TW202214850A - Methods and vectors for enhancing expression and/or inhibiting degradation of protein - Google Patents

Abstract

The present disclosure provides a method for increasing expression and/or inhibiting degradation of an interested polypeptide in a host cell with the steps of constructing an expression vector comprising a polynucleotide coding an intrinsically disordered region (IDR) with high S/T and/or Q content and a polypeptide coding the interested polypeptide, transforming the expression vector to a host cell and culturing the host cell under conditions that allow for expression of the interested polypeptide.

Description

Methods and vectors for enhancing protein expression and/or inhibiting its degradation

The present invention relates to the field of recombinant protein expression. The present invention relates to expression vectors comprising intrinsically disordered regions (IDRs) with high S/T and/or Q content or S/T-Q cluster domains (SCDs) and methods of using the expression vectors to enhance protein expression .

Recombinant fusion protein methods have been widely used in biological research and therapeutics. This method typically involves joining two or more cDNA sequences in frame via ligation or overlapping polymer chain reaction (PCR). Next, the DNA sequence thus obtained encodes a single polypeptide with a fusion carrier or a protein tag including an expression carrier, a soluble carrier, a fluorescent tag, a targeting tag, a reporter Body tags, chromatography (or affinity) tags and epitope tags, etc. US 20160009779 provides a fusion tag with 20 to 50 amino acids and an expression vector system comprising the 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 generating a peptide of interest as a fusion protein.

There is a need to improve translation, folding or stability of biologically or pharmacologically active proteins.

In one aspect, the present invention provides a method for enhancing the expression and/or inhibiting degradation of a polypeptide of interest in a host cell, the method comprising the steps of: constructing an expression vector comprising an encoding intrinsically disordered region ( IDR) polynucleotides and polynucleotides encoding the polypeptide of interest; transform the expression vector into the host cell; and culture the host cell under conditions that allow expression of the polypeptide of interest; wherein S, The content of T and Q as a percentage of the total amino acid content of the IDR is higher than 10%.

In one embodiment, the IDR contains one or more S/T-Q cluster domains (SCDs).

In some embodiments of the invention, one or more of the S, T or Q residues in the IDR are replaced with an amino acid having a hydrophobic side chain. In some embodiments of the invention, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 residues in the IDR are replaced with amino acids having hydrophobic side chains.

In some embodiments of the invention, one or more of the S, T or Q residues in the SCD are replaced with an amino acid having a negatively charged side chain. In some embodiments of the invention, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 residues in the IDR are replaced with amino acids having negatively charged side chains.

In some embodiments of the invention, one or more of the S, T or Q residues in the IDR are replaced with another amino acid of S, T and Q. In some embodiments of the invention, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 residues in the IDR are via another amine group in S, T and Q Acid replacement.

In another aspect, the present invention provides a vector comprising an intrinsically disordered region operably linked to a polypeptide encoding a polypeptide of interest, wherein the content of S, T and Q accounts for 30% of the total amino acid content of the IDR The overall percentage is above 10%.

In another aspect, the present invention provides a host cell comprising a vector of the present invention.

In one embodiment, the SCD or IDR is fused to the N-terminus of the polypeptide of interest.

In one embodiment, IDR or SCD is used as a fusion tag to enhance expression and/or inhibit degradation of the polypeptide of interest.

In one embodiment, the polypeptide of interest is a heterologous protein.

In one embodiment, the SCD or IDR is phosphorylated.

In one embodiment, the content of S, T and Q as a percentage of the total amino acid content of the IDR or SCD is greater than 15%. In some embodiments, the content of S, T and Q is higher than 15%, 20%, 25% or 30% of the total amino acid content of the IDR or SCD.

In one embodiment, the content of S, T, and Q ranges from about 30% to about 40% of the overall percentage of the total amino acid content of the IDR or SCD.

In one embodiment, the SCD is an IDR with high S/T and Q content (Traven A. and Heierhorst J. (2006) Bioessays 27, 397-407).

In some embodiments, the IDR or SCD is as listed in Table 1. Table 1. Amino acid sequences of SCD or IDR used in the present invention Name of SCD or IDR sequence SEQ ID NO. Hop1-SCD (258-324 aa) MEKAGNTNFIRVDPFDLILQQQEENKLEESAPTKPQNFVTSQTTNVLGNLLNSSQASIQPTQFVSNN 1 Rad51-NTD (1-66 aa) MSQVQEQHISESQLQYGNGSLMSTVPADLSQSVVDGNGNGGSEDIEATNGSGDGGGLQEQAEAQGE 2 Rad51-NTD-3SA (Rad51-NTD-3A) M A QVQEQHISE A QLQYGNGSLMSTVPADL A QSVVDGNGNGGSEDIEATNGSGDGGGLQEQAEAQGE 3 Rad51-NTD-3SD (Rad51-NTD-3D) M D QVQEQHISE D QLQYGNGSLMSTVPADL D QSVVDGNGNGGSEDIEATNGSGDGGGLQEQAEAQGE 4 Rad51-NTD-S2A M A QVQEQHISESQLQYGNGSLMSTVPADLSQSVVDGNGNGGSEDIEATNGSGDGGGLQEQAEAQGE 5 Rad51-NTD-S12A MSQVQEQHISE A QLQYGNGSLMSTVPADLSQSVVDGNGNGGSEDIEATNGSGDGGGLQEQAEAQGE 6 Rad51-NTD-S30A MSQVQEQHISESQLQYGNGSLMSTVPADL A QSVVDGNGNGGSEDIEATNGSGDGGGLQEQAEAQGE 7 Rad51-NTD-6SQA M AA VQEQHISE AA LQYGNGSLMSTVPADL AA SVVDGNGNGGSEDIEATNGSGDGGGLQEQAEAQGE 8 Rad51-NTD-9SQA M AA V A E A HISE AA L A YGNGSLMSTVPADL AA SVVDGNGNGGSEDIEATNGSGDGGGLQEQAEAQGE 9 Rad51-NTD-12SQA M AA V A E A HISE AA L A YGNGSLMSTVPADL AA SVVDGNGNGGSEDIEATNGSGDGGGL A E A AEA A GE 10 Rad53-SCD1 (1-29 aa) MENITQPTQQSTQATQRFLIEKFSQEQIG 11 Rad53-SCD1-5STA MENI A QP A QQS A QA A QRFLIEKF A QEQIG 12 Rad53-SCD1-7QA MENIT A PT AA ST A AT A RFLIEKFS A E A IG 13 Rad53-SCD1-12STQA MENI AA P AAA S AA A AA RFLIEKF AA E A IG 14 Sml1-NTD (1-27 aa) MQNSQDYFYAQNRSQQQQAPSTLRTVT 15 Sml1-NTD (1-50 aa) MQNSQDYFYAQNRSQQQQAPSTLRTVTMAEFRRVPLPPMAEVPMLSTQNS 16 Sup35-PND (1-39 aa) MSDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQA 17 Sup35-PND-S ^17A (Sup35-NTD-S ^17A ) MSDSNQGNNQQNYQQY A QNGNQQQGNNRYQGYQAYNAQA 18 Sup35-PND-3SA M A D A NQGNNQQNYQQY A QNGNQQQGNNRYQGYQAYNAQA 19 Sup35-PND-3QA MSDSNQGNNQQNYQQYSQNGN AAA GNNRYQGYQAYNAQA 20 Sup35-PND-5QA MSDSNQGNN AA NY AA YS A NGNQQQGNNRYQGYQAYNAQA twenty one Sup35-PND-8QA MSDSNQGNN AA NY AA YS A NGN AAA GNNRYQGYQAYNAQA twenty two Sup35-PND-15SQA M A D A N A GNN AA NY AA Y AA NGN AAA GNNRY A GY A AYNA A A twenty three Sup35-PND-9NA MSDS A QG AA QQ A YQQYSQ A G A QQQG AA RYQGYQAY A AQA twenty four Sup35-PND-24SQNA M A D AAA G AAAAA Y AA Y AAA G AAAA G AA RY A GY A AY A A A A 25 Sup35-PFD (1-114 aa) MSDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGYQQYNPDAGYQQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNSLQGYQ 26 New1-NPD (1-156 aa) MPPKKFKDLNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQRNWKQGGNYQQGGYQSYNSNYNNYNNYNNYNNYNNYNNYNNYNKYNGQGYQKSTYKQSAVTPNQSGTPTPSASTTSLTSLNEKLSNLELTPISQFLSKIPECQS 27 Ure2-UPD (1-91 aa) MMNNNGNQVSNLSNALRQVNIGNRNSNTTTDQSNINFEFSTGVNNNNNNNSSSNNNNVQNNNSGRNGSQNNDNENNIKNTLEQHRQQQQAF 28 Vps64-NTD (1-152 aa) MVELEKRRRPPPQLQHSPYVRDQSNSQGMTKTPETSPPKRPMGRARSNSRSSGSRSNVDIDQYTIPPGLDLLPTASSPPSVHQVSQQQQLSPILANKIRSPFENQSQDQNDNSIDPTPAGQVTIPVEAVSPPALDELSKFQNGSTETLFRTG 29 Ssk2-NTD (1-191 aa) MSHSDYFNYKPYGDSTEKPSSSKMRQSSSSSSSRLRSESLGRNSNTTQARVASSPISPGLHSTQYFRSPNAVYSPGESPLNTVQLFNRLPGIPQGQFFHQNAISGSSSSSARSSRRPSNIGLPLPKNPQQSLPKLSTQPVPVHKKVEASKTESEIIKKPAPVNSNQDPLLTTPTLVISPELASLNTTNTSI 30 Kel1-NTD (1-69 aa) MAGFSFAKKFTHKKHGKTPSDASISDQSREASLSTPPNEKFFTKQETPQKGRQFSQGYHSNVNKTSSPP 31

In some embodiments, the host cell line is a prokaryotic cell. In some embodiments, the host cell is a mammalian cell, yeast cell, fungal cell, insect cell, plant cell, or protozoan cell. In another embodiment, the host cell line is Saccharomyces cerevisiae .

The present inventors have unexpectedly found that when an SCD has an _NH2 -terminal fusion tag naturally present or an artificially designed _NH2 -terminal fusion tag, it has autonomous expression enhancing activity. The present invention discloses two novel roles of SCD in the regulation of protein homeostasis in vivo, indicating that SCD can be used as a fusion tag for high-level protein production and/or protein degradation inhibition.

As used herein, the term "expression vector" refers to any molecule used to transfer coding information to a host cell.

As used herein, the term "host cell" refers to a cell transformed, transfected, or transduced with an expression vector carrying a gene of interest, and then the expression vector is expressed by the cell.

As used herein, the term "polynucleotide" is a polymer of nucleotides usually linked from one deoxyribose or ribose sugar to another deoxyribose or ribose sugar, and refers to DNA as well as RNA, as appropriate. The term "polynucleotide" does not contain any size limitation and also encompasses polynucleotides comprising modifications, specifically, modified nucleotides.

As used herein, a "polypeptide of interest" refers to a polypeptide to be expressed in a host cell.

As used herein, "polypeptide" refers to a molecule comprising a polymer of amino acids linked together by one or more peptide bonds. Polypeptides include polypeptides of any length, including proteins (eg, having more than 50 amino acids) and peptides (eg, having 2-49 amino acids). Polypeptides include proteins and/or peptides having any activity or biological activity.

Intrinsically disordered regions (IDRs) are protein sequences that lack a fixed or ordered three-dimensional structure. Many IDRs have important molecular functions such as physical interactions, post-translational modifications or solubility enhancement. Thus, the present invention unexpectedly discloses that several biologically important IDRs can be used as N-terminal fusion vehicles to facilitate target protein folding or protein quality control, thereby enhancing protein expression, and are compatible with the high S/T/T/T in IDRs. Q/N amino acid content is strongly correlated and is tunable (eg, via phosphorylation) to regulate protein homeostasis.

Several studies revealed that high levels of Ser(S), Thr(T), Gln(Q), Asn(N), Pro, Gly or charged amino acids [Arg(R), Lys(K) and His(H) ] is a common feature of many IDRs ( Macossay-Castillo M, Marvelli G, Guharoy M, Jain A, Kihara D, Tompa P et al ., The balancing act of intrinsically disordered proteins: enabling functional diversity while minimizing promiscuity. J Mol Biol. 2019 ;431:1650-70 ). Because IDRs have the potential to associate with many partners due to their multiple possible metastable configurations, they are functionally key components of subcellular machinery and signaling pathways. Many 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. "IDR" has been evolutionarily retained as a toolkit to generate complex regulatory networks and it provides multiple mechanisms for cell-type or tissue-specific signaling, such as solubility, physical interactions, post-translational modifications, or Liquid-liquid phase transition. IDRs offer many advantages for proteins, including: (1) mediate protein-protein or protein-peptide interactions by adopting different conformations; (2) facilitate protein regulation through different post-translational modifications; and (3) regulate as a proteasome The half-life of the protein targeted for degradation.

In one embodiment, the present invention provides several IDRs with high STQN amino acid content that exhibit autonomous expression enhancing activity when fused in vivo with an exogenous target protein, such as β-galactosidase (LacZ) Produces natural protein at a high level. In some embodiments, most, if not all, IDRs described herein have one or more ATR ^Mec1 /ATM ^Tel1 DNA damage checkpoint kinase phosphorylation sites, indicating that their function and/or stability may be mediated by ATR ^Mec1 Or ATM ^Tel1 is tuned by protein phosphorylation.

In one embodiment, the SCD of the budding yeast Saccharomyces cerevisiae was previously defined as a region with at least three S/TQs within a 50-residue stretch (Cheung HC et al., BMC Genomics 2012 13:664). The conserved DNA damage response (DDR) checkpoint kinases Tel1 and Mec1 in Saccharomyces cerevisiae and their human homologues ATM and ATM phosphorylation are well-known DDR proteins at the S/TQ common site (Traven A. and Heierhorst J. ( 2006) Bioessays 27, 397-407). SCD may have a broader and more comprehensive role in eukaryotic cells due to the unexpected abundance of S. cerevisiae and mammalian SCD-containing proteins that belong to DDR-independent functional schemes such as endocytosis, microtubules, myocardium Actin cytoskeleton, and in pathways related to nervous system development. The present inventors have unexpectedly discovered that SCD and/or its phosphorylation are involved in the regulation of essential functions in protein homeostasis.

High S, T and Q content in the IDR or SCD and the overall number of S/T-Q motifs are extremely important for high levels of protein production and/or functional inhibition of protein degradation. In one embodiment, the content of S, T and Q is higher than 10% of the total amino acid content of the IDR or SCD. In some embodiments, the content of S, T and Q is greater than 15%, 15%, 20%, 25% or 30% of the total amino acid content of the IDR or SCD domain. In one embodiment, the content of S, T and Q is in the range of about 30% to about 40% of the total amino acid content of the IDR or SCD (eg, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%). In one embodiment, the SCD is an IDR with high S/T and Q content. In some embodiments, the IDR or SCD is Hop1-SCD (258-324 amino acids), Rad51-NTD (1-66 amino acids), Rad51-NTD-3SA, Rad51-NTD-6SQA, Rad51- NTD-9SQA, Rad51-NTD-12SQA, Rad53-SCD1 (1-29 amino acids), Rad53-SCD1-5STA, Rad53-SCD1-7QA, Rad53-SCD1-12STQA, Sml1-NTD (1-27 amino acids) amino acids), Sml1-NTD (1-50 amino acids), Sup35-PND (1-39 amino acids), 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 amino acids), New1-NPD (1-156 amines) amino acids), Ure2-UPD (1-91 amino acids), Vps64-NTD (1-152 amino acids), Ssk2-NTD (1-191 amino acids) or Kell-NTD (1-69 amino acids) amino acids), as shown in SEQ ID Nos: 1 to 31.

In a specific embodiment, the _NH2 -terminal domain (NTD; residues 1-66) of S. cerevisiae Rad51 is a bona fide with 3 SQ motifs (S2Q, S12Q ^{and S30Q )} SCD. All three S/Q motifs can be phosphorylated by Mec1 ^ATR and Tel1 ^ATM during vegetative growth and meiosis. Rad51-NTD has two distinct functions in regulating the steady-state content of Rad51. First, Rad51-NTD has autonomous expression enhancing activity. Second, Mec1 ^ATR /Tel1 ^ATM -dependent phosphorylation of Rad51-NTD antagonizes the proteasomal degradation pathway, thereby increasing the stability of the in vivo expressed protein Rad51. All three S/Q motifs in Rad51 are direct target sites for Mec1 ^ATR and Tel1 ^ATM not only in vegetative cells exposed to the DNA damaging agent methyl methanesulfonate (MMS) but also during normal meiosis .

In a specific embodiment, Sup35 (translation termination factor eRF3) is a well-characterized yeast prion protein that aggregates to form [ PSI ⁺ ]prion protein. Sup35-PND is an S-rich and Q/N-rich domain with 3 serines, 9 asparagine and 12 glutamic acids and only one S ¹⁷ Q motif.

In a specific embodiment, Rad53-SCD1 contains four TQ motifs and one SQ motif.

The prion (nucleation) domains of the three yeast prion proteins (Sup35, Ure2 and New1) share two structural features in common with DDR-SCD. Not only does it have a high S/T/Q/N amino acid content, but it also contains at least one S/TQ site that can be phosphorylated by Mec1 ^ATR or Tel1 ^ATM .

In a specific embodiment, the Ure2 prion protein domain (UPD) (residues 1-91) of the Ure2 nitrogen catabolite-repressing transcriptional regulator is the basis of the prion protein [ URE3 ⁺ ]. UPD is critical for the in vivo function of Ure2, as removal of UPD in Ure2 reduces protein stability and steady-state protein content (but not transcript content) of the corresponding Ure2-ΔUPD mutant. Like Sup35-PND, Ure2-UPD has a high STQN content (>63%; 10 serine, 5 threonine, 10 glutamic acid, 33 aspartamine among 91 amino acids) ) and adopts a completely disordered structure. In addition, Ure2-UPD also has the S ⁶⁸ Q phantom.

In a specific embodiment, the New1 prion protein domain (NPD) supports [ NU ⁺ ] and is susceptible to [ PSI+ ]prion protein induction. New1 is a non-essential ATP-binding cassette F-type protein that fine-tunes the efficiency of translation termination or ribosome recycling. New1-NPD also has high STQN content (>44%; 19 serine, 8 threonine, 14 glutamic acid, 26 aspartamine out of 156 amino acids) and S ¹⁴⁵ Q Motif.

It is also worth noting that the three (N-terminal IDR) N-IDR proteins (Vps64, Ssk2 and Kell) do not have known DDR-related functions. Vps64 (also known as Far9) is required for cytoplasmic to vacuolar targeting of proteins. Interestingly, the vps64Δ mutant showed increased aneuploidy tolerance. Kel1 (Kelch repeat 1) is required for proper cell fusion and cell morphology, and acts in conjunction with Kel2 (ohnolog of Kel1) to negatively regulate mitotic exit. Suppression of sensor kinase 2 (Ssk2) is a mitogen-activated protein kinase (MAPK) kinase of the Hog1 osmotically regulated signaling pathway. Activation of Ssk2 mediates actin cytoskeleton recovery from osmotic stress, respectively.

While not intending to be bound by any theory, it is believed that the effect of IDR or SCD itself in promoting high steady state levels of the polypeptide of interest is greater than the effect of IDR or SCD phosphorylation. In addition, while not intending to be bound by any theory, it is believed that IDR or SCD phosphorylation stabilizes the polypeptide of interest by preventing proteasomal degradation during the DNA damage response, preferably a negatively charged IDR or SCD is sufficient to confer interest Peptide stabilization.

In some embodiments of the invention, one or more of the S, T or Q residues in the IDR are replaced with an amino acid having a hydrophobic side chain. Examples of amino acids with hydrophobic side chains include, but are not limited to, alanine, valine, isoleucine, leucine, or methionine. Preferably, the amino acid with a hydrophobic side chain is alanine. Examples of IDRs in which one or more S, T, or Q residues are replaced by amino acids with hydrophobic side chains include, but are not limited to, Rad51-NTD-3SA, Rad51-NTD-6SQA, Rad51-NTD-9SQA, Rad51- NTD-12SQA, Rad53-SCD1-5STA, Rad53-SCD1-7QA, or Sup35-PND-1SA.

In some embodiments of the invention, one or more of the S, T or Q residues in the SCD are replaced with an amino acid having a negatively charged side chain. Examples of amino acids with negatively charged side chains include, but are not limited to, both aspartic acid or glutamic acid. Preferably, the amino acid with a negatively charged side chain is aspartic acid.

In some embodiments of the invention, one or more of the S, T or Q residues in the IDR are replaced with another amino acid of S, T and Q.

In various aspects, the expression vector is a nucleic acid, plastid, cosmid, virus or artificial chromosome. An expression vector comprises a polynucleotide sequence encoding a polypeptide of interest (polypeptide coding sequence) operably linked to or under the control of a promoter capable of effecting expression in a mammalian cell line of interest. An expression vector may contain one or more polypeptide coding sequences. Thus, the expression vector comprises at least a first, and optionally a second, third, fourth, etc. polypeptide coding sequence. Thus, in some embodiments, the expression cassette comprises first and second polypeptide coding sequences, which may be under the control of a single promoter or separate promoters.

The choice of vector will depend on several factors, including compatibility of the vector with the mammalian cell into which the vector is to be introduced (eg, mammalian cells that can be used to propagate or amplify the vector, or host cells such as bacterial cells) and integration of the vector into the genome of mammalian cells. The vector can be a viral vector, phage, phage, cosmid, fosmid, bacteriophage, artificial chromosome, colony vector, shuttle vector, plastid (linear or closed circular) or the like . Vectors can include chromosomal, non-chromosomal and synthetic DNA sequences. A large number of suitable vectors are known to those skilled in the art and are commercially available. Examples of suitable vectors are provided in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (2nd Edition), Vols. 1-3, Cold Spring Harbor Laboratory (1989).

Expression vectors may include additional features or elements useful for expressing one or more polypeptides of interest in mammalian host cells 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 that are important for nuclear export, translation, and/or stability of mRNA.

The host cell is preferably a eukaryotic host cell. The eukaryotic cells are preferably selected from the group consisting of mammalian cells, yeast cells, fungal cells, insect cells, plant cells and protozoan cells, and progeny of the parent cell, regardless of the morphology or genetic composition of the progeny. Whether the original parent is the same or not, as long as the selected gene is present. In general, any vector can be used in the methods of the invention and, in one aspect, the selection of an appropriate vector is based on the selection of a host cell for protein expression.

In various aspects, the host cell is a prokaryotic or eukaryotic cell. In various aspects, the host cell is a bacterial cell, a protist cell, a fungal cell, a plant cell or an animal cell.

Products of interest, eg, polypeptides produced according to the present invention, can be recovered, further purified, isolated and/or modified by methods known in the art. For example, the product can be recovered from the culture medium by conventional procedures including, but not limited to, centrifugation, filtration, ultrafiltration, extraction or precipitation. Purification can be performed by a variety of procedures known in the art, including but not limited to chromatography (eg, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatographic focusing and size exclusion chromatography), electrophoretic procedures ( such as preparative isoelectric focusing), differential solubility (eg ammonium sulfate precipitation) or extraction.

The product of interest can be any biological product that can be produced by any other event of transcription, translation, or expression of the genetic information encoded by the polynucleotide. In this regard, the product will be the expression product. The product may be a pharmaceutically active or therapeutically active compound, or a research tool to be used in an assay. In a particularly preferred embodiment, the product is a polypeptide, preferably a pharmaceutically active or therapeutically active polypeptide, or a research tool to be used in diagnosis or other assays. Thus, a polypeptide is not limited to any particular protein or group of proteins, but on the contrary, it can be any protein of any size, function or origin desired to be selected and/or expressed by the methods described herein. Thus, several different polypeptides of interest can be expressed/produced.

While the following examples provide further detailed descriptions of certain aspects and embodiments of the present invention, they should be considered illustrative only and in no way limit the scope of the claimed scope. example

Materials and Methods

Yeast strain and two-hybrid analysis : All meiotic experiments were performed using the diploid isogenic SK1 strain. Quantitative yeast two-hybrid analysis, tetrad dissection, immunostaining of chromosome smears, cycloheximide blocking experiments, and physical analysis were performed 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. cerevisiae Mre11 recruits SUMO moietie to facilitate The assembly and function of the Mre11-Rad50-Xrs2 complex. Nucleic Acids Research 2016 44, 2199-2213 . ) were performed.

Antiserum, immunoblotting and cytology : using synthetic phosphorylated peptides M ¹ S ^[P] QVQEQHISESQL ¹⁴ , E ⁶ QHI SES ^[P] QLQYGNGS ²⁰ , T ²⁴ VPADLS ^[P] QSVVDGNGN ³⁹ and E ¹⁸² LFGEFRTGKS ^[P] QLCHT ¹⁹⁷ was used as an antigen to generate rabbit antisera against phosphorylated Rad51-S ² Q, Rad51-S ¹² Q, Rad51-S ³⁰ Q and Rad51-S ¹⁹² Q, respectively, wherein S ^[P] is phosphorylated serine. Antisera were pre-cleaned with the corresponding non-phosphorylated peptides coupled to agarose beads by peptide-specific affinity chromatography. Phosphorylated peptide synthesis and animal immunization were performed by LTK BioLaboratories (Taiwan). Rabbit antiserum against Hop1, rabbit antiserum against phosphorylated Hop1-T ³¹⁸ Q, and goat antiserum against Zip1 were as previously described ( Chuang CN, Cheng YH, Wang TF Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis. Nucleic Acids Res. 2012;40:11416-11427 ). Goat anti-Rad51 antibody (yN-19), goat anti-Clb1 antibody (yS-19) and rabbit anti-Sic1 antibody (FL-284) for Western blotting were purchased from Santa Cruz Biotechnology (CA, USA). The rabbit anti-Dmcl antibody system was a gift from Douglas Bishop (University of Chicago, IL, USA). Rat anti-HA antibody system was purchased from Roche (Basel, Switzerland). Mouse anti-V5 antibody system was purchased from Bio-Rad (CA, USA). Rabbit anti-Hsp104 and anti-hexokinase antisera were kindly provided by Chung Wang (Academia Sinica, Taiwan). Western blot analysis was performed as described ( 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: 586-596 ) implementation. Protein signals on Western blot membranes were exposed to X-ray film (Fujifilm Medical, Tokyo, Japan) or visualized and captured using ImageQuant LAS 4000 (GE Healthcare, IL, USA) and quantified using ImageJ (NIH, MD, USA). Cytological analysis using rabbit anti-Dmc1 and guinea pig anti-Rad51 was as previously described ( Shinohara M., Gasior SL, Bishop DK, Shinohara A. Tid1/Rdh54 promotes colocalization of Rad51 and Dmc1 during meiotic recombination. Proc. Natl. Acad. Sci. USA 2000;97:10814-10819 ). The stained samples were observed using an epifluorescence microscope (BX53, Olympus) with a 100X objective (NA1.4). Images were captured by a CCD camera (CoolSNAP HQ2, Teledyne Photometrics) at room temperature and then processed using iVision software (BioVision Technologies).

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. Rabbit anti-Sml1 antibody system was purchased from Agrisera (Vännäs, SE, AS10847). Rabbit anti-Hsp104 antiserum was kindly provided by Chung Wang (Academia Sinica, Taiwan). Rabbit antiserum against phosphorylated Sup35- ^S17Q was generated using the synthetic phosphorylated peptide ^{N12YQQYS [P] QNGNQQQGNNR28} as antigen, where S ^[P] is phosphorylated serine. Phosphorylated peptide synthesis and animal immunization were performed by LTK BioLaboratories (Taiwan). Western blot analysis was performed as described ( Chuang CN, Cheng YH, Wang TF. Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis. Nucleic Acids Res. 2012;40:11416-27 ; 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 ). Quantitative β-galactosidase activity assay was performed 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. Identification of ion channel-associated proteins using the yeast two-hybrid system. Methods Enzymol. 1998;293:104-22 ).

Example 1 Saccharomyces cerevisiae Rad51 short N end area (NTD ; 1-66 indivual amino acid residue ) Has unexpected expression enhancing activity

The NTD of Rad51 is a true SCD with ³ SQ motifs (S2Q, ^S12Q and ^S30Q ) that can be phosphorylated by Mec1 ^ATR and Tel1 ^ATM in vivo. Biochemical and genetic analysis showed that Rad51-NTD phosphorylation significantly promotes the stability of Rad51 by preventing proteasome-mediated degradation. The order of half-lives (t1/2) was wild-type Rad51 (>3 hours) to Rad51-3D >> Rad51-3A (20-30 minutes). Rad51-3D and ^Rad51-3A are mimic phosphorylation mutants and phosphorylation deficient mutants, and all ^three of these serine residues (S2, ^S12 and S30) are mutated to aspartic acid (D) and alanine (A).

Notably, the steady-state protein content of Rad51 ^ΔN (the NTD truncated form of Rad51; residues 67-400) was <5% compared to the steady-state protein content of wild-type Rad51 protein in vivo (Fig. 1A and 1). 1B). Under nutrient conditions and upon MMS treatment to induce the expression of Rad51 protein in WT, the steady-state level of Rad51-ΔN protein in rad51 - ΔN cells was only about 3% relative to the steady-state level of Rad51 in WT (Figure 1C). ). Rad51 ^ΔN still has the ability to repair DNA double-strand breaks because rad51 -ΔN cells are resistant to DNA damaging agents (i.e., hydroxyurea and methyl methanesulfonate) during vegetative growth (Fig. About 50% viable spores were produced during division (Fig. 1E).

Rad51-NTD contains 66 amino acid residues. To characterize biochemical properties, the S. cerevisiae pYC2/CT/ lacZ -V5 expression vector was modified by replacing the GAL1 promoter with the promoter of the wild-type RAD51 gene (P _RAD51 ) and inserting the SV40 nuclear localization signal (NLS) peptide before V5 (Invitrogen, USA) to give _PRAD51 - lacZ -NLS-V5. This new vector retains the C-terminal V5 epitope for detection of LacZ-V5 or LacZ-V5 fusion proteins by immunoblotting. Next, three corresponding vectors were constructed: _PRAD51 -NTD-lacZ-NLS-V5, _PRAD51 - ^NTD3A -lacZ-NLS-V5, and _PRAD51 - ^NTD3D -lacZ-NLS-V5. All four of these vectors were transformed into the SK1 yeast cell line. Transformants were vegetatively propagated to log phase and collected for the preparation of denatured lysates. Immunoblotting analysis using antisera against phosphorylated Rad51-S ¹² Q indicated that Tel1 ^ATM and Mec1 ^ATR phosphorylated NTD-LacZ-V5, but not NTD ^3A -LacZ-V5 or NTD ^3D -LacZ-V5 phosphorylation (Figure 2A). All three of these NTD-LacZ-V5 fusion proteins can be recognized in immunoblotting using anti-Rad51 antiserum and the order of steady state protein content is NTD-LacZ-V5≈NTD ^3D -LacZ-V5>NTD ^3A -LacZ -V5 (Fig. 2A), all of which showed significantly higher protein content than LacZ-V5 (Fig. 2B). Therefore, Rad51-NTD can enhance the expression of its fusion partner, and given the presence of more NTD ^3A -LacZ-V5 degradation products in the ink dots (Fig. 2B, marked by black arrows), NTD ^3A -LacZ-V5 may Not as stable as NTD-LacZ-V5 and NTD ^3D -LacZ-V5.

The beta-galactosidase activity of these four fusion proteins was also determined. The stratified and normalized relative activity levels were NTD ^3D -LacZ-V5 (13.2-fold)=NTD-LacZ-V5 (13.2-fold)>NTD ^3A -LacZ-V5 (9.6-fold)>>LacZ-V5 (1-fold)( Figure 2C). These results indicate that Rad51-NTD can be used as an expression vehicle to facilitate expression, folding or stabilization of its fusion partner. The function of the expression vehicle for Rad51-NTD was further stabilized by Tel1 ^ATM -dependent and Mec1 ^ATR -dependent phosphorylation or by mimetic phosphorylation.

The expression enhancing activity of SCD is autonomous. When Rad51-NTD alone was fused to the NH ₂ terminus of lacZ-V5-His ₆ (ie, β-galactosidase with a V5 epitope tag and a hexahistidine (His ₆ ) affinity tag), It not only increased (>10-fold) the steady-state content of the target protein (ie, lacZ-V5-His ₆ ) (Fig. 3A), but also caused significantly (>10-fold) higher β-galactosidase activity (Figure 3B). It has also been shown that the N-terminal SCD1 of Rad53 (1-29 amino acid residues) and the intermediate SCD of Hopl (258-324 amino acid residues) also have such expression enhancing activity (Figure 3). Next, the expression-promoting activity of Rad53-SCD1 was determined using four different target proteins, including β-galactosidase (LacZ), green fluorescent protein (GFP), glutathione S-transferase (GST) and Non-dimerized GST (GST-(nd)). In GST (nd), two important residues (Arg73 and Asp77) that contribute to the dimer stability of GST were mutated to proline and lysine. In the case of the N-terminal Rad53-SCD1 fusion, all four target proteins were found to show dramatically increased expression compared to the untagged protein (Figure 4).

example 2 Rad51-NTD exist during vegetative growth and meiosis Mec1 ^ATR dependency and Tel1 ^ATM dependency phosphorylation

Rad51-NTD (residues 1-66) contains three SQ motifs ( ^S2Q , ^S12Q and ^S30Q ) (Fig. 5A). To reveal the function of Rad51-NTD itself and phosphorylation of NTD, antisera specific for phosphorylated Rad51 ^- S2Q, Rad51- ^S12Q and Rad51- ^S30Q proteins were first generated (FIG. 5B). Next, a set of mutants was generated including NTD deletion mutants ( rad51 - ΔN or rad51 ^67-440 ), mec1-kd (kinase inactive) sml1Δ double mutant, mec1-kd (kinase inactive) sml1Δ tel1Δ triple mutant, three monoamino acid substitution mutants ( rad51-S2A , rad51-S12A , and rad51-S30A ), phosphorylation-deficient mutant ( rad51-3A ), and a phosphorylation-mimicking mutant ( rad51-3D ) ) (Figure 5A). The results of immunoblotting analysis (Figure 5B-D) indicated that all three S/Q motifs in Rad51 were not only in vegetative cells exposed to the DNA damaging agent methyl methanesulfonate (MMS) (Figure 5C) It is also a direct target site for Mec1 ^ATR and Tel1 ^ATM during normal meiosis (Fig. 5D).

Like several other eukaryotic organisms, such as higher plants and mammals, S. cerevisiae has two RecA-like recombinases, Rad51 and Dmcl. Rad51 is ubiquitous in all eukaryotic cells, whereas Dmc1 is meiosis-specific. In Saccharomyces cerevisiae, neither Rad51 nor Dmc1 is required for meiosis. However, the catalytic function of Rad51 is inhibited by direct interaction with the meiosis-specific protein Hed1, which makes Rad51 a cofactor for Dmc1 during meiosis. Deficiency of the meiosis-specific recombinase Dmc1 causes a very strong cell cycle arrest phenotype in meiotic prophase I. Sporulation and spore viability were significantly improved in the dmc1Δhed1Δ mutant , which may be attributed to efficient interhomologous recombination by Rad51 in the absence of inhibition exerted by Hed1.

It was found that Mec1 ^ATR /Tel1 ^ATM -dependent Rad51-NTD phosphorylation was associated with meiotic recombination in dmc1Δhed1Δ mutants (Fig. 5E ) and exposure to higher concentrations of DNA damaging agents (ie, 0.02% and Essential for DNA repair (Fig. 5F) in vegetative cells other than 0.01% MMS). Notably, the mimetic phosphorylation mutant ( rad51-3D ) can functionally replace phosphorylated Rad51 in dmc1Δhed1Δ (Fig. 5E ). In contrast, the dmc1Δhed1Δrad51-3A triple mutant behaved similarly to dmc1Δ , exhibiting a phenotype associated with strong cell cycle arrest in prophase I. It has also been shown that the meiotic defect of the dmc1Δhed1Δrad51-3A triple mutant can be rescued (at least in part) by introducing a high-copy number vector expressing the full-length Rad51 or the Rad51-3A mutant (FIG. 6).

example 3 Rad51-NTD for Promotes vegetative growth and high homeostasis during meiosis Rad51 Protein content matters

Rad51-NTD itself and Rad51-NTD phosphorylation have distinct roles in regulating the overall steady-state level of Rad51 protein in vivo. The rad51 - ΔN ( rad51 ^67-440 ) dual gene encodes the NTD truncation mutant Rad51-ΔN (residues 67-400) under the control of the native RAD51 promoter. First, rad51-ΔN diploid cells displayed an approximately 50% reduction in spore viability (Fig. 5E). Second, rad51-ΔN grew as fast as wild-type cells on YPD dishes without or with only 0.01% MMS. In contrast, it exhibited slower vegetative growth on YPD disks containing 0.02% MMS (Fig. 5F). These results indicate that Rad51-ΔN still has (at least in part) the ability to promote HDRR during mitosis and meiosis. The meiotic cell cycle arrest phenotype of the rad51 - ΔN dmc1Δhed1Δ triple mutant can be rescued (at least in part) by introducing high-copy number vectors expressing full-length Rad51 or Rad51-3A mutants, but introducing Rad51- The high copy number vector of the ΔN mutant was unable to rescue (Figure 6). The immunoblotting time course experiment further revealed that the steady-state content of Rad51-ΔN in rad51 - ΔN cells was significantly lower than that in WT cells during vegetative growth (Fig. 1A and Fig. 1B) or meiosis (Fig. 7A) Steady-state content of Rad51 protein. This suggests that NTD itself has a more profound effect on promoting high steady-state levels of Rad51 during vegetative growth and meiosis than NTD phosphorylation.

Regarding sml1Δ repression, rad51 - ΔN is phenotypically similar to mec1-kd . Since the steady-state levels of Rad51-ΔN in rad51 - ΔN cells were not significantly different from those in rad51 -ΔN sml1Δ cells during vegetative growth and meiosis, we concluded that Rad51 may be involved in Saccharomyces cerevisiae. Rich targets for Mec1 ^ATR and Tel1 ^ATM . Consistent with this hypothesis, it has been reported (Yeast Genome Database: https://www.yeastgenome.org) that the median abundance of Rad51 (6070±2766 molecules/cell) in vegetative cells is higher than known Mec1 ^ATRs and median abundances of Tel1 ^ATM target proteins such as Rad9 (1279±701 molecules/cell), Rad53 (1533±520 molecules/cell) and Dun1 (1729±971 molecules/cell).

Phosphorylated Rad51 was shown to be prone to foci on chromosomes during the thick filament phase of meiosis (FIG. 8B). The Rad51 foci detected using antisera targeting native Rad51 and phosphorylated Rad51 were not fully colocalized, implying partial phosphorylation of Rad51 or possible competition for antigen recognition in this assay. Nonetheless, foci formation of Rad51 in the phosphorylation-deficient rad51-3A mutant was indistinguishable from that of WT controls (Fig. 8B to D), although it was delayed chromosomally (Fig. 8E), which may be due to Due to the low protein expression level of Rad51 (see below). In addition, Dmc1 foci formation was not significantly perturbed in the rad51-3A mutant (Figure 8C and D), suggesting that the function of Rad51 as a Dmc1 cofactor may be retained by the phosphorylation-deficient Rad51-3A.

Example 4 NTD and Mec1 ^ATR /Tel1 ^ATM dependency Rad51-NTD Phosphorylation dmc1 Δ hed1 Δ Essential for meiotic recombination in mutants

The results revealed that the dmc1Δhed1Δ double mutant and the dmc1Δhed1Δrad51-3D triple mutant exhibited meiotic progression and spore viability similar to each other (Fig. 8A ), indicating that the Rad51-3D protein has meiotic DSB repair Features. Additionally, rad51-3D in the dmc1Δhed1Δ mutant behaved similarly to WT RAD51 , as delayed meiotic progression and a meiotic I nondisjunctive phenotype were observed (most tetrads contained two or zero viable spores) ) (Figure 8A). Strikingly, like the dmc1Δ mutant, the dmc1Δhed1Δrad51-3A and dmc1Δhed1Δrad51 - ΔN triple mutants hardly sporulate and exhibit strong meiotic progression in prophase I Stalled phenotype (Figure 8A). Thus, Rad51-NTD and Rad51-NTD phosphorylation are critical for Rad51-mediated meiotic recombination in the dmc1Δhed1Δ mutant, and the mimic phosphorylation mutant Rad51-3D can functionally replace Rad51-3D in the same genetic background Phosphorylated Rad51. This situation was further confirmed by the extremely low spore viability following meiosis in the dmc1Δhed1Δmec1 -kd sml1Δ strain , where Rad51-NTD phosphorylation was solely dependent on Tel1 ^ATM (Fig. 5B, right panel).

Example 5 Phosphorylation deficient Rad51 exist Insufficiently maintained protein levels during meiosis

Immunoblotting experiments further revealed that in DMC1 HED1 and dmc1 Δ hed1 Δ meiotic cells, the steady-state protein content of Rad51-3A was lower than that of Rad51 and Rad51-3D (Figure 9 A and B). In DMC1 HED1 meiotic cells, the maximal steady-state content of Rad51-3A (after 7 hours in SPM) was found to be approximately 30% of that of WT Rad51 (Figure 9B). Comparison of WT Rad51, Rad51-3A and Rad51-3D protein levels in 1 hr or 5 hr meiotic cultures in the same immunoblot revealed that Rad51-3A levels stagnated and remained below 30% of WT Rad51 levels, On the other hand, Rad51-3D was extremely abundant and maintained an expression level of about 1.4-fold relative to WT Rad51 since early meiosis (Fig. 9 C and D). We performed immunoblotting analysis of Hop1 and Zip1 to rule out the possibility that the meiotic defect in the dmc1Δhed1Δrad51-3A strain was indirectly caused by aperiodic meiotic progression. No significant differences in the overall protein content pattern of Hopl or Zipl were observed in all six strains examined (Figure 9 A and B). Given that phosphorylated Hop1 protein migrates slower in SDS polyacrylamide gels than unphosphorylated Hop1, it is worth noting that all three strains in the dmc1Δhed1Δ background accumulated more protein even after 12 hours in SPM. Hyperphosphorylated Hop1 (Figure 9 A and B). Similarly, higher phosphorylation was also observed in the dmc1Δhed1Δ mutant in immunoblots using phospho-specific Rad51 ^- S2Q, Rad51- ^S12Q , and Rad51- ^S30Q antisera , respectively Rad51 content (Figure 9 A and B). These results may reflect long-term checkpoint activation by Mec1 ^ATR and Tel1 ^ATM in the absence of Dmc1.

example 6 Rad51-NTD Phosphorylation produces a more stable Rad51 protein

Next, cycloheximide blocking experiments were performed to compare the protein stability of Rad51, Rad51-3A and Rad51-3D. Protein synthesis was inhibited by adding 200 μg/ml cycloheximide to the meiotic cultures at the 4 hour time point (Figure 10A). Samples were taken at 0, 30, 60, 90, 120 and 180 minutes after the addition of cycloheximide. Immunoblotting analysis revealed that in the presence of cycloheximide, the levels of Rad51 and Dmc1 remained stable for up to 180 minutes. In contrast, in the rad51-3A and dmc1Δhed1Δrad51-3A strains, the protein content of the Rad51-3A mutant was significantly reduced within 30 minutes of the addition of cycloheximide and little after 60 minutes. detected (Fig. 10A, bottom). These results indicate that the half-life ( t _1/2 ) of Rad51-3A is ≤ 30 minutes, while the t _1/2 of Rad51 and Dmc1 is ≥ 180 minutes.

In time-course experiments performed on the WT strain, with a 30-minute interval between sample collections, native Rad51 protein content was found to increase gradually as meiosis progresses, with progressive phosphorylation of the three SQ motifs (Fig. 10 B and C). We speculate that during early meiosis (ie, at the 1 hr meiotic time point), the degree of instability at the 4 hr time point of unphosphorylated native Rad51 (but not Rad51-3D) is similar to that of Rad51-3D. Rad51-3A is similar ( t _1/2 ≤ 30 min) and becomes stable upon phosphorylation of Rad51-NTD upon entry into subsequent meiosis. Using aliquots of the same cultures shown in Figure 10B, we performed cycloheximide blocking assays at different time points and compared WT Rad51, Rad51 from 1 hour and 5 hour meiotic cultures - Stability of 3A and Rad51-3D. The results showed that native Rad51 ( t _1/2 ≥ 180 min) from 5 hr meiotic cultures was actually more stable than that from 1 hr meiotic cultures (t _1/2 ≈ 180 min) (Fig. 10D and E). We further show that the mock phosphorylated mutant (Rad51-3D) in 1 hr or 5 hr meiotic cultures is as stable as native Rad51 from 5 hr meiotic cultures ( t _1/2 ≥ 180 min) ( Figure 10 D and E), showing that negatively charged NTDs are sufficient to stabilize Rad51 during meiosis.

Therefore, Rad51-NTD phosphorylation plays an important role in enhancing the protein stability of Rad51. When Dmc1 was present in the rad51-3A strain, low steady-state levels of Rad51-3A were apparently sufficient to promote Dmc1-mediated meiotic recombination, consistent with cytological observations (Figure 8C and D). In contrast, when Rad51 is the only "active" recombinase in meiotic cells (ie, in the dmc1Δhed1Δ strain ), Rad51-NTD phosphorylation is essential for ensuring higher steady-state levels of Rad51 for repair DSB induced by Spo11 is critical. Additionally, phosphorylated Rad51 can be functionally substituted with Rad51-3D.

example 7 Not phosphorylated Rad51 Proteins are degraded via the proteasome pathway

It has been shown that the 26S protease system is required for HDRR in S. cerevisiae. Next, the proteasome inhibitor MG132 was used to determine whether WT Rad51 and Rad51-3A were degraded by the proteasome during the DNA damage response. To promote MG132 uptake, these strains deleted PDR5 to reduce efflux pump activity. After 30 min of MMS treatment, Rad51-3A disappeared faster under cycloheximide treatment than Rad51 (Figure 11 A to C). In contrast, simultaneous addition of MG132 and cycloheximide delayed the degradation of Rad51-3A, indicating that Rad51-NTD phosphorylation stabilizes Rad51 protein during the DNA damage response by preventing proteasomal degradation.

To determine which SQ motif phosphorylation in Rad51-NTD is most important for its stability, cycloacetimide blocking experiments were performed to determine the relative protein stability of Rad51-S2A, Rad51-S12A, and Rad51-S30A (Figure 11). D). The results show that all three of these Rad51 protein variants are less stable than WT Rad51, but these variants are much more stable than Rad51-3A, with the least stable mutant protein Rad51-S2A exhibiting a half-life of about 3 hours (Figure 11E ). Thus, phosphorylation of each SQ motif in Rad51-NTD synergistically contributes to Rad51 protein stabilization.

The results obtained for Examples 1 to 7 are summarized herein:

(a) Removal of NTD from Rad51 resulted in low steady-state Rad51-ΔN protein content and higher sensitivity to MMS.

(b) It has been suggested that Rad51-NTD may have autonomous activity to promote Rad51-ΔN protein expression. Interestingly, this novel function of Rad51-NTD can be replaced by Rad53-SCD1 and the prion nucleation domain (PND; residues 1-39) of the yeast Sup35 protein. Rad53-SCD1 contains four TQ motifs and one SQ motif. Sup35 (translation termination factor eRF3) is a well-characterized yeast prion protein that aggregates to form [ PSI ⁺ ]prion protein. It is an S-rich and Q/N-rich domain with only one ^S17Q motif. Therefore, Sup35-PND is an IDR, not an SCD. The results also revealed that Mec1 ^ATR -dependent and Tel1 ^ATM -dependent phosphorylation did not affect the protein expression enhancement function of Rad51-NTD, Rad53-SCD1 and Sup35-PND, because their phosphorylation-deficient mutants could induce the corresponding fusion proteins, That is, high steady state levels of Rad51-3A (or Rad51-NTD-3A-Rad51-ΔN), Rad53-SCD1-5A-Rad51-ΔN, and Sup35-PND-S ¹⁷ A-Rad51-ΔN.

(c) The results demonstrate that Rad51-NTD can act autonomously to promote the expression of β-galactosidase (LacZ). Saccharomyces cerevisiae midsection expression vector pYC2/CT was modified by replacing the GAL1 promoter with the promoter of the native RAD51 gene ( _PrRAD51 ) and inserting the LacZ gene before the V5 epitope tag and the hexahistidine (His6) affinity tag / Pr _GAL1 -LacZ-V5-His6 (Invitrogen, USA) to give Pr _RAD51 -LacZ-V5-His6. Next, three corresponding mutant vectors were constructed: Pr _RAD51 -NTD-LacZ-V5-His6, Pr _RAD51 -NTD-3A-LacZ-NLS-V5-His6 and Pr _RAD51 -NTD-3D-LacZ-V5-His6 . All four of these vectors were transformed into the SK1 yeast cell line. Transformants were vegetatively propagated to log phase and then collected for the preparation of denatured lysates. Immunoblotting analysis using antiserum against phosphorylated Rad51-S ¹² Q indicated that Tel1 ^ATM and Mec1 ^ATR phosphorylated NTD-LacZ-V5-His6, but not NTD-3A-LacZ-V5-His6 or NTD -3D-LacZ-V5-His6 phosphorylation. All three of these fusion proteins were recognized in immunoblotting using anti-Rad51 antiserum. The order of steady-state protein content is NTD-LacZ-V5-His6≈NTD-3D-LacZ-V5-His6>NTD-3A-LacZ-V5-His6, all of which were significantly higher than LacZ-NLS-V5 _- His6 the protein content. Thus, NTD, NTD-3A and NTD-3D all have the ability to enhance the expression of their fusion partner LacZ-V5-His6. It is worth noting that NTD-3A-LacZ-V5-His6 is inferior to NTD-LacZ-V5-His6 and NTD-3D-LacZ- V5-His6 is stable. The beta-galactosidase activity of these four fusion proteins was also determined. The relative activity levels of stratification and normalization are NTD-3D-LacZ-V5 _- His6 (13.2 times) ~ NTD-LacZ-V5-His6 (13.2 times) > NTD-3A-LacZ-V5-His6 (9.6 times) >>LacZ-V5-His6 (control; 1.0-fold). These results also show that most, if not all, NTD-LacZ-V5-His6 fusion proteins are phosphorylated like the native WT Rad51 protein.

(d) Rad51-NTD is not the only SCD capable of promoting high-level LacZ-V5-His6 protein production. Here, it is also reported that Rad53-SCD1 (residues 1-29), Hop1-SCD (residues 258-324) or Sml1-SCD (residues 1-27 or residues 1-50) can all be used as _NH2 , respectively A terminal fusion tag to enhance LacZ-V5-His6 expression (upper panel) resulted in a 6-10-fold increase in beta-galactosidase activity (lower panel).

(e) Rad53-SCD1 enhances not only LacZ but also three other target proteins, namely green fluorescent protein (GFP), glutathione S-transferase (GST) and non-dimerized GST [GST-(nd )] of high-level manufacturing (Fig. 10H). β-Galactosidase is a tetrameric protein. GFP is a monomeric protein. GST is a dimeric protein. In GST (nd), two important residues (Arg73 and Asp77) that contribute to the dimer stability of GST were mutated to proline and lysine. These results indicate that the quaternary structure of the target protein is unexpectedly associated with the expression enhancing function of SCD.

(f) SCD is defined as at least three S/TQ sites in a segment of <50 amino acids in S. cerevisiae ( Cheung, HC, San Lucas, FA, Hicks, S., Chang, K., Bertuch, AA and Ribes-Zamora, A. (2012) An S/TQ cluster domain census unveils new putative targets under Tel1/Mec1 control. BMC Genomics, 13 , 664 ) ). The enrichment of S/TQ motifs in SCD means that it exhibits lower sequence complexity. Low sequence complexity and high S/T and Q content are characteristic features of intrinsically disordered regions (IDRs) in proteins. It remains to be seen whether the IDR properties of SCD are critical to its ability to promote high-level protein production. Therefore, the role of S/T or Q in different SCDs was examined using the alanine scanning method. First, the normalized protein steady state content of the Rad51-NTD-LacZ-V5-His6 variant was stratified in the line NTD~NTD-3A>NTD-6SQA>NTD-9SQA>NTD-12SQA~control. The respective normalized β-galactosidase activities of Rad51-SCD-LacZ-V5 variants were stratified by NTD (11.3 times) ~ NTD-3A (11.3 times) > NTD-6SQA (7.7 times) >> NTD-9SQA (2.5 times)>NTD-12SQA (1.3 times)～control group (1.0 times). These results indicate that for high levels of protein production by Rad51-NTD, the first six glutamic acids in the Rad51-SCD sequence are more influential than the last three glutamic acids and three serines (S ² , S ¹² and S ³⁰ ) have far-reaching effects. These results also show that the normalized protein steady state content and β-galactosidase activity of the Rad53-SCD1-LacZ-V5-His6 fusion variant is stratified by SCD1 (6.5-fold) > SCD1-5STA (5.3-fold) >SCD1-7QA (4.3 times) >>SCD1-12STQA (1.1 times) ~ control control group (1.0 times). It is concluded from this that high S/T and/or Q content is more important for high-level protein manufacturing function than the overall number of S/TQ motifs.

(g) The results further confirmed that Sup35-PND could also induce high steady-state levels of Rad51-ΔN and LacZ-V5-His6. The Sup35-PND line has S-rich and Q/N-rich domains of 3 serine, 9 asparagine, and 12 glutamic acid. Stratification of normalized LacZ activity of Sup35-NTD-LacZ-V5-His6 fusion variants Sup35-NTD (8.6-fold) ~ Sup35-NTD-S ¹⁷ A (8.1-fold) >> control control (1.0-fold).

(h) Using all LacZ fusion proteins analyzed in Figure 6, a noteworthy correlation was found between relative LacZ activity and the percentage of overall STQ in the total amino acid content of SCD or IDR. To achieve more than 8-fold optimal LacZ activity, the overall STQ content of the SCD or IDR should be in the range of about 30% to about 40%.

example 8 four DDR-SCD and three prion protein domains can promote target protein expression

As recently reported ( 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 ), Removal of NTDs from Rad51 reduced the protein content of the corresponding Rad51-ΔN protein by approximately 97% relative to wild type (Fig. 12A), making yeast highly sensitive to the DNA damaging agent methyl methanesulfonate (MMS) (Fig. 12B). ). This nanny function of Rad51-NTD can be rescued in Rad51-ΔN by N-terminal fusion of Rad53-SCD1, Rad53-SCD1-5STA (all five S/TQ motifs become AQ), or Sup35-PND, respectively (nanny function)(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. BioRxiv . 2020.12.08.407247; doi : https://doi.org/10.1101/2020.12.08.407247).

Next, different X-LacZ-NVH fusion proteins, including Rad51-NTD, _Rad53 - SCD1 , Hop1 -SCD ( Residues 258-324), Sml1-NTD (residues 1-27), Sml1-NTD (residues 1-50), Sup35-PFD, Sup35-PND, Sup35-PND- ^S17A , Ure2-UPD and New1 -NPD (Figure 12C). The NVH tag contains the SV40 nuclear localization signal ( N LS) peptide (Woo TT, Chuang CN, Higashide M, Shinohara _A , Wang TF. Dual ) before the V5 epitope tag and the hexahistidine ( His6 ) affinity tag roles of yeast Rad51 N-terminal domain in repairing DNA double-strand breaks. Nucleic Acids Res. 2020;48:8474-89 ). In Sup35-PND-S ¹⁷ A, the S ¹⁷ Q motif becomes AQ. Sml1 is a potent inhibitor of ribonucleotide reductase and in the SK1 S. cerevisiae strain, it has three S/TQ motifs ( ^S4Q , ^S14Q and ^T47Q ). We found that the β-galactosidase activity of all X-LacZ-NVH fusion proteins was 4.1-11.6-fold higher than that of LacZ-NVH (Figure 12C). Interestingly, the use of anti-phosphorylated Rad51-S ¹² Q, Rad51-S ³⁰ Q, Hop1-T ³¹⁸ Q and Sup35-S ¹⁷ Q antisera, respectively, found that Rad51-NTD-LacZ-NVH, Hop1-SCD-LacZ -NVH and Sup35-PND-LacZ-NVH were phosphorylated in response to MMS treatment, but not Sup35-PND- ^S17A -LacZ-NVH. As recently 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 ), The nanny functions of these IDRs occur in the presence or absence of MMS-induced DNA damage responses.

Sup35 is mainly located in the cytosol, while Mec1 ^ATR and Tel1 ^ATM are nuclear proteins. The NVH tag contains the SV40 NLS peptide. Sup35-PND-LacZ-NVH can target the nucleus and become accessible to Mec1 ^ATR and Tel1 ^ATM for phosphorylation. Then we determined that MMS only expresses wild-type Sup35 and expresses GFP-tagged Sup35 (Sup35-GFP) respectively ( 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 ) and expressing Sup35-S ¹⁷ A-GFP mutants (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. BioRxiv . 2020.12.08.407247; doi: https://doi.org/10.1101/2020.12.08.407247) Saccharomyces cerevisiae cannot induce phosphorylation of Sup35-PND-S ¹⁷ Q .

example 9 high STQN content for IDR The babysitter function is very important

A common feature of DDR-SCDs, Sup35-PND, Ure2-UPD and New1-NPD is their high STQN amino acid content (Figure 13A). We wanted to know if high STQN content in IDRs is critical for promoting high levels of protein expression. We used alanine scanning mutagenesis to test this hypothesis. By definition, alanine scanning increases the relative amount of alanine residues in a given sequence that are known to favor alpha helix formation ( Marqusee S, Robbins VH, Baldwin RL. Unusually stable helix formation in short alanine- based peptides. Proc Natl Acad Sci US A. 1989;86:5286-90 ) and thus facilitates the transition from disorder to order.

The results revealed a positive correlation between relative β-galactosidase activity or LacZ-NVH fusion protein content and the percentage of total STQN amino acids in the IDR (Figures 13A-D). For each individual IDR examined, a linear relationship (R2 = 0.86-0.94) was observed between its ^STQN amino acid content and its LacZ activity (Figure 13 B-D). Although the overall lengths of these IDRs vary, the results indicate that in order to achieve optimal LacZ activity over 5-fold relative to wild type, the STQN amino acid content in the IDR should be at least ≥30% (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. BioRxiv . 2020.12.08.407247; doi: https://doi.org/10.1101/2020.12. 08.407247).

Notably, the thresholds for STQN content were different in the three cases presented here (Figures 13B-D), indicating that the percentage of STQN residues in the IDR is not the only factor contributing to protein expression. Therefore, we performed a more in-depth analysis of the amino acid composition of all N-IDRs analyzed here.

Figures 1 A to D show that NTD, a canonical SCD, is essential for promoting high steady-state protein content of S. cerevisiae Rad51 in vivo. Figure 1 A shows that total cell lysates were prepared from vegetative cells under methyl methanesulfonate (MMS) treatment and subjected to immunoblotting at the indicated time points. Hsp104 was used as an internal reference. Immunoblotting was performed using goat anti-Rad51 antibody (yN-19) purchased from Santa Cruz Biotechnology (CA, USA). This antibody system was generated at the N-terminus of yeast Rad51 using a peptide (sc-8936) map. Figure 1B shows that immunoblotting was performed using guinea pig anti-Rad51 antibody. The predicted molecular weight of Rad51-ΔN is 36,270 Daltons. Figure 1C shows that total cell lysates from wild-type (WT) mitotic cells under MMS treatment were diluted with rad51 - ΔN or empty sample buffer at the indicated titers to estimate the relative steady state of WT Rad51 and Rad51-ΔN protein content. Figure 1 D shows that NTDs are not essential for DNA damage tolerance in vegetative growth. Spot analysis showed five-fold serial dilutions of the indicated haploid or diploid strains growing on YPD plates and YPD plates containing the indicated concentrations of MMS. The ploidy of the different strains is indicated on the right. Figure 1 E shows that spore viability was analyzed after 3 days at 30°C on sporulation medium. To score spore viability, only tetrads (but not dyads or triads) were dissected on the YPD.

Figures 2A to C show that Rad51-NTD phosphorylation not only promotes protein expression, but also enhances protein stability. Using the indicated yeast expression vector, i.e., _PRAD51 - lacZ -V5 (control), _PRAD51 -NTD- lacZ -V5, _PRAD51 - ^NTD3A - lacZ -V5 or _PRAD51 - ^NTD3D - lacZ -V5, The wild-type SK1 haploid strain was transformed. In Figure 2C, total cell lysates were prepared from exponentially growing mitotic cells and visualized by immunoblotting and quantitative yeast LacZ analysis with the corresponding antisera. Figure 2A shows that the NTD-LacZ-V5 fusion protein (approximately 128 kilodaltons) is marked with a white arrow. Endogenous Rad51 (approximately 43 kilodaltons) is indicated by black arrows. The numbers below the anti-Rad51 immune dots indicate the fold change in NTD-LacZ-V5 fusion protein content normalized to endogenous Rad51 protein content in the same dot. Figure 2B shows that immunoblotting was performed using anti-V5 antiserum. Putative degradation products are indicated by black arrows. Hsp104 was used as an internal reference. Figure 2C shows that quantitative yeast LacZ assay was performed as previously described ( Chuang CN, Cheng YH, Wang TF Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis. Nucleic Acids Res. 2012; 40:11416- 11427 ) is executed. Error bars indicate the standard deviation between experiments. Asterisks indicate significant differences, where p -values were calculated using a two-tailed t-test (*, p -value &lt; 0.05 and ***, p -value &lt; 0.001).

Figures 3 A and B show that the SCD of three different S. cerevisiae proteins has "expression-promoting" activity. The indicated yeast expression vectors with the endogenous _RAD51 promoter were used, i.e., _PRAD51 - lacZ -V5-His6 (control), _PRAD51 -Rad51-SCD- lacZ -V5-His6, _PRAD51 - _Rad53 -SCD1- lacZ -V5-His6 or _PRAD51 - _Hop1 -SCD- lacZ -V5 _- His6 to transform wild-type haploid strains. Rad51-SCD and Rad53-SCD1 contain N-terminal 1-66 and 1-29 amino acid residues of Rad51 and Rad53, respectively. Hopl-SCD contains the intermediate region of Hopl (258-324 amino acid residues). Total cell lysates were prepared from exponentially growing mitotic cells and visualized by immunoblotting and quantitative yeast LacZ analysis with the corresponding antisera. Figure 3A shows that immunoblotting was performed using anti-V5 antiserum. Hsp104 was used as an internal reference. Figure 3B shows that a quantitative yeast LacZ (beta-galactosidase) assay was performed as previously described. Error bars indicate the standard deviation between experiments. Asterisks indicate significant differences, where p -values were calculated using a two-tailed t-test (*, p -value &lt; 0.05 and ***, p -value &lt; 0.001).

Figure 4 shows that Rad53-SCD1 expresses four different target proteins on a large scale in S. cerevisiae. The indicated yeast expression vectors, namely, _PRAD51 -lacZ-V5-His6 (control), _PRAD51 -Rad53-SCD1-lacZ-V5 _{- His6} , _PRAD51 - GFP -V5 _- His6 (control) group), _PRAD51 -Rad53-SCD1-GFP-V5-His6, _PRAD51 - GST _- V5-His6 (control group), _PRAD51 -Rad53-SCD1-GST-V5 _{- His6} , _PRAD51 -GST (nd) _-V5 -His6 (control) or P _RAD51 -Rad53-SCD1-GST(nd) _-V5 -His6, wild-type haploid strains were transformed. Beta-galactosidase is a tetrameric protein, while GST is a dimeric protein. GFP is known to be in monomeric form in vivo. GST(nd) is a non-dimerizing GST mutant. Immunoblotting was performed using anti-V5 antiserum. Hsp104 was used as an internal reference.

Figures 5A-F show that the NTD of budding yeast Rad51 is a direct target of Mec1 ^ATR and Tel1 ^ATM . Figure 5A shows the amino acid sequences (1-66 residues) of Rad51-NTD, a phosphorylation-deficient mutant (Rad51-3A) and a phosphorylation-mimicking mutant (Rad51-3D). Figure 5 B, C and D show phosphorylation of Rad51-NTD performed during vegetative growth and meiosis. Rad51-NTD performs ATR ^Mec1 -dependent and ATM ^Tel1 -dependent phosphorylation during vegetative growth and meiosis. Figure 5B shows demonstration of the specificity of anti-phosphorylated Rad51 ^- S2Q, Rad51- ^S12Q and Rad51- ^S30Q antisera. Total cell lysates were prepared from mitotic cells (as shown in Figure 5C) under MMS treatment or from meiotic cells (as shown in Figure 5D) at the indicated sporulation time points, and then by using the corresponding Antiserum immunoblotting method was used for observation. Hsp104 was used as an internal reference. The sizes of standard protein markers in kilodaltons are indicated to the left of the dots. Figure 5E shows that spore viability was analyzed after 3 days on sporulation medium at 30°C. To score spore viability, only tetrads (but not dyads or triads) were dissected on the YPD. Figure 5F shows spot analysis showing that five-fold serial dilutions of the indicated strains grew on YPD plates and YPD plates containing 0.01% or 0.02% MMS.

Figure 6 shows tetrad anatomy analysis. Yeast diploid cells were transformed with the indicated 2μ overexpression vector. RAD51 , rad51-3A and rad51-ΔN were overexpressed under the native RAD51 promoter.

Figures 7 A to B show that Rad51-ΔN is expressed at low levels in meiotic cells. Total cell lysates were prepared from mitotic cells under MMS treatment or at indicated time points from meiotic cultures and subjected to immunoblotting as described in Figure 1. Hsp104 was used as an internal reference. Figure 7A shows that immunoblotting was performed using goat anti-Rad51 antibody (yN-19) purchased from Santa Cruz Biotechnology (CA, USA). This antibody system was generated at the N-terminus of yeast Rad51 using a peptide (sc-8936) map. The 2μ -based overexpression vector carrying the WT RAD51 or rad51-ΔN dual gene was transformed into diploid strains of the indicated genotypes (as shown in Figure ID) and immunoblotting time course using guinea pig anti-Rad51 antibody analyze. Figure 7B shows that the effect of sml1Δ on Rad51-ΔN expression in the indicated meiotic cells was analyzed as described in Figure 7A.

Figures 8 A to E show that Rad51-NTD and its phosphorylation line are essential for only Rad51 meiotic recombination during dmc1Δhed1Δmeiosis . Figure 8A shows that meiotic progression was monitored by nuclear staining with DAPI (4',6-diamidino-2-phenylindole). Cell lines collected from SPM with two or four nuclei (as determined by fluorescence microscopy) were assessed as having completed meiosis I (MI), and relative to each time point such cells are plotted Percentage of total cell count ( n =200). Figures 8B and 8C show cytology. Representative images of meiotic nuclear surface diffusion 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 the indicated sporulation time points. Scale bar, 5 μm. Figure 8D shows quantification of the number of Rad51 and Dmc1 foci in WT and rad51-3A strains. The number of foci in each foci-positive chromosomal smear (with more than five foci) was counted and plotted as indicated. The size of the circle is proportional to the number of nuclei with a given number of foci. The average number of foci per nucleus is shown in red (bottom) and is also shown as a red bar in the figure. The standard deviation of the number of foci is shown in parentheses. N represents the number of nuclei for focus count analysis. P values were calculated using the two-tailed Mann-Whitney's U - test. Figure 8E shows the kinetics of Rad51 foci in meiosis of WT and rad51-3A strains. For each replicate ( n =3), 60 chromosomal smears were examined for Rad51 foci-positive nuclei (with more than five foci). Error bars indicate the standard deviation between experiments. Asterisks indicate significant differences between WT and rad51-3A strains, where P -values were calculated using a two-tailed t-test (* P -value<0.05; ** P -value<0.01 and *** P value < 0.001).

Figures 9 A to D show that phosphorylation-deficient Rad51 maintains insufficient protein levels during meiosis. Figure 9A shows that immunoblotting time course analysis of wild-type and mutant strains was performed as previously described ^21,45 . Total cell lysates were prepared from meiotic cells at the indicated sporulation time points and then visualized by immunoblotting with the corresponding antisera. Antisera to Zip1 and Hopl were included as references for meiotic progression. Hsp104 was used as an internal reference. The sizes of standard protein markers in kilodaltons are indicated to the left of the dots. Asterisks indicate nonspecific bands. Figure 9B shows that, at each time point, the quantification of the protein bands in Figure 9A was normalized to the quantification of Hsp104 using ImageJ software and displayed as relative steady-state levels of the indicated proteins. The highest level of immune dot signal of each dot was used as a comparison standard. Figure 9C shows the estimation of Rad51 protein content of rad51 mutants at 1 hour and 5 hours in SPM using two-fold serial dilutions of WT cell lysates. Figure 9D shows that the protein content of the Rad51 variant from the undiluted lysate shown in Figure 9C was quantified and normalized to Hsp104 content. The Rad51 content of WT in SPM for 1 hour was used as a standard.

Figures 10 A to E show the determination of the half-life of different Rad51 proteins. Figure 10A shows a cycloheximide-shutoff experiment. by 4 hours (as shown in Figure 10A) or 1 hour and 5 hours (as shown in Figure 10D) after the transfer of meiotic cells into the sporulation medium at the indicated time points 200 μg/ml cycloheximide was added to meiotic cultures to inhibit protein synthesis. Untreated (upper panel) or cycloheximide-treated (lower panel) samples were taken for immunoblot analysis at 0, 30, 60, 90, 120, and 180 minutes after cycloheximide addition. Figure 10B shows that immunoblotting time course analysis of WT meiosis revealed the phosphorylation status of native Rad51 and Hop1. Hsp104 was used as an internal reference. Figure 10C shows the relative steady state protein content in Figure 10B is plotted compared to the relative steady state protein content at ₅ hours ( T5 ) after normalization for Hsp104 at each time point (TSPM) in _SPM . Figure 10E shows a graph showing the relative protein content in Figure 10D. Error bars indicate standard deviation between experiments ( n =3). Asterisks indicate _values at TC ₍ hours of cycloheximide treatment) = 3 hours, which are significantly different from the values at TC = 3 hours for WT [ T ₀ =5 hours], where the P value is Calculated using a two-tailed t-test (** P -value<0.01 and *** P -value<0.001).

Figures 11 A to E show that Rad51-3A is degraded by the 26S proteasome. Exponential cultures of the indicated strains were treated with MMS (0.02%) 30 minutes prior to the addition of cycloheximide (200 μg/ml) and/or MG132 (25 μM). As described in Figure 6, protein samples were collected at the indicated time points and immunoblotting was performed. To reduce MG132 efflux, the PDR5 gene encoding the ABC transporter was deleted. Another nutritional housekeeping protein, hexokinase, was analyzed for protein content by immunoblotting using an anti-hexokinase antibody and shown in Figure 11A as an internal reference. Quantification of immunoblotting results in Figures 11 A and D is plotted in Figures 11 C and E, respectively, where total Rad51 protein content normalized to Hsp104 content at that time point is plotted. Quantitative results for WT and rad51-3A samples in the presence of cycloheximide from Figure 11 C are included in Figure 11 E for comparison.

Figures 12A-C show that N-terminal fusions of SCD or prion protein (nucleation) domains promote high level expression of the target protein. Figure 12A shows native Rad51 (NTD-Rad51-ΔN), Rad51-ΔN and Rad51-ΔN fusion proteins visualized by immunoblotting. Hsp104 was used as an internal reference. The sizes of standard protein markers in kilodaltons are indicated to the left of the dots. Black arrows indicate the protein band of Rad51-ΔN. Figure 12B shows MMS sensitivity. Spot analysis showed five-fold serial dilutions of the indicated strains grown on YPD plates in the presence or absence of the indicated concentrations (w/v) of MMS. Figure 12C shows quantitative beta-galactosidase assay. Error bars indicate the standard deviation between experiments (n > 3). Asterisks indicate significant differences, where P values were calculated using a two-tailed t-test (***, P value < 0.001).

Figures 13 A and D show that relative beta-galactosidase (LacZ) activity correlates with the percent STQN amino acid content of IDRs. Figure 13A shows a list of IDRs with respective length, number of S/T/Q/N amino acids, overall STQN percentage and relative β-galactosidase activity. Figures 13B-D show the relative β-galactogens of Rad51-NTD (shown in Figure 13B), Sup35-PND (shown in Figure 13C), and Rad53-SCD1 (shown in Figure 13D) Linear regression between glycosidase activity and overall STQN percentage. The coefficient of determination (R ² ) for each simple linear regression is indicated.

         <![CDATA[<110> ACADEMIA SINICA]]> <![CDATA[<120> Methods and vectors for enhancing protein expression and/or inhibiting its degradation]]> <![CDATA[<130 > none]]> <![CDATA[<160> 31 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211 > 67]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Saccharomyces cerevisiae]]> <![CDATA[<400> 1]]> Met Glu Lys Ala Gly Asn Thr Asn Phe Ile Arg Val Asp Pro Phe Asp 1 5 10 15 Leu Ile Leu Gln Gln Gln Glu Glu Asn Lys Leu Glu Glu Ser Ala Pro 20 25 30 Thr Lys Pro Gln Asn Phe Val Thr Ser Gln Thr Thr Asn Val Leu Gly 35 40 45 Asn Leu Leu Asn Ser Ser Gln Ala Ser Ile Gln Pro Thr Gln Phe Val 50 55 60 Ser Asn Asn 65 <![CDATA[<210> 2]]> <![CDATA[<211> 66]]> <![ CDATA[<212> PRT]]> <![CDATA[<213> Saccharomyces cerevisiae]]> <![CDATA[<400> 2]]> Met Ser Gln Val Gln Glu Gln His Ile Ser Glu Ser Gln Leu Gln Tyr 1 5 10 15 Gly Asn Gly Ser Leu Met Ser Thr Val Pro Ala Asp Leu Ser Gln Ser 20 25 30 Val Val Asp Gly Asn Gly Asn Gly Gly Ser Glu Asp Ile Glu Ala Thr 35 40 45 Asn Gly Ser Gly Asp Gly Gly Gly Leu Gln Glu Gln Ala Glu Ala Gln 50 55 60 Gly Glu 65 <![CDATA [<210> 3]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[ <220>]]> <![CDATA[<223> Artificial Mutants]]> <![CDATA[<400> 3]]> Met Ala Gln Val Gln Glu Gln His Ile Ser Glu Ala Gln Leu Gln Tyr 1 5 10 15 Gly Asn Gly Ser Leu Met Ser Thr Val Pro Ala Asp Leu Ala Gln Ser 20 25 30 Val Val Asp Gly Asn Gly Asn Gly Gly Ser Glu Asp Ile Glu Ala Thr 35 40 45 Asn Gly Ser Gly Asp Gly Gly Gly Leu Gln Glu Gln Ala Glu Ala Gln 50 55 60 Gly Glu 65 <![CDATA[<210> 4]]> <![CDATA[<211> 66]]> <![CDATA[<212> PRT]]> <! 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Thr Met Ala Glu Phe Arg 20 25 30 Arg Val Pro Leu Pro Pro Met Ala Glu Val Pro Met Leu Ser Thr Gln 35 40 45 Asn Ser 50 <![CDATA[<210> 17]]> <![CDATA[< 211> 39]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Saccharomyces cerevisiae]]> <![CDATA[<400> 17]]> Met Ser Asp Ser Asn Gln Gly Asn Asn Gln Gln Asn Tyr Gln Gln Tyr 1 5 10 15 Ser Gln Asn Gly Asn Gln Gln Gln Gly Asn Asn Arg Tyr Gln Gly Tyr 20 25 30 Gln Ala Tyr Asn Ala Gln Ala 35 <![CDATA[<210> 18] ]> <![CDATA[<211> 39]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Artificial Mutants]]> <![CDATA[<400> 18]]> Met Ser Asp Ser Asn Gln Gly Asn Asn Gln Gln Asn Tyr Gln Gln Tyr 1 5 10 15 Ala Gln Asn Gly Asn Gln Gln Gln Gly Asn Asn Arg Tyr Gln Gly Tyr 20 25 30 Gln Ala Tyr Asn Ala Gln Ala 35 <![CDATA[<210> 19]]> <![CDATA[<211> 39]]> <![ CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Artificial mutant]]> <![CDATA[<400> 19]]> Met Ala Asp Ala Asn Gln Gly Asn Asn Gln Gln Asn Tyr Gln Gln Tyr 1 5 10 15 Ala Gln Asn Gly Asn Gln Gln Gln Gly Asn Asn Arg Tyr Gln Gly Tyr 20 25 30 Gln Ala Tyr Asn Ala Gln Ala 35 <![CDATA[<210> 20]]> <![CDATA[<211> 39]]> <![CDATA[<212> PRT]] > <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Artificial Mutant]]> <![CDATA[<400> 20]] > Met Ser Asp Ser Asn Gln Gly Asn Asn Gln Gln Asn Tyr Gln Gln Tyr 1 5 10 15 Ser Gln Asn Gly Asn Ala Ala Ala Gly Asn Asn Arg Tyr Gln Gly Tyr 20 25 30 Gln Ala Tyr Asn Ala Gln Ala 35 <! [CDATA[<210> 21]]> <![CDATA[<211> 39]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![ CDATA[<220>]]> <![CDATA[<223> Artificial Mutants]]> <![CDATA[<400> 21]]> Met Ser Asp Ser Asn Gln Gly Asn Asn Ala Ala Asn Tyr Ala Ala Tyr 1 5 10 15 Ser Ala Asn Gly Asn Gln Gln Gln Gly Asn Asn Arg Tyr Gln Gly Tyr 20 25 30 Gln Ala Tyr Asn Ala Gln Ala 35 <![CDATA[<210> 22]]> <![CDATA[<211 > 39]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Manual Mutant]]> <![CDA TA[<400> 22]]> Met Ser Asp Ser Asn Gln Gly Asn Asn Ala Ala Asn Tyr Ala Ala Tyr 1 5 10 15 Ser Ala Asn Gly Asn Ala Ala Ala Gly Asn Asn Arg Tyr Gln Gly Tyr 20 25 30 Gln Ala Tyr Asn Ala Gln Ala 35 <![CDATA[<210> 23]]> <![CDATA[<211> 39]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequences]]> <![CDATA[<220>]]> <![CDATA[<223> Artificial Mutants]]> <![CDATA[<400> 23]]> Met Ala Asp Ala Asn Ala Gly Asn Asn Ala Ala Asn Tyr Ala Ala Tyr 1 5 10 15 Ala Ala Asn Gly Asn Ala Ala Ala Gly Asn Asn Arg Tyr Ala Gly Tyr 20 25 30 Ala Ala Tyr Asn Ala Ala Ala 35 <![CDATA[<210> 24]] > <![CDATA[<211> 39]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> < ![CDATA[<223> Artificial Mutants]]> <![CDATA[<400> 24]]> Met Ser Asp Ser Ala Gln Gly Ala Ala Gln Gln Ala Tyr Gln Gln Tyr 1 5 10 15 Ser Gln Ala Gly Ala Gln Gln Gln Gly Ala Ala Arg Tyr Gln Gly Tyr 20 25 30 Gln Ala Tyr Ala Ala Gln Ala 35 <![CDATA[<210> 25]]> <![CDATA[<211> 39]]> <![CDATA [<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Artificial Mutant]]> <![CDATA [<400> 25]]> M et Ala Asp Ala Ala Ala Gly Ala Ala Ala Ala Ala Tyr Ala Ala Tyr 1 5 10 15 Ala Ala Ala Gly Ala Ala Ala Ala Gly Ala Ala Arg Tyr Ala Gly Tyr 20 25 30 Ala Ala Tyr Ala Ala Ala Ala 35 <![ CDATA[<210> 26]]> <![CDATA[<211> 114]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Saccharomyces cerevisiae]]> <![CDATA [<400> 26]]> Met Ser Asp Ser Asn Gln Gly Asn Asn Gln Gln Asn Tyr Gln Gln Tyr 1 5 10 15 Ser Gln Asn Gly Asn Gln Gln Gln Gly Asn Asn Arg Tyr Gln Gly Tyr 20 25 30 Gln Ala Tyr Asn Ala Gln Ala Gln Pro Ala Gly Gly Tyr Tyr Gln Asn 35 40 45 Tyr Gln Gly Tyr Ser Gly Tyr Gln Gln Gly Gly Tyr Gln Gln Tyr Asn 50 55 60 Pro Asp Ala Gly Tyr Gln Gln Gln Tyr Asn Pro Gln Gly Gly Tyr Gln 65 70 75 80 Gln Tyr Asn Pro Gln Gly Gly Tyr Gln Gln Gln Phe Asn Pro Gln Gly 85 90 95 Gly Arg Gly Asn Tyr Lys Asn Phe Asn Tyr Asn Asn Ser Leu Gln Gly 100 105 110 Tyr Gln <![CDATA[ <210> 27]]> <![CDATA[<211> 156]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Saccharomyces cerevisiae]]> <![CDATA[< 400> 27]]> Met Pro Pro Lys Lys Phe Lys Asp Leu Asn Ser Phe Leu Asp Asp Gln 1 5 10 15 Pro Lys Asp Pro Asn Leu Val Ala Ser Pro Phe Gly Gly Tyr Phe Lys 20 25 30 Asn Pro Ala Ala Asp Ala Gly Ser Asn Asn Ala Ser Lys Lys Ser Ser 35 40 45 Tyr Gln Gln Gln Arg Asn Trp Lys Gln Gly Gly Asn Tyr Gln Gln Gly 50 55 60 Gly Tyr Gln Ser Tyr Asn Ser Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn 65 70 75 80 Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Lys 85 90 95 Tyr Asn Gly Gln Gly Tyr Gln Lys Ser Thr Tyr Lys Gln Ser Ala Val 100 105 110 Thr Pro Asn Gln Ser Gly Thr Pro Thr Pro Ser Ala Ser Thr Thr Ser 115 120 125 Leu Thr Ser Leu Asn Glu Lys Leu Ser Asn Leu Glu Leu Thr Pro Ile 130 135 140 Ser Gln Phe Leu Ser Lys Ile Pro Glu Cys Gln Ser 145 150 155 <![CDATA[<210> 28]]> <![CDATA[<211> 91]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Saccharomyces cerevisiae]]> <![CDATA[<400> 28]]> Met Met Asn Asn Asn Gly Asn Gln Val Ser Asn Leu Ser Asn Ala Leu 1 5 10 15 Arg Gln Val Asn Ile Gly Asn Arg Asn Ser Asn Thr Thr Asp Gln 20 25 30 Ser Asn Ile Asn Phe Glu Phe Ser Thr Gly Val Asn Asn Asn Asn Asn 35 40 45 Asn Asn Ser Ser Ser Asn Asn Asn Asn Val Gln Asn Asn Asn Ser Gly 50 55 60 Arg Asn Gly Ser Gln Asn Asn Asp Asn Glu Asn Asn Ile Lys Asn Thr 65 70 75 80 Leu Glu Gln His Arg Gln Gln Gln Gln Gln Ala Phe 85 90 <![CDATA[<210> 29]]> <![CDATA[<211> 152]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Saccharomyces cerevisiae]]> <![CDATA[<400> 29]]> Met Val Glu Leu Glu Lys Arg Arg Arg Pro Pro Pro Gln Leu Gln His 1 5 10 15 Ser Pro Tyr Val Arg Asp Gln Ser Asn Ser Gln Gly Met Thr Lys Thr 20 25 30 Pro Glu Thr Ser Pro Pro Lys Arg Pro Met Gly Arg Ala Arg Ser Asn 35 40 45 Ser Arg Ser Ser Gly Ser Arg Ser Asn Val Asp Ile Asp Gln Tyr Thr 50 55 60 Ile Pro Pro Gly Leu Asp Leu Leu Pro Thr Ala Ser Ser Pro Pro Ser 65 70 75 80 Val His Gln Val Ser Gln Gln Gln Gln Leu Ser Pro Ile Leu Ala Asn 85 90 95 Lys Ile Arg Ser Pro Phe Glu Asn Gln Ser Gln Asp Gln Asn Asp Asn 100 105 110 Ser Ile Asp Pro Thr Pro Ala Gly Gln Val Thr Ile Pro Val Glu Ala 115 120 125 Val Ser Pro Pro Ala Leu Asp Glu Leu Ser Lys Phe Gln Asn Gly Ser 130 135 140 Thr Glu Thr Leu Phe Arg Thr Gly 145 150 <![ CDATA[<210> 30]]> <![CDATA[<211> 191]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Saccharomyces cerevisiae]]> <![CDATA [<400> 30]]> Met Ser His Ser Asp Tyr Phe Asn Tyr Lys Pro Tyr Gly Asp Ser Thr 1 5 10 15 Glu Lys Pro Ser Ser Ser Lys Met Arg Gln Ser Ser Ser Ser Ser Ser 20 25 30 Ser Arg Leu Arg Ser Glu Ser Leu Gly Arg Asn Ser Asn Thr Thr Gln 35 40 45 Ala Arg Val Ala Ser Ser Pro Ile Ser Pro Gly Leu His Ser Thr Gln 50 55 60 Tyr Phe Arg Ser Pro Asn Ala Val Tyr Ser Pro Gly Glu Ser Pro Leu 65 70 75 80 Asn Thr Val Gln Leu Phe Asn Arg Leu Pro Gly Ile Pro Gln Gly Gln 85 90 95 Phe Phe His Gln Asn Ala Ile Ser Gly Ser Ser Ser Ser Ser Ala Arg 100 105 110 Ser Ser Arg Arg Pro Ser Asn Ile Gly Leu Pro Leu Pro Lys Asn Pro 115 120 125 Gln Gln Ser Leu Pro Lys Leu Ser Thr Gln Pro Val Pro Val His Lys 130 135 140 Lys Val Glu Ala Ser Lys Thr Glu Ser Glu Ile Ile Lys Lys Pro Ala 145 150 155 160 Pro Val Asn Ser Asn Gln Asp Pro Leu Leu Thr Thr Pro Thr Leu Val 165 170 175 Ile Ser Pro Glu Leu Ala Ser Leu Asn Thr Thr Asn Thr Ser Ile 180 185 190 <![CDATA[<210> 31]]> <![CDATA[<211> 69] ]> <![CDATA[<212> PRT]]> <![CDATA[<213> Saccharomyces cerevisiae]]> <![CDATA[<400> 31]]> Met Ala Gly Phe Ser Phe Ala Lys Lys Phe Thr His Lys Lys His Gly 1 5 10 15 Lys Thr Pro Ser Asp Ala Ser Ile Ser Asp Gln Ser Arg Glu Ala Ser 20 25 30 Leu Ser Thr Pro Asn Glu Lys Phe Phe Thr Lys Gln Glu Thr Pro 35 40 45 Gln Lys Gly Arg Gln Phe Ser Gln Gly Tyr His Ser Asn Val Asn Lys 50 55 60 Thr Ser Ser Pro Pro 65
      

Claims (19)

A method for enhancing the expression of a polypeptide of interest in a host cell and/or inhibiting its degradation, the method comprising the steps of: constructing an expression vector comprising a polynucleotide encoding an intrinsic disordered region (IDR) and encoding The polynucleotide of the polypeptide of interest; transforming the expression vector into the host cell; and culturing the host cell under conditions that allow expression of the polypeptide of interest; wherein the content of S, T and Q accounts for the IDR The overall percentage of total amino acid content is higher than 10%. The method of claim 1, wherein the IDR includes one or more S/T-Q cluster domains (SCDs). A vector comprising an IDR operably linked to a polypeptide encoding a polypeptide of interest, wherein the content of S, T and Q is greater than 10% of the total amino acid content of the IDR. The carrier of claim 3, wherein the IDR comprises one or more SCDs. A host cell comprising the vector of claim 3. A host cell comprising the vector of claim 4. The method of claim 1 or 2, the vector of claim 3 or 4, or the host cell of claim 5 or 6, wherein the SCD or the IDR is fused to the N-terminus of the polypeptide of interest. The method of claim 1 or 2, the vector of claim 3 or 4, or the host cell of claim 5 or 6, wherein the SCDs or the IDRs are used to enhance the expression and/or inhibit the polypeptide of interest Degraded fusion tags. The method of claim 1 or 2, the vector of claim 3 or 4, or the host cell of claim 5 or 6, wherein the polypeptide of interest is a heterologous protein. The method of claim 1 or 2, the vector of claim 3 or 4, or the host cell of claim 5 or 6, wherein the SCD or the IDR is phosphorylated. The method of claim 1 or 2, the vector of claim 3 or 4, or the host cell of claim 5 or 6, wherein the content of S, T and Q as a percentage of the total amino acid content of the IDR is high at 15%. The method of claim 1 or 2, the vector of claim 3 or 4, or the host cell of claim 5 or 6, wherein the content of S, T and Q as a percentage of the total amino acid content of the IDR is high at 20%. The method of claim 1 or 2, the vector of claim 3 or 4, or the host cell of claim 5 or 6, wherein the content of S, T and Q as a percentage of the total amino acid content of the IDR is high at 30%. The method of claim 1 or 2, the vector of claim 3 or 4, or the host cell of claim 5, wherein the content of S, T and Q as a percentage of the total amino acid content of the IDR is about 30% % to about 40%. The method of claim 1 or 2, the vector of claim 3 or 4, or the host cell of claim 5, wherein one or more S, T or Q residues in the IDR are via an amine having a hydrophobic side chain base acid, amino acid with negatively charged side chain or replaced by another amino acid of S, T and Q. The method of claim 1 or 2, the vector of claim 3 or 4 or the host cell of claim 5 or 6, wherein the IDR or the SCD is selected from the group consisting of SEQ ID Nos: 1 to 31. The method of claim 1 or 2 or the host cell of claim 5 or 6, wherein the host cell is a prokaryotic cell. The method of claim 1 or 2 or the host cell of claim 5 or 6, wherein the host cell is a mammalian cell, a yeast cell, a fungal cell, an insect cell, a plant cell or a protozoan cell. The method of claim 1 or 2 or the host cell of claim 5 or 6, wherein the host cell is Saccharomyces cerevisiae .

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