZ-V5-His 6 , P RAD51 - GFP-V5-His 6 (mock), P RAD51 -Rad53-SCD1-GFP-V5-His 6 , P RAD51 -GST-V5-His 6 (mock), P RAD51 - Rad53-SCD1-GST-V5-His 6 , P RAD51 -GST(nd)-V5-His 6 (mock) or P RAD51 -Rad53-SCD1-GST(nd)- V5-His 6 .
  • FIGS. 5 A to F show that the NTD of budding yeast Rad51 is a direct target of Mec1 ATR and Tel1 ATM .
  • Figure 5 A shows the amino acid sequences (1-66 residues) of Rad51- NTD, the phosphorylation-defective mutant (Rad51-3A) and the phosphomimetic mutant (Rad51-3D).
  • Figures 5 B, C and D show that phosphorylation of Rad51-NTD during vegetative growth and meiosis was performed. ATR Mec1 - and ATM Tel1 -dependent phosphorylation of Rad51-NTD during vegetative growth and meiosis was performed.
  • Figure 5 B shows demonstration of the specificity of anti-phosphorylated Rad51-S 2 Q, Rad51-S 12 Q and Rad51- S 30 Q antisera.
  • Total cell lysates were prepared from mitotic cells under MMS treatment (as shown in Figure 5 C) or from meiotic cells at indicated sporulation time-points (as shown in Figure 5 D), and then visualized by immunoblotting with the corresponding antisera. Hsp104 was used as a loading control.
  • Figure 5 E shows that spore viability was analyzed after 3 days on sporulation media at 30 °C. To score spore viability, only tetrads (but not dyads or triads) were dissected on YPD.
  • Figure 5 F demonstrates that spot assay showing five-fold serial dilutions of indicated strains were grown on YPD plates and YPD plates containing 0.01% or 0.02% MMS.
  • Figure 6 shows tetrad dissection analysis. Yeast diploid cells were transformed with the indicated 2 ⁇ overexpression vector.
  • FIGS 7 A to B show low-level expression of Rad51- ⁇ N in meiotic cells.
  • Total cell lysates were prepared from mitotic cells under MMS treatment or from meiotic cultures at indicated time-points and subjected to immunoblotting as described in Figure 1.
  • Hsp104 was used as a loading control.
  • Figure 7 A shows that immunoblotting using goat anti-Rad51 antibody (yN-19) was purchased from Santa Cruz Biotechnology (CA, USA). This antibody was generated using a peptide (sc-8936) mapping at the N-terminus of yeast Rad51.
  • Figure 8 A shows that meiotic progression was monitored by DAPI (4',6-diamidino-2-phenylindole)-staining of nuclei.
  • DAPI 4',6-diamidino-2-phenylindole
  • Figures 8 B and 8 C show cytology. Representative images of meiotic nuclear surface spreading experiments using guinea pig anti- Rad51 (green) and anti-phosphorylated Rad51-S 30 Q (red) or anti-Dmc1 (red) antisera, respectively.
  • Meiotic chromosomes were stained with DAPI (blue) at indicated sporulation time- points. Scale bars, 5 pm.
  • Figure 8 D shows quantification of the numbers of Rad51 and Dmc1 foci in the WT and rad51-3A strains. The numbers of foci in each foci-positive chromosome spread (with more than five foci) were counted and plotted as shown. The sizes of circles are proportional to the numbers of nuclei with a given number of foci. The mean number of foci per nucleus is shown in red (bottom), and also as a red bar in the graph. Standard deviations of numbers of foci are shown in parentheses. N represents the number of nuclei analyzed for foci- counting.
  • Figures 9 A to D show that Phosphorylation-defective Rad51 sustains scant protein levels during meiosis.
  • Figure 9 A shows that immunoblotting time-course analyses of wild-type and mutant strains were performed as previously described 21,45 .
  • Total cell lysates were prepared from meiotic cells at indicated sporulation time-points and then visualized by immunoblotting with the corresponding antisera.
  • Antisera against Zip1 and Hop1 were included as references for meiotic progression.
  • Hsp104 was used as a loading control. Size in kilodaltons of standard protein markers is labeled to the left of the blots. The asterisk indicates non-specific bands.
  • Figure 9 B shows that quantifications of the protein bands in Figure 9 A were normalized to those of Hsp104 at each time-point using ImageJ software and are shown as the relative steady- state levels of indicated proteins. The highest level of immunoblot signal in each blot was used as the standard for comparison.
  • Figure 9 C shows that two-fold serial dilutions of WT cell lysates were used to estimate the Rad51 protein levels in rad51 mutants at 1 h and 5 h in SPM.
  • Figure 9 D shows that the protein levels of Rad51 variants from non-diluted lysates shown in Figure 9 C were quantified and normalized to Hsp104 levels. The Rad51 level of WT at 1 h in SPM was used as the standard.
  • Figures 10 A to E show determination of the half-lives of different Rad51 proteins.
  • Figure 10 A shows cycloheximide-shutoff experiments. Protein synthesis was inhibited by adding 200 ⁇ g/ml cycloheximide to the meiotic cultures at the indicated time-points, i.e. 4-h (as shown in Figure 10 A) or 1-h and 5-h (as shown in Figure 10 D) after the meiotic cells had been transferred into the sporulation medium. Samples untreated (upper panels) or treated (lower panels) with cycloheximide were taken at 0, 30, 60, 90, 120 and 180 min after the addition of cycloheximide for immunoblotting analysis.
  • Figure 10 B shows that Immunoblotting time-course analyses of WT meiosis reveal the phosphorylation status of native Rad51 and Hop1. Hsp104 was used as a loading control.
  • Figure 10 C shows that after normalization to Hsp104 at each time-point in SPM (T SPM ), the relative steady-state protein levels in Figure 10 B were plotted as compared to those at 5-h (T 5 ).
  • FIGS 11 A to E show Rad51-3A is degraded by the 26S proteasome. Exponential cultures of indicated strains were treated with MMS (0.02%) 30 min prior to the addition of cycloheximide (200 ⁇ g/ml) and/or MG132 (25 ⁇ M). Protein samples were harvested at indicated time-points and immunoblotting was performed as described in Figure 6. To decrease MG132 efflux, we deleted the PDR5 gene, which encodes an ABC transporter. The protein levels of another vegetative house-keeping protein, hexokinase, were analyzed by immunoblotting using anti-hexokinase antibodies and are shown as the loading control in Figure 11 A.
  • FIGs 11 A and D Quantification of immunoblotting results in Figures 11 A and D are plotted in Figures 11 C and E, respectively, in which total Rad51 protein levels normalized to Hsp104 levels at that time-point are plotted. Quantification results of samples of WT and rad51-3A with cycloheximide from Figure 11 C were included in Figure 11 E for comparison.
  • Figures 12 A to C show that N-terminal fusion of SCD or the prion (nucleation) domain promotes high-level expression of target proteins.
  • Figure 12 A shows visualization of native Rad51 (NTD-Rad51- ⁇ N), Rad51- ⁇ N, and the Rad51- ⁇ N fusion proteins by immunoblotting. Hsp104 was used as a loading control.
  • Figures 13 A and D show Relative ⁇ -galactosidase (LacZ) activities are correlated with the percentage STQN amino acid content of IDRs.
  • Figure 13 A shows list of IDRs with their respective length, numbers of S/T/Q/N amino acids, overall STQN percentage, and relative ⁇ -galactosidase activity.
  • Figures 13 B to D show linear regressions between relative ⁇ - galactosidase activities and overall STQN percentages for Rad51-NTD (as shown in Figure 13 B), Sup35-PND (as shown in Figure 13 C), and Rad53-SCD1 (as shown in Figure 13 D).
  • the coefficients of determination (R 2 ) are indicated for each simple linear regression, Detailed Description of the Invention
  • SCDs possess autonomous expression- enhancing activity when they occur naturally or are artificially designed as NH 2 -terminal fusion tags.
  • the present disclosure reveals two novel roles for SCDs in the regulation of protein homeostasis in vivo , suggesting that SCDs can be used as fusion tags for high-level protein production and/or inhibition of protein degradation.
  • expression vector refers to any molecule used to transfer coding information to a host cell.
  • the term "host cell” refers to a cell that has been transformed, transfected, or transduced by an expression vector bearing a gene of interest, which is then expressed by the cell.
  • polynucleotide is a polymer of nucleotides which are usually linked from one deoxyribose or ribose to another and refers to DNA as well as RNA, depending on the context.
  • polynucleotide does not comprise any size restrictions and also encompasses polynucleotides comprising modifications, in particular modified nucleotides.
  • an “interested polypeptide” refers to the polypeptide to be expressed in a host cell.
  • a polypeptide refers to a molecule comprising a polymer of amino acids linked together by a peptide bond(s).
  • Polypeptides include polypeptides of any length, including proteins (for example, having more than 50 amino acids) and peptides (for example, having 2-49 amino acids). Polypeptides include proteins and/or peptides of any activity or bioactivity.
  • Intrinsically disordered regions are protein sequences lacking fixed or ordered three-dimensional structures. Many IDRs are endowed with important molecular functions such as physical interactions, posttranslational modifications or solubility enhancement.
  • the present disclosure surprisingly reveals that several biologically important IDRs can act as N- terminal fusion carriers to promote target protein folding or protein quality control, thereby enhancing protein expression, and has a strong correlation with high S/T/Q/N amino acid content in IDRs and it is tunable (e.g., via phosphorylation) to regulate protein homeostasis.
  • IDRs are key components of subcellular machineries and signaling pathways because they have the potential to associate with many partners due to their multiple possible metastable conformations.
  • IDRs are regulated by alternative splicing and post-translational modifications. Some IDRs are involved in the formation of various membraneless organelles via intracellular liquid-liquid phase separation.
  • the "IDRs" have been evolutionarily retained as toolkits to create complexity in regulatory networks and they provide multiple mechanisms for cell-type or tissue-specific signaling, e.g., solubility, physical interactions, post-translational modifications or liquid-liquid phase transition.
  • IDRs provide many advantages to proteins, including: (1) mediating protein-protein or protein- peptide interactions by adopting different conformations; (2) facilitating protein regulation via diverse posttranslational modifications; and (3) regulating the half-lives of proteins that have been targeted for proteasomal degradation.
  • the present disclosure provides several IDRs with high STQN amino acid contents exhibit autonomous expression-enhancing activity for high-level production of native protein and when fused to exogenous target proteins, e.g., ⁇ -galactosidase (LacZ) in vivo.
  • target proteins e.g., ⁇ -galactosidase (LacZ) in vivo.
  • most (if not all) IDRs described herein possess one or more ATR Mec1 /ATM Tel1 DNA damage checkpoint kinase phosphorylation sites, suggesting their functions and/or stability may be tunable by ATR Mec1 or ATM Tel1 via protein phosphorylation.
  • cerevisiae was previously defined as a region with at least three S/T-Q within a stretch of 50 residues (Cheung H. C. et al. BMC Genomics 2012 13:664).
  • the conserved DNA damage response (DDR) checkpoint kinases Tell and Mec1 in S. cerevisiae and their human homologs ATM and ATM phosphorylate are well- known DDR proteins at S/T-Q consensus sites (Traven A. and Heierhorst J. (2006) Bioessays 27, 397-407).
  • SCDs might possess much broader and overarching roles in eukaryotic cells because there is an unexpected abundance of S.
  • SCDs and/or their phosphorylation are involved in the essential functions in regulating protein homeostasis.
  • the high S, T and Q content in an IDR or SCD and the overall number of S/T-Q motifs are important for the high-level protein production and/or inhibition of protein degradation function.
  • the content of S, T and Q in the total amino acid content of the IDR or SCD is higher than 10%.
  • the S, T and Q content in the total amino acid content of the IDR or SCD domain is higher than 15%, 15%, 20%, 25% or 30%.
  • the overall percentage of the content of S, T and Q in the total amino acid content of the IDR or SCD ranges from about 30% to about 40% (e.g.31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%).
  • the SCD is an IDR with high S/T and Q content.
  • the IDR or SCD is Hop1-SCD (258-324 a.a.), Rad51-NTD (1-66 a.a.), Rad51-NTD-3SA, Rad51-NTD-6SQA, Rad51-NTD-9SQA, Rad51-NTD-12SQA, Rad53- SCD1 (1-29 a.a.), Rad53-SCD1-5STA, Rad53-SCD1-7QA, Rad53-SCD1-12STQA, Sml1-NTD (1-27 a.a.), Sml1-NTD (1-50 a.a.), Sup35-PND (1-39 a.a.), Sup35-PND-S17A, Sup35-PND-3SA, Sup35-PND-3QA, Sup35-PND-5QA, Sup35-PND-8QA, Sup35-PND-15SQA, Sup35-PND-9NA, Sup35-PND-24SQNA, Sup35-PFD (1-114 a.
  • the NH 2 -terminal domain (NTD; residues 1-66) of S. cerevisiae Rad51 is a bona fide SCD with 3 SQ motifs (S 2 Q, S 12 Q and S 30 Q). All three S/Q motifs can be phosphorylated by Mec1 ATR and Tel1 ATM during both vegetative growth and meiosis.
  • Rad51-NTD has two distinct functions in regulating the steady-state levels of Rad51. Firstly, Rad51-NTD possesses autonomous expression-enhancing activity.
  • Mec1 ATR /Tel1 ATM ⁇ dependent phosphorylation of Rad51-NTD antagonizes the proteasomal degradation pathway, thereby increasing the stability of the expressed protein Rad51 in vivo.
  • All three S/Q motifs in Rad51 are direct target sites of Mec1 ATR and Tel1 ATM not only in vegetative cells exposed to the DNA damage agent methyl methanesulfonate (MMS) but also during normal meiosis.
  • Sup35 transformation termination factor eRF3
  • eRF3 is a well- characterized yeast prion protein that aggregates to form the [PSI + ] prion.
  • Sup35-PND is a S-rich and Q/N-rich domain with 3 serines, 9 asparagines and 12 glutamines with only one S 17 Q motif.
  • Rad53-SCD1 contains four TQ motifs and one SQ motif.
  • the prion (nucleation) domains of three yeast prion-causing proteins (Sup35, Ure2 and New1) share two common structural features with DDR-SCDs. They not only have high S/T/Q/N amino acid contents, but also contain at least one S/T-Q site that might be phosphorylated by Mec1 ATR or Tel1 ATM .
  • the Ure2 prion domain (UPD) (residues 1-91) of the Ure2 nitrogen catabolite repression transcriptional regulator is the basis of the prion [URE3 + ].
  • UPD is critical for Ure2's in vivo function because removal of UPD from Ure2 results in reduced protein stability and steady-state protein levels (but not transcript levels) of the corresponding Ure2- ⁇ UPD mutants.
  • Ure2-UPD has a high STQN content (>63%; 10 serines, 5 threonines, 10 glutamines, 33 asparagines in 91 amino acids) and adopts a completely disordered structure.
  • Ure2-UPD also possesses an S 68 Q motif.
  • the New1 prion domain supports [NU + ] and is susceptible to [PSI+] prion induction.
  • New1 is a non-essential ATP-binding cassette type F protein that fine-tunes the efficiency of translation termination or ribosome recycling.
  • Newl- NPD also has a high STQN content (>44%; 19 serines, 8 threonines, 14 glutamines, 26 asparagines in 156 amino acids) and an S 145 Q motif.
  • Vps64 (also called Far9) is required for cytoplasm-to-vacuole targeting of proteins. Intriguingly, the vps64 ⁇ mutant shows increased aneuploidy tolerance.
  • Kel1 Kelch repeat 1 is required for proper cell fusion and cell morphology, and functions with Kel2 (the ohnolog of Kell) to negatively regulate mitotic exit.
  • Ssk2 suppression of sensor kinase 2 is a mitogen-activated protein kinase (MAPK) kinase of the Hogl osmoregulatory signaling pathway. Activation of Ssk2 mediates actin cytoskeleton recovery from osmotic stress respectively.
  • MAPK mitogen-activated protein kinase
  • IDR or SCD itself has more profound impacts than IDR or SCD phosphorylation in promoting high steady- state-levels of the interested polypeptide.
  • IDR or SCD phosphorylation stabilizes the interested polypeptide by preventing proteasomal degradation during the DNA damage response, preferably, negatively-charged IDR or SCD is sufficient to stabilize the interested polypeptide.
  • one or more S, T or Q residues in the IDR are replaced with an amino acid with a hydrophobic side chain.
  • the amino acid with a hydrophobic side chain include, but are not limited to, alanine, valine, isoleucine, leucine, or methionine.
  • the amino acid with a hydrophobic side chain is alanine.
  • Examples of the IDR with one or more S, T or Q residues replaced with an amino acid with a hydrophobic side chain include, but are not limited to,, Rad51-NTD-3SA, Rad51-NTD-6SQA, Rad51-NTD-9SQA, Rad51 -NTD- 12 SQ A, Rad53-SCD1-5STA, Rad53-SCD1-7QA, or Sup35-PND-1SA.
  • one or more S, T or Q residues in the SCD are replaced with an amino acid with a negative-charged side chain.
  • the amino acid with a negative-charged side chain include, but are not limited to, two aspartic acid or glutamic acid.
  • the amino acid with a negative-charged side chain is aspartic acid.
  • one or more S, T or Q residue in the IDR are replaced with another amino acid of S, T and Q.
  • the expression vector is a nucleic acid, a plasmid, a cosmid, a virus, or an artificial chromosome.
  • Expression vectors comprise a polynucleotide sequence encoding a polypeptide of interest (polypeptide coding sequence) operably linked, or under the control of, a promoter capable of effecting expression in a mammalian cell line of interest.
  • Expression vectors can comprise one or more polypeptide coding sequence(s). Accordingly, expression vectors comprise at least a first, and optionally a second, third, fourth, etc., polypeptide coding sequence.
  • expression cassettes comprise first and second polypeptide coding sequences, which can be under the control of a single promoter or separate promoters.
  • the choice of the vector will depend on several factors, including the compatibility of the vector with the mammalian cell into which the vector is to be introduced (e.g., a mammalian cell, or a host cell such as a bacterial cell, useful for propagating or amplifying the vector), and the ability of the vector to integrate into the mammalian cell genome.
  • the vector can be a viral vector, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, an artificial chromosome, a cloning vector, a shuttle vector, a plasmid (linear or closed circular), or the like.
  • Vectors can include chromosomal, non-chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art and are commercially available. Examples of suitable vectors are provided in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (1989). [0059] Expression vectors may include additional features or elements useful for expressing the polypeptide(s) of interest in mammalian host cells which are well-known in the art. Such features include enhancer elements, transcription initiation sequences and ribosome binding sites, polyadenylation signal sequences, termination sequences and other features important for nuclear export, translation, and/or stability of the mRNA.
  • the host cell is preferably a eukaryotic host cell.
  • Said eukaryotic cell is, preferably, selected from the group consisting of a mammalian cell, a yeast cell, a fungal cell, an insect cell, a plant cell and a protozoan cell, and the progeny of the parent cell, regardless of whether the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present.
  • any vector can be used in the methods of the invention and the selection of an appropriate vector is, in one aspect, based on the host cell selected for expression of the protein.
  • a host cell is, in various aspects, a prokaryotic or eukaryotic cell.
  • the host cell is a bacteria cell, a protist cell, a fungal cell, a plant cell, or an animal cell.
  • the product of interest for example a polypeptide produced in accordance with the invention, may be recovered, further purified, isolated and/or modified by methods known in the art.
  • the product may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, ultra-filtration, extraction or precipitation.
  • Purification may be performed by a variety of procedures known in the art including, but not limited to, chromatography (e.g. ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g. ammonium sulfate precipitation) or extraction.
  • chromatography e.g. ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g. ammonium sulfate precipitation
  • the product of interest can be any biological product capable of being produced by transcription, translation or any other event of expression of the genetic information encoded by said polynucleotide.
  • the product will be an expression product.
  • the product can be a pharmaceutically or therapeutically active compound, or a research tool to be utilized in assays.
  • the product is a polypeptide, preferably a pharmaceutically or therapeutically active polypeptide, or a research tool to be utilized in diagnostic or other assays.
  • a polypeptide is accordingly not limited to any particular protein or group of proteins, but may on the contrary be any protein, of any size, function or origin, which one desires to select and/or be expressed by the methods described herein. Accordingly, several different polypeptides of interest may be expressed/produced.
  • Yeast strains and two-hybrid assay All meiotic experiments were performed using diploid isogenic SKI strains. Quantitative yeast two-hybrid assays, tetrad dissection, immunostaining of chromosome spreads, cycloheximide-shutoff experiments, and physical analyses were carried out as previously described ⁇ Chen. Y.-J, Chuang, Y.-C., Chuang, C.-N, Cheng, Y.-H, Chang, C.-R., Leng, C.-H., and Wang, T.-F., S.
  • the antisera were pre-cleaned by peptide-specific affinity chromatography using the corresponding non-phosphorylated peptides coupled to agarose beads. Phosphopeptide synthesis and animal immunization were conducted by LTK BioLaboratories, Taiwan. The rabbit antisera against Hop1, rabbit antisera against phosphorylated Hop1-T 318 Q and the goat antisera against Zip1 were described previously (Chuang C.N., Cheng Y.H., Wang T.F. Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis. Nucleic Acids Res.2012; 40:11416–11427).
  • the goat anti-Rad51 antibody (yN-19), goat anti-Clb1 antibody (yS-19) and rabbit anti-Sic1 antibody (FL- 284) for western blot were purchased from Santa Cruz Biotechnology (CA, USA).
  • the rabbit anti-Dmc1 antibody was a gift from Douglas Bishop (University of Chicago, IL, USA).
  • the rat anti-HA antibody was purchased from Roche (Basel, Switzerland).
  • the mouse anti-V5 antibody was purchased from Bio-Rad (CA, USA).
  • the rabbit anti-Hsp104 and anti-hexokinase antisera were kindly provided by Chung Wang (Academia Sinica, Taiwan).
  • the mouse anti-GFP antibody and anti-GST antibody were purchased from Bio-Rad (CA, USA, MCA1360), Clontech (SF, USA, 632381) and GenScript (NJ, USA, A00865), respectively.
  • the rabbit anti-Smll antibody was purchased from Agrisera (Vannas, SE, AS 10847).
  • the rabbit anti-Hsp104 antiserum was kindly provided by Chung Wang (Academia Sinica, Taiwan). Rabbit antisera against phosphorylated Sup35-S 17 Q were raised using the synthetic phosphopeptide N 12 YQQYS [P] QNGNQQQGNNR 28 as an antigen, where S [P] is phosphorylated serine.
  • Quantitative ⁇ -galactosidase activity assays were carried out as previously described (Woo TT, Chuang CN, Higashide M, Shinohara A, Wang TF. Dual roles of yeast Rad51 N -terminal domain in repairing DNA double-strand breaks. Nucleic Acids Res. 2020;48:8474-89; Lin FM, Lai YJ, Shen HJ, Cheng YH, Wang TF. Yeast axial-element protein, Red1, binds SUMO chains to promote meiotic interhomologue recombination and chromosome synapsis. EMBO J. 2010;29:586-96; Niethammer M, Sheng M.
  • NTD N-terminal domain
  • Rad51-3D and Rad51-3A are the phosphomimetic mutant and the phosphorylation-defective mutant, and all three of these serine residues (S 2 , S 12 and S 30 ) are mutated into aspartic acids (D) and alanines (A).
  • S 2 , S 12 and S 30 serine residues
  • A aspartic acids
  • A alanines
  • the steady-state protein levels of Rad51 ⁇ N (the NTD-truncated form of Rad51; residues 67-400) are ⁇ 5% as compared to those of wild-type Rad51 proteins in vivo ( Figures 1 A and 1 B).
  • Rad51-NTD comprises 66 amino acid residues. To characterize its biochemical properties, a S.
  • P RAD51 -lacZ-NLS-V5 expression vector (Invitrogen, USA) was modified into P RAD51 -lacZ-NLS-V5 by replacing the GAL1 promoter with the promoter of the wild-type RAD51 gene (P RAD51 ) and inserting an SV40 nuclear localization signal (NLS) peptide preceding V5.
  • This new vector retains a C-terminal V5 epitope for detection of LacZ-V5 or LacZ-V5 fusion proteins by immunoblotting.
  • NTD-LacZ-V5 fusion proteins can be recognized in immunoblotting using the anti-Rad51 antiserum and the order of steady-state protein levels is NTD-LacZ-V5 ⁇ NTD 3D -LacZ-V5 > NTD 3A -LacZ-V5 ( Figure 2 A), all of which showed apparently higher protein levels than that of LacZ-V5 ( Figure 2 B).
  • NTD 3A -LacZ-V5 may be more labile than NTD- LacZ-V5 and NTD 3D -LacZ-V5, given that there were more degraded NTD 3A -LacZ-V5 products in our blot ( Figure 2 B, marked by the black arrowhead).
  • Figure 2 B marked by the black arrowhead.
  • Rad51-NTD is phosphorylated in a Mec1 ATR - and Tel1 ATM - dependent manner during vegetative growth and meiosis
  • Rad51-NTD contains three SQ motifs (S 2 Q, S 12 Q and S 30 Q) ( Figure 5 A). To reveal the functions of Rad51-NTD itself and NTD phosphorylation, we first generated antisera specific to phosphorylated Rad51-S 2 Q, Rad51-S 12 Q and Rad51-S 30 Q proteins ( Figure 5 B).
  • Mec1 ATR /Tel1 ATM -dependent Rad51-NTD phosphorylation is indispensable for meiotic recombination in a dmc1 ⁇ hed1 ⁇ mutant ( Figure 5 E) and for DNA repair in vegetative cells exposed to higher concentrations of a DNA damage agent, i.e., 0.02% not 0.01% MMS ( Figure 5 F).
  • the phosphomimetic mutant (rad51-3D) can be functionally substituted for phosphorylated Rad51 in dmc1 ⁇ hed1 ⁇ ( Figure 5 E).
  • the dmc1 ⁇ hed1 ⁇ rad51-3A triple mutant behaved like dmc1 ⁇ , exhibiting phenotypes related to strong cell cycle arrest at prophase I. It was also illustrated that the meiotic defects of the dmc1 ⁇ hed1 ⁇ rad51-3A triple mutant could be rescued (at least in part) by introducing high-copy number vectors expressing the full-length Rad51 or the Rad51-3A mutant ( Figure 6).
  • Example 3 Rad51-NTD is critical for promoting high steady-state Rad51 protein levels during vegetative growth and meiosis
  • Rad51-NTD itself and Rad51-NTD phosphorylation apparently have different roles in regulating the overall steady-state levels of Rad51 protein in vivo.
  • the rad51- ⁇ N (rad51 67-440 ) allele encodes an NTD-truncation mutant Rad51- ⁇ N (residues 67-400) under the control of the native RAD51 promoter.
  • the rad51- ⁇ N diploid cells displayed ⁇ 50% reduction in spore viability ( Figure 5 E).
  • Rad51- ⁇ N Since the steady-state levels of Rad51- ⁇ N in rad51- ⁇ N cells are not significantly different from those in rad51- ⁇ N sml1 ⁇ cells during vegetative growth and meiosis, we reason that Rad51 might be an abundant target of Mec1 ATR and Tel1 ATM in S. cerevisiae.
  • Rad51 foci detected using antisera targeting native Rad51 and phosphorylated Rad51 did not completely co-localize, implying partial phosphorylation of Rad51 or possible competition for antigen recognition in this assay. Nevertheless, formation of Rad51 foci in the phosphorylation-defective rad51-3A mutant was indistinguishable from that of the WT control ( Figures 8 B to D), despite their delayed appearance on chromosomes ( Figure 8 E), likely due to low Rad51 protein expression levels (see below).
  • rad51- 3D behaved like WT RAD51 in the dmc1 ⁇ hed1 ⁇ mutant in that delayed meiotic progression and meiosis I non-dysjunction phenotypes (a preponderance of tetrads containing two or zero viable spores) were seen ( Figure 8 A). Strikingly, both dmc1 ⁇ hed1 ⁇ rad51-3A and dmc1 ⁇ hed1 ⁇ rad51- ⁇ N triple mutants, like the dmc1 ⁇ mutant, hardly sporulated and exhibited a strong meiotic progression arrest phenotype at prophase I ( Figure 8 A).
  • both Rad51-NTD and Rad51-NTD phosphorylation are essential for Rad51-mediated meiotic recombination in the dmc1 ⁇ hed1 ⁇ mutant, and the phosphomimetic mutant Rad51-3D can functionally substitute for phosphorylated Rad51 in the same genetic background.
  • This scenario is further supported by the very low spore viability following meiosis of the dmc1 ⁇ hed1 ⁇ mec1-kd sml1 ⁇ strain, in which Rad51-NTD phosphorylation is solely dependent on Tel1 ATM ( Figure 5 B, right panel).
  • Immunoblotting experiments further revealed that the steady-state protein levels of Rad51-3A were lower than those of Rad51 and Rad51-3D in both DMC1 HED1 and dmc1 ⁇ hed1 ⁇ meiotic cells ( Figures 9 A and B).
  • DMC1 HED1 meiotic cells we found the maximum steady-state levels of Rad51-3A (after 7 h in SPM) to be ⁇ 30% those of WT Rad51 ( Figure 9 B).
  • Rad51-NTD can be replaced by Rad53-SCD1 and the prion nucleation domain (PND; residues 1-39) of yeast Sup35 protein.
  • Rad53-SCD1 contains four TQ motifs and one SQ motif.
  • Sup35 transcription termination factor eRF3
  • eRF3 translation termination factor 3
  • Sup35- PND is an IDR but not an SCD.
  • Pr RAD51 -LacZ-V5-His6 (Invitrogen, USA) was modified into Pr RAD51 -LacZ-V5-His6 by replacing the GAL1 promoter with the promoter of the native RAD51 gene (Pr RAD51 ) and inserting a LacZ gene preceding a V5 epitope tag and a hexahistidine (His6) affinity tag.
  • Pr RAD51 -NTD-LacZ-V5-His6 Pr RAD51 - NTD-3A-LacZ-NLS-V5-His6
  • Pr RAD51 -NTD-3D-LacZ-V5-His6 All four of these vectors were transformed into an SK1 yeast cell line. The transformants were vegetatively propagated to reach logarithmic phase and then harvested for denatured lysate preparation.
  • NTD, NTD-3A, and NTD-3D all possess the capability to enhance the expression of their fusion partner, LacZ-V5-His6.
  • NTD-3A- LacZ-V5-His6 was more labile than NTD-LacZ-V5-His6 and NTD-3D-LacZ-V5-His6, given that there were more degraded NTD-3A-LacZ-V5-His6 products in the immunoblot.
  • the ⁇ - galactosidase activities of these four fusion proteins were also determined.
  • Rad53-SCD1 (residues 1-29), Hop1-SCD (residues 258-324) or Sml1-SCD (either residues 1-27 or residues 1-50) can all act as an NH 2 - terminal fusion tag to enhance LacZ-V5-His6 expression (upper panels), resulting in 6-10-fold increases of ⁇ -galactosidase activity (lower panels), respectively.
  • Rad53-SCD1 enhanced higher production of not only LacZ, but also of three other target proteins, i.e., green fluorescent protein (GFP), glutathione S-transferase (GST) and a non- dimerizing GST [GST-(nd)] ( Figure 10H).
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • GST-(nd) a non- dimerizing GST
  • Figure 10H Figure 10H.
  • ⁇ -galactosidase is a tetrameric protein.
  • GFP is a monomeric protein.
  • GST is a dimeric protein.
  • the two important residues (Arg73 and Asp77) contributing to GST dimer stability are mutated into proline and lysine.
  • SCD was defined to be at least three S/T-Q sites in a stretch of ⁇ 50 amino acids in S. cerevisiae (Cheung, H.C., San Lucas, F.A., Hicks, S., Chang, K., Bertuch, A.A. and Ribes- Zamora, A. (2012) An S/T-Q cluster domain census unveils new putative targets under Tel1/Mec1 control. BMC Genomics, 13, 664)).
  • Enrichment of S/T-Q motifs in SCDs means they exhibit low sequence complexity. Low sequence complexity and high S/T and Q content are characteristic features of intrinsically disordered regions (IDRs) in proteins.
  • IDRs intrinsically disordered regions
  • Sup35-PND is an S-rich and Q/N-rich domain with 3 serines, 9 asparagines and 12 glutamines.
  • the hierarchy of normalized LacZ activities for the Sup35-NTD-LacZ-V5-His6 fusion variants was Sup35-NTD (8.6-fold) ⁇ Sup35-NTD-S 17 A (8.1-fold) >> mock (1.0-fold).
  • the overall content of STQ of SCDs or IDRs should range from about 30% to about 40%.
  • Example 8 Four DDR-SCDs and three prion domains can all promote target protein expression [00104] As reported recently (Woo TT, Chuang CN, Higashide M, Shinohara A, Wang TF. Dual roles of yeast Rad51 N-terminal domain in repairing DNA double-strand breaks. Nucleic Acids Res.
  • the NVH tag contains an SV40 nuclear localization signal (NLS) peptide preceding a V5 epitope tag and a hexahistidine (His 6 ) affinity tag (Woo TT, Chuang CN, Higashide M, Shinohara A, Wang TF. Dual roles of yeast Rad51 N-terminal domain in repairing DNA double-strand breaks. Nucleic Acids Res. 2020;48:8474-89). The S 17 Q motif was changed to AQ in Sup35-PND-S 17 A.
  • Sml1 is a potent inhibitor of ribonucleotide reductase and, in the SK1 S.
  • Sup35 mainly located in cytosol, whereas Mec1 ATR and Tel1 ATM are nuclear proteins.
  • the NVH-tag contains an SV40 NLS peptide.
  • Sup35-PND-LacZ-NVH might be targeted to nuclei and became accessible to Mec1 ATR and Tel1 ATM for phosphorylation. We then confirm that MMS could not induce phosphorylation of Sup35-PND-S 17 Q in S.
  • Sup35-GFP GFP-tagged Sup35
  • Franzmann TM Jahnel M, Pozniakovsky A, Mahamid J, Holehouse AS, Nuske E, et al. Phase separation of a yeast prion protein promotes cellular fitness. Science. 2018;359
  • Sup35-S 17 A-GFP mutant respectively (Chuang CN, Woo TT, Tsai, SY, Li, WC, Chen CL, Liu HC, Chen CY, Hsueh YP, Wang TF.
  • Intrinsic disorder codes for leaps of protein expression.
  • Example 9 High STQN content is critical for IDR's nanny functions
  • a common feature of DDR-SCDs, Sup35-PND, Ure2-UPD and New1-NPD is their high STQN amino acid contents ( Figure 13 A). We investigated if the high STQN content of IDRs is critical for promoting high-level protein expression. We employed an alanine-scanning mutagenesis approach to test this hypothesis.
  • Alanine scanning by definition increases the relative content of a given sequence in alanine residues that are known to favor the formation of alpha helices (Marqusee S, Robbins VH, Baldwin RL. Unusually stable helix formation in short alanine-based peptides. Proc Natl Acad Sci U S A. 1989;86:5286-90 ) and hence to favor a transition from disorder to order. [00109]
  • Our results reveal a positive correlation between relative ⁇ -galactosidase activities or

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Abstract

La présente invention concerne un procédé permettant d'augmenter l'expression et/ou d'inhiber la dégradation d'un polypeptide intéressé dans une cellule hôte avec les étapes consistant à construire un vecteur d'expression comprenant un polynucléotide codant pour une région intrinsèquement désordonnée (IDR) avec une teneur élevée en S/T et/ou en Q et un polypeptide codant pour le polypeptide intéressé, à transformer le vecteur d'expression en une cellule hôte et à cultiver la cellule hôte dans des conditions qui permettent l'expression du polypeptide intéressé.
PCT/US2021/036247 2020-06-09 2021-06-07 Procédés et vecteurs permettant d'améliorer l'expression et/ou d'inhiber la dégradation d'une protéine WO2021252391A1 (fr)

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US20050272130A1 (en) * 2004-03-17 2005-12-08 Chin-Yen King Methods and compositions relating to prion-only transmission of yeast strains
US20180228908A1 (en) * 2015-08-04 2018-08-16 Duke University Genetically encoded intrinsically disordered stealth polymers for delivery and methods of using same
US20190317111A1 (en) * 2016-04-29 2019-10-17 Forschungszentrum Juelich Gmbh Method for identifying inhibitors of primary nucleation of amyloid-beta aggregation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050272130A1 (en) * 2004-03-17 2005-12-08 Chin-Yen King Methods and compositions relating to prion-only transmission of yeast strains
US20180228908A1 (en) * 2015-08-04 2018-08-16 Duke University Genetically encoded intrinsically disordered stealth polymers for delivery and methods of using same
US20190317111A1 (en) * 2016-04-29 2019-10-17 Forschungszentrum Juelich Gmbh Method for identifying inhibitors of primary nucleation of amyloid-beta aggregation

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Title
CHUANG ET AL.: "Intrinsic disorder codes for leaps of protein expression", BIORXIV, 8 December 2020 (2020-12-08), XP055885325, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.12.08.407247v1.full> *
TRAVEN ET AL.: "SQ/TQ cluster domains: concentrated ATM/ATR kinase phosphorylation site regions in DNA-damage-response proteins", BIOESSAYS, vol. 27, no. 4, 30 April 2005 (2005-04-30), pages 397 - 407, XP055885326 *

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