WO2012087835A2 - Compositions et procédés pour favoriser le repliement d'une protéine - Google Patents

Compositions et procédés pour favoriser le repliement d'une protéine Download PDF

Info

Publication number
WO2012087835A2
WO2012087835A2 PCT/US2011/065563 US2011065563W WO2012087835A2 WO 2012087835 A2 WO2012087835 A2 WO 2012087835A2 US 2011065563 W US2011065563 W US 2011065563W WO 2012087835 A2 WO2012087835 A2 WO 2012087835A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
bag
predicted
polypeptide
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2011/065563
Other languages
English (en)
Other versions
WO2012087835A3 (fr
Inventor
Shinichi Takayama
Akinori HISHIYA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boston Biomedical Research Institute Inc
Original Assignee
Boston Biomedical Research Institute Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boston Biomedical Research Institute Inc filed Critical Boston Biomedical Research Institute Inc
Publication of WO2012087835A2 publication Critical patent/WO2012087835A2/fr
Publication of WO2012087835A3 publication Critical patent/WO2012087835A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag

Definitions

  • the present invention features compositions and methods that enhance protein folding.
  • the invention features a fusion polypeptide contains a BAG domain that interacts with an Hsp70 molecular chaperone linked to a heterologous polypeptide.
  • the BAG domain is derived from a mammalian BAG1 or BAG3 polypeptide.
  • the BAG domain comprises amino acids 419-536 or 388-500 of human BAG3.
  • the heterologous polypeptide is cystic fibrosis transmembrane conductance regulator (CFTR), IL13 receptor a2 protein (IL13Ra2), or antitrypsin (al-AT).
  • the invention provides a polynucleotide encoding a BAG domain that interacts with an Hsp70 molecular chaperone.
  • the invention provides an expression vector contains a promoter suitable for expression in a mammalian cell operably linked to a polynucleotide sequence encoding a fusion polypeptide contains a BAG domain that interacts with an HSP70 molecular chaperone linked to a heterologous polypeptide.
  • the polynucleotide sequences encoding the BAG domain and the heterologous polypeptide are linked by a nucleic acid linker contains between about 5 and 25 nucleic acids.
  • the BAG domain is attached to the amino terminus or the carboxy terminus of the heterologous polypeptide, or wherein the BAG domain is positioned between the amino and carboxy termini of the heterologous polypeptide.
  • the BAG domain is linked to the heterologous polypeptide by a linker contains a protease-sensitive site.
  • the invention features a host cell contains the expression vector of a previous aspect.
  • the invention features a method of producing a BAG fusion protein, the method involving the steps of expressing in a host cell, a polynucleotide encoding a fusion protein of a previous aspect; and culturing the host cell under conditions appropriate for production of the fusion protein.
  • the invention features a method of treating a subject having a disease associated with the expression of a misfolded protein, the method involving expressing in a cell of the subject, a polynucleotide encoding a fusion protein of a previous aspect; and expressing the protein in the cell under conditions appropriate for production of the fusion protein, thereby treating the subject.
  • compositions featuring a BAG family protein and methods of using such compositions to enhance protein folding or otherwise stabilize an exogenously expressed protein.
  • Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims. Definitions
  • agent any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
  • analog is meant a molecule that is not identical, but has analogous functional or structural features.
  • a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical
  • An analog may include an unnatural amino acid.
  • BAG domain is meant a protein interaction domain having at least about 85% amino acid sequence identity or having similarity to a BAG domain of BAGl, 2, 3, 4, 5, or 6, or a fragment thereof capable of mediating protein -protein interactions.
  • a BAG domain has at least 85%, 95%, or even 100% identity to a BAG domain set forth in Figure 11.
  • a BAG domain mediates interactions with Hsp70 molecular chaperones, and includes any BAG domains recognized within members of the BAG polypeptide super family.
  • a "BAG domain” may refer to a BAG domain with a WW domain (similar to that found in the BAG3 polypeptide family), a BAG domain with a BAGl type sequence for BAGl N-terminal ubiquitin like domain (similar to that found in the BAGl polypeptide family), a BAG domain with an LMBR1 domain (present only in non-mammalian BAG super family members), or a BAG domain with no known representative protein domain.
  • An exemplary "BAG domain” is represented by amino acids 419-500 of the human BAG3 protein (GenBank Accession GL38502170), having the sequence shown below:
  • a "BAG3 family polypeptide” is a polypeptide having at least 85% amino acid sequence identity or similarity to human BAG3 protein (GenBank Accession GL38502170) or a fragment thereof capable of mediating protein -protein interactions.
  • a BAG3 polypeptide has the exemplary sequence shown below:
  • BAG1 family polypeptide is a polypeptide having at least 85% amino acid sequence identity or similarity to GenBank Accession GL288915525 or a fragment thereof capable of mediating protein-protein interactions.
  • a BAG1 polypeptide has the exemplary sequence shown below:
  • Bagl polypeptides are described, for example, in Takayama et al., Cell 80: 279-284, 1995; Takayama et al., Genomics 35: 494-498, 1996; Takayama et al., J. Biol. Chem. 274: 781-786, 1999; and Takayama et al., J Biol Chem. 1999 Jan 8;274(2):781-6.
  • Detect refers to identifying the presence, absence or amount of the analyte to be detected.
  • detectable label is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • an effective amount is meant the amount of an active compound required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • the invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein.
  • the methods of the invention provide a facile means to identify therapies that are safe for use in subjects.
  • the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high- volume throughput, high sensitivity, and low complexity.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • Hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
  • inhibitory nucleic acid is meant a double- stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene.
  • a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule.
  • an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.
  • isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • linker is meant an amino acid sequence that joins a protein of interest and a bag domain.
  • marker any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
  • obtaining as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
  • Primer set means a set of oligonucleotides that may be used, for example, for PCR.
  • a primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.
  • reference is meant a standard or control condition.
  • a “reference sequence” is a defined sequence used as a basis for sequence
  • a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.
  • the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
  • siRNA is meant a double stranded RNA.
  • an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end.
  • These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream.
  • Such siRNAs are used to downregulate mRNA levels or promoter activity.
  • telomere binding By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.
  • Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule.
  • hybridize is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
  • BLAST program may be used, with a probability score between e "3 and e "100 indicating a closely related sequence.
  • subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
  • the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • Figure 1 shows the sequence of human BAG3 protein (GenBank Accession
  • Figure 2 shows the sequence of BAG domain containing subfragments 419-536 (Panel A) and 388-500 (Panel B) of human BAG3 protein (GenBank Accession
  • Figure 3 shows the alignment of BAG domains present in exemplary BAG1 polypeptide family members.
  • Figure 4 shows the alignment of BAG domains present in exemplary Hsp70 regulators.
  • Figure 5 shows alignment data for BAG domains that display high levels of identity to the BAG domain of human BAG3 protein (GenBank Accession GL38502170).
  • FIG. 6 is a schematic diagram illustrating the Folding Tag system for enhancing protein folding.
  • the gene coding the amino acid sequence of the molecular chaperone interacting domain of BAG protein (FOLDING TAG) must be attached to genes of interest, therefore, expressed proteins are fusion proteins with the FOLDING TAG.
  • the expressed protein correctly folded in cells, to escape from the degradation by proteasome, thus leading to an enhancement of stability and an increased number of functional proteins.
  • Figure 7 is a Western blot showing CFTR protein expression in human cells.
  • Figure 8 shows the intracellular localization of CFTR in human cells. Nuclei are visualized with DAPI.
  • Figure 9 is a graph showing CFTR activity as measured by fluorescence intensity in response to forskolin and KSCN. The activity of the cells that recombinantly express CFTR is shown relative to wild-type and mock transfected.
  • Figure 10 is a Western blot showing expression of IL3Ra2 in human cells.
  • Figure 11 is a Western blot showing expression of alpha 1 anti-trypsin in human cells.
  • Figure 12 is the amino acid alignment of human BAG family protein (BAG1-6) using clustalW alignment software.
  • Figure 13 provides amino acid and polynucleotide sequences of N-terminal (F) and C- terminal (R) Folding Tags.
  • Figure 14 provides amino acid and polynucleotide sequences of CFTR, IL13 receptor a2 protein (IL13Ra2), or anti-trypsin (al-AT).
  • the invention features compositions and methods that are useful for enhancing protein folding.
  • the invention is based, at least in part, on the discovery that BAG3 functions in the proper folding and stabilization of binding proteins in vivo through the acceleration of protein folding. Stability and functionality of interacting proteins are regulated by the C- terminus of BAG3, where Hsp70/Hsc70 interacts.
  • the invention provides a fusion protein comprising a protein of interest linked to a BAG domain, wherein the BAG domain enhances both the stabilization and function of the protein.
  • Compositions and methods described herein are useful for generating recombinant proteins that can be used for research or therapeutic purposes.
  • vectors encoding such proteins are useful in polynucleotide therapy in vivo.
  • BAG family proteins contain a BAG domain that interacts with Hsp70 molecular chaperones, thereby accelerating the protein-folding cycle of Hsp70 molecular chaperones.
  • BAG family proteins also contain a diverse array of additional domains, which allow them to interact with specific target proteins or which target them to specific locations within cells. Based on the results reported herein, BAG family proteins likely act as bridging molecules that recruit molecular chaperones to target proteins, presumably modulating protein function through alterations in their conformation.
  • the Hsp70/Hsc70 interacting motif in BAG family proteins regulates protein folding by recruiting molecular chaperone complex, Hsp70/Hsc70 and co-chaperone proteins.
  • BAG family proteins associate with the ubiquitin proteasome system to regulate the folding or degradation decision.
  • the tag sequence accelerates folding and/or prevents activation of the ubiquitin proteasome degradation pathway.
  • the amino acid sequence of BAG protein is attached to the protein of interest, and the protein is expressed as a fusion protein with a part of BAG sequence (termed a "FOLDING TAG").
  • the fusion protein is expressed in cells, and specifically folded with the aid of the FOLDING TAG peptides.
  • Figure 13 provides Folding Tag sequences. One is for the N-terminal tag (F) and the other is for the C-terminal tag (R). BAG domain in nucleotides (font with blue background) and in amino acid (green
  • the present invention is useful not only for in vitro methods of enhancing the expression of recombinant proteins, but is also useful for enhancing protein conformation in diseases associated with abnormal protein folding.
  • Abnormal protein folding is observed in many conformational diseases or degenerative diseases. Misfolded or Unfolded proteins causes problems in which 1) the protein loses an original function, 2) the protein is unstable, or 3) the protein has an unexpected function, which is toxic to cells. These problems cause serious outcomes, especially when the protein is expressed exogenously.
  • molecular chaperones have been co-expressed with the target proteins.
  • molecular chaperone proteins are involved in protein folding and stressed condition induces molecular chaperones
  • exogenous expression of molecular chaperones or pre-treatment of cells with stress have been used for increasing protein folding 2"6 .
  • the protein of interest is not successfully folded, however, because of problems in the specificity of recognition by exogenously expressed chaperones or stress-induced chaperones.
  • Many tumor cells and cancer cells express more chaperone proteins than under normal conditions, and the expression of chaperone proteins in those cells may make the cells resistant to various stresses such as apoptosis.
  • activation of molecular chaperone itself may in some cases have harmful side effects, such as tumorigenesis when used as gene therapy 7- " 9.
  • the present invention advantageously avoids these drawbacks because the direct attachment of the FOLDING TAG to a protein of interest increases correct folding by recruiting the
  • proteasome inhibitors were sometimes used to prevent degradation of exogenously over-expressed proteins.
  • the inhibitor of the proteasome was used in the cell culture medium 10 .
  • Unfolded or abnormally folded proteins are degraded by a protein complex called proteasome.
  • An inhibitor of proteasome such as MG132, increases the stability of proteins.
  • using a proteasome inhibitor to increase production of a particular protein is not very effective.
  • Proteasome inhibitors are not specific to specific target proteins and are sometimes very toxic to cells, because of inhibition of cell division related proteins.
  • MG132 only inhibits protein degradation, it does not increase proper protein folding.
  • proteasome inhibitor was used to inhibit the degradation of CFTR protein in vitro, but stabilized CFTR protein doesn't work properly in cells 11 ' 12.
  • the present invention directly accesses the protein folding problem, and in fact, as reported in more detail below CFTR protein expressed with a Folding Tag was functional.
  • the invention provides compositions and methods to enhance function and stabilization of a given protein, and therefore has broad application in many fields. For example, in 1) gene therapy, 2) the production of recombinant protein, 3) experimental protein expression.
  • Polynucleotide therapy is useful to treat not only a recessive genetic disorder, but also various other diseases such as cancer.
  • a given protein is expressed in a human tissue or in specific cells.
  • Most of the effort has been put into developing a delivery system and high expression system in the target tissue or cells.
  • polynucleotide therapy research has focused on the "posttranslational problem".
  • many proteins are degraded due to inappropriate protein conformation at the post-translational level.
  • increasing protein folding is one of the most important problems to be solved in polynucleotide therapy.
  • the present invention enhances the expression of the given protein, since the fusion protein recruits the molecular chaperone complex to enhance proper folding.
  • the expressed protein is a fusion protein and not "a native protein", the TAG sequence itself is derived from human cytoplasmic protein, BAG3. Therefore, the fusion polypeptide is unlikely to be immunogenic.
  • a recombinant protein is a protein, which is translated from a recombinant DNA.
  • Molecular biology techniques enable the production of recombinant proteins in a variety of cell types (e.g., bacteria, yeast, insect cells and mammalian cells).
  • Virtually any recombinant protein can be produced using the methods of the invention, including but not limited to antibodies, antigens, hormones, therapeutic drugs, enzymes, probes for molecular screening of interacting partners or small molecules modifying the activity of the recombinant protein in vitro. Since the posttranslational modification of proteins depends on each species, bacterial recombinant protein derived from mammalian DNA has some limitation of bioactivity due to physiological modification. Over the last two decades, much effort has been made to enhance the productivity of recombinant protein in mammalian cells.
  • proteasome inhibitors do not solve the folding problem, since the proteasome degradation pathway is activated by inappropriate folding.
  • proteasome inhibitors are not appropriate for "secreted proteins” and for “stable cell lines” which express the recombinant protein permanently.
  • the present invention solves the protein folding problem of recombinant protein in a physiological way and enables the stabilization of the protein.
  • the fusion protein is purified using methods known in the art. Such methods include, but are not limited to affinity purification using an antibody against bag domain or an additional tag (myc or flag etc).
  • the recombinant protein is affinity purified on a protein column conjugating ATPase domain of Hsc70 because of high affinity interaction between ATPase domain of Hsc70 and BAG domain.
  • the TAG protein can be cleaved off with a protease recognizing the peptides sequence between the TAG and the protein of interest, for separation of only target proteins.
  • the ATPase domain of Hsc70 is highly stable as a bacterial recombinant protein and is purified using a GST fusion protein or equivalent methods (His tag etc). Following purification, the purified protein is cross linked on sepharose or agarose beads for column production. After affinity purification of BAG domain (FOLDING TAG) with this column, the addition of ATP will wash BAG domain (FOLDING TAG) off of the column and regenerate the Hsc70ATPase column. To make the column efficient, short fragments of Hsc70ATPase (mini-domain), which is sufficient for interacting with BAG domain may be used 21.
  • the Hsc70ATPase column is an inexpensive, specific association with BAG TAG and easy to reuse. This provides significant advantages relative to antibody based purification methods.
  • the present invention is useful for stable protein expression, and accordingly provides expression vectors, which contain the nucleotide sequence for the TAG with the multi cloning restriction enzyme sites inserted in front or after the TAG. These vectors may be used for expression of the TAG in bacteria, yeast, insects and mammalian cells.
  • the invention further provides methods of optimizing the expression vector constructs to ensure stable protein production. Methods for adjusting the number of nucleotides between the FOLDING TAG and the target protein are known in the art.
  • the present invention provides methods of treating diseases characterized by the presence of misfolded protein or a reduction in the level of correctly folded protein, and/or disorders or symptoms thereof.
  • Methods of the invention comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a recombinant polynucleotide or protein of the invention to a subject (e.g., a mammal such as a human).
  • a subject e.g., a mammal such as a human.
  • a subject e.g., a mammal such as a human
  • a subject e.g., a mammal such as a human.
  • a method of treating a subject suffering from or susceptible to a disease characterized by the presence of misfolded protein or a reduction in the level of correctly folded protein or disorder or symptom thereof includes the step of administering to the mammal a therapeutic amount of an amount of a polynucleotide or polypeptide described herein in an amount sufficient to treat
  • the methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
  • the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • the therapeutic methods of the invention in general comprise administration of a therapeutically effective amount of the polynucleotides or polypeptides described herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human.
  • a subject e.g., animal, human
  • Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).
  • the compounds herein may be also used in the treatment of any other disorders in which protein folding defects may be implicated.
  • the invention provides a method of monitoring treatment progress.
  • the method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with the presence of misfolded protein or a reduction in the level of correctly folded protein, in which the subject has been administered a therapeutic amount of a protein or polynucleotide herein sufficient to treat the disease or symptoms thereof.
  • the level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status.
  • a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy.
  • a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.
  • Expressed proteins are fusion proteins, part of which contain BAG3, which recruits the protein folding machinery (Hsp70/Hsc70 and co-chaperone, Hdj proteins).
  • the distance between a BAG3 tag and the protein of interest was important for the stabilization of the fusion protein. Since all BAG domain proteins bind to the Hsc70 molecular chaperone and regulate folding activity, all BAG family proteins are likely to have similar activity.
  • the present invention provides fusion proteins comprising a BAG family protein or fragment thereof.
  • BAG proteins and fragments thereof may be derived from mammalian or non- mammalian BAG family proteins.
  • Example I An Hsc70 interacting domain of BAG3 for use in generating a fusion protein.
  • cystic fibrosis transmembrane conductance regulator CFTR
  • the amino acid and polynucleotide sequences of CFTR are provided at Figure 14A
  • the amino acid and polynucleotide sequences of IL13 receptor a2 protein IL13Ra2 protein
  • the amino acid and polynucleotide sequences of al anti-trypsin (al-AT) are provided at Figure 14C.
  • the polynucleotide sequence encoding CFTR was inserted into Myc-(F)- pcDNA3 in frame using two restricted enzyme sites, Not I and Xho I (Myc-(F)-CFTR- pcDNA3).
  • the nucleic acid sequence encoding IL13Ra2 and al-AT was inserted into (F)-Flag- pcDNA3 in frame with Kpn I and Xho I or EcoR I and Xho I, respectively (IL13Ra2-(F)- Flag-pcDNA3 and al-AT-(F)-Flag-pcDNA3).
  • the Myc tag or Flag tag was also conjugated to the N-terminus or C-terminus of the amino acid sequence to provide for detection of the fusion protein product. If desired, such tags (Flag and Myc) can be omitted.
  • the antibody against the FOLDING TAG sequence can also be used to detect the expressed protein.
  • the position of the FOLDING TAG and a linker size between the FOLDING TAG and a given protein should be considered may be varied with each protein.
  • Figure 13 provides an exemplary sequence of a folding tag. In one
  • 33-38 amino acids is added to the BAG domain.
  • the linker may be between 15-20, 20-30, 30-40, 40-50, or 50-60 amino acids in length.
  • a linker is an additional sequence that links an insert and a BAG domain.
  • the length may be varied to optimize the folding function of Hsp70 as it relates to accessibility of the protein to be folded.
  • the FOLDING TAG length can be varied. In one embodiment, the
  • FOLDING TAG is shortened to generate a minimum peptide capable of recruiting a folding complex.
  • the sequence is varied to create a FOLDING TAG that can be cleaved from the fusion protein.
  • the position of the tag is varied. For certain applications, it may be preferred to have the FOLDING TAG at the C- terminus.
  • a C-terminal tag may be preferred for a protein with an N- terminal signal sequence or other important function at N-terminus (see example #3).
  • alternate BAG family proteins from different species may be used.
  • candidate BAG family members may include, but are not limited to, those shown in Figures 3, 4, and 5.
  • Additional candidate BAG family members may include, but are not limited to, those listed in Tables 1-4.
  • a BAG protein is selected from the same species as the host cell used to express the recombinant protein and different target proteins.
  • the amino acid sequence of the FOLDING TAG is optimized to modify the FOLDING TAG's activity. An overview of this process is shown in Figure 6.
  • PREDICTED LOW QUALITY PROTEIN: BAG family molecular chaperone regulator 3- like [Pongo abelii] 575 aa protein XP_002821250.1 GL297687501
  • BAG family molecular chaperone regulator 3 [Macaca mulatta] 575 aa protein XP_001104160.2 GL297301961
  • BCL2-associated athanogene 3 [Bos taurus] 585 aa protein DAA14707.1 GL296472592
  • BCL2-associated athanogene 3 [Xenopus (Silurana) tropicalis] 562 aa protein
  • BAG family molecular chaperone regulator 3 [Bos taurus] 585 aa protein
  • BAG family molecular chaperone regulator 3 [Mus musculus] 577 aa protein
  • BCL2-associated athanogene 3 variant [Homo sapiens] 575 aa protein BAD96520.1 GL62897159
  • BAG family molecular chaperone regulator 3 [Rattus norvegicus] 574 aa protein NP_001011936.1 GL58865450
  • BAG family molecular chaperone regulator 3 [Homo sapiens] 575 aa protein
  • Bcl-2-binding protein BIS [Homo sapiens] 575 aa protein AAF26839.1 GL6724086
  • BAG-family molecular chaperone regulator-3 [Homo sapiens] 575 aa protein
  • melanoleuca 279 aa protein XP_002926223.1 GI:301781606
  • BCL-2-associated athanogene 2 [Arabidopsis lyrata subsp. lyrata] 282 aa protein
  • Os08g0546100 [Oryza sativa Japonica Group] 501 aa protein NP_001062419.2
  • Os06g0126500 [Oryza sativa Japonica Group] 339 aa protein
  • BAG family molecular chaperone regulator 1 isoform 1 [Macaca mulatta] 232 aa protein XP_001090384.2 GL297270768
  • BAG family molecular chaperone regulator 1 isoform BAG- IS [Homo sapiens] 230 aa protein NP_001165886.1 GL288915527
  • BAG family molecular chaperone regulator 1 isoform BAG-1L [Homo sapiens] 345 aa protein NP_004314.5 GL288915525
  • protein binding protein [Zea mays] 320 aa protein NP_001151280.1 GL226495281
  • BAG domain protein [Penicillium marneffei ATCC 18224] 421 aa protein
  • BAG-1-associated athanogene 1 BAG-1 (Glucocorticoid receptor-associated protein RAP46) [Equus caballus] 320 aa protein XP_001917762.1 GL194224914
  • ATBAG4 (ARABIDOPSIS THALIANA BCL-2- ASSOCIATED ATHANOGENE 4); protein binding [Arabidopsis thaliana] 269 aa protein NP_190746.2 GL186510943
  • ATBAG1 (ARABIDOPSIS THALIANA BCL-2- ASSOCIATED ATHANOGENE 1); protein binding [Arabidopsis thaliana] 342 aa protein NP_200019.2 GI: 145359142
  • hypothetical protein CNBB2190 [Cryptococcus neoformans var. neoformans B-3501A] 276 aa protein XP_777418.1 GI:134108072
  • Os09g0524800 [Oryza sativa Japonica Group] 334 aa protein NP_001063716.1
  • Os04g0619900 [Oryza sativa Japonica Group] 272 aa protein NP_001053903.1
  • Os01g0831200 [Oryza sativa Japonica Group] 262 aa protein NP_001044700.1
  • BAG1 protein [Homo sapiens] 310 aa protein AAH14774.2 GI:110611785
  • BAG domain-containing protein [Oryza brachyantha]
  • BAG family molecular chaperone regulator 1 [Suberites domuncula] 258 aa protein CAJ65915.1 GL110162114
  • BAG-family molecular chaperone regulator- 1 BCL-2 binding athanogene-1
  • BAG-1 Glucocorticoid receptor-associated protein RAP46
  • BCL2-associated athanogene [Homo sapiens] 207 aa protein CAH72518.1 GL55661642
  • ATBAG2 (ARABIDOPSIS THALIANA BCL-2- ASSOCIATED ATHANOGENE 2); protein binding [Arabidopsis thaliana] 285 aa protein NP_568950.2 GL30697630
  • BAG family molecular chaperone regulator BaglOl Schizosaccharomyces pombe
  • BAG1 human homolog family member [Caenorhabditis elegans] 210 aa protein NP_491893.1 GL 17507755
  • ATBAG3 (ARABIDOPSIS THALIANA BCL-2- ASSOCIATED ATHANOGENE 3); protein binding [Arabidopsis thaliana] 303 aa protein NP_196339.1 GI: 15240726
  • glucocortoid receptor-associated protein RAP46 Homo sapiens 274 aa protein
  • LMBR1 domain-containing protein [Paracoccidioides brasiliensis PbOl] 1035 aa protein XP_002795957.1 GL295670820 2. conserved hypothetical protein [Uncinocarpus reesii 1704] 1098 aa protein XP_002582789.1 GL258564088
  • LMBRl domain protein [Aspergillus flavus NRRL3357] 771 aa protein XP_002383137.1 GI:238503810
  • BAG domain-containing protein [Glomerella graminicola Ml.001] 323 aa protein
  • BAG domain-containing protein [Verticillium albo-atrum VaMs.102] 381 aa protein XP_003000558.1 GL302405443
  • melanoleuca 569 aa protein XP_002918888.1 GL301766948
  • melanoleuca 545 aa protein XP_002915457.1 GL301759215
  • BAG family molecular chaperone regulator 1 [Xenopus (Silurana) tropicalis] 224 aa protein XP_002939543.1 GL301620355
  • BCL-2-associated athanogene 5 [Arabidopsis lyrata subsp. lyrata] 212 aa protein
  • BCL-2-associated athanogene 6 [Arabidopsis lyrata subsp. lyrata] 1050 aa protein XP_002880227.1 GL297824689
  • BAG domain-containing protein [Ajellomyces dermatitidis SLH14081] 482 aa protein XP_002622007.1 GL261191198
  • protein binding protein putative [Ricinus communis] 152 aa protein XP_002530491.1 GL255579292
  • BAG domain protein [Talaromyces stipitatus ATCC 10500] 326 aa protein
  • BAG protein [Ajellomyces capsulatus H143] 492 aa protein EER45017.1 GL240281514
  • BAG domain-containing protein [Ajellomyces dermatitidis ER-3] 464 aa protein EEQ83829.1 GL239606842
  • BAG domain-containing protein [Ajellomyces capsulatus G186AR] 488 aa protein EEH04941.1 GL225556653
  • BCL-2 associated athanogene 3-like protein [Piriformospora indica] 279 aa protein ACN76856.1 GL224995894
  • BAG family molecular chaperone regulator 5 [Salmo salar] 252 aa protein ACN58723.1 GL224587836
  • BAG family molecular chaperone regulator 1 A [Schizosaccharomyces japonicus yFS275] 203 aa protein XP_002175892.1 GL213410244
  • BAG family molecular chaperone regulator IB [Schizosaccharomyces japonicus yFS275] 238 aa protein XP_002172980.1 GL213404416
  • BCL2-associated athanogene 5 [Xenopus (Silurana) tropicalis] 450 aa protein
  • GK17074 [Drosophila willistoni] 602 aa protein XP_002061647.1 GL195427161
  • GJ13715 [Drosophila virilis] 615 aa protein XP_002047962.1 GL195378380
  • GI13926 [Drosophila mojavensis] 603 aa protein XP_002009228.1 GL195129569
  • BAG family molecular chaperone regulator 2 [Rattus norvegicus] 210 aa protein NP_001121667.1 GL189491875
  • BAG domain protein [Aspergillus fumigatus A1163] 393 aa protein EDP55944.1 GL159130831
  • BAG domain protein [Aspergillus fumigatus Af293] 393 aa protein XP_001481711.1 GI: 146322491
  • BCL2-associated athanogene 2 [Homo sapiens] 108 aa protein CAM28256.1
  • BAG domain protein [Aspergillus clavatus NRRL 1] 430 aa protein XP_001269553.1 GI: 121702577
  • BAG domain protein [Neosartorya fischeri NRRL 181] 393 aa protein XP_001265061.1 GI: 119496575
  • BAG-2 isoform 1 [Pan troglodytes] 175 aa protein XP_001158291.1 GI:114607983
  • BAG family molecular chaperone regulator 2 isoform 3 [Macaca mulatta] 211 aa protein XP_001110620.1 GL109071591
  • BAG family molecular chaperone regulator 5 [Bos taurus] 447 aa protein
  • BAG family molecular chaperone regulator 2 [Bos taurus] 211 aa protein
  • UNCoordinated family member [Caenorhabditis elegans] 457 aa protein NP_001024011.1 GL71997311
  • BAG family molecular chaperone regulator 5 isoform a [Homo sapiens] 488 aa protein NP_001015049.1 GL62548856 200.
  • BAG family molecular chaperone regulator 5 [Mus musculus] 447 aa protein
  • BAG family molecular chaperone regulator 5 [Rattus norvegicus] 447 aa protein NP_001008526.1 GL56606102
  • BCL2-associated athanogene 2 [Xenopus (Silurana) tropicalis] 213 aa protein
  • BCL2-associated athanogene [Homo sapiens] 60 aa protein CAH72520.1 GL55661644
  • Chain A Structural Genomics Of Caenorhabditis Elegans: Structure Of Bag-1 Protein 137 aa protein 1T7S_A GL49259265
  • BAG2 Homo sapiens
  • 211 aa protein CAG38527.1 GL49065418
  • UNCoordinated family member [Caenorhabditis elegans] 399 aa protein NP_872142.1 GL32566728
  • BCL2-associated athanogene 5 [Homo sapiens] 447 aa protein AAH50551.1
  • starvin, isoform B [Drosophila melanogaster] 516 aa protein NP_729912.1 GL24663868 219.
  • starvin, isoform C [Drosophila melanogaster] 542 aa protein NP_729911.1 GL24663865
  • BAG family molecular chaperone regulator 2 [Mus musculus] 210 aa protein
  • BAG family molecular chaperone regulator Bagl02 [Schizosaccharomyces pombe 972h-] 206 aa protein NP_595316.1 GL19112108
  • UNCoordinated family member [Caenorhabditis elegans] 458 aa protein NP_505307.1 GI: 17564906
  • BAG6 BCL-2-ASSOCIATED ATHANOGENE 6
  • calmodulin binding / protein binding [Arabidopsis thaliana] 1043 aa protein NP_182147.1 GL15225945
  • ATBAG5 (ARABIDOPSIS THALIANA BCL-2- ASSOCIATED ATHANOGENE 5); protein binding [Arabidopsis thaliana] 215 aa protein NP_172670.1 GI:15221182
  • Chain B Crystal Structure Of A Bag Domain In Complex With The Hsc70 Atpase Domain 114 aa protein 1HX1_B GL13399492
  • BAG family molecular chaperone regulator 5 isoform b [Homo sapiens] 447 aa protein NP_004864.1 GL6631077
  • BAG family molecular chaperone regulator 2 [Homo sapiens] 211 aa protein
  • KIAA0873 protein [Homo sapiens] 466 aa protein BAA74896.1 GL4240235
  • a pcDNA3 with a Myc tag (Myc-pcDNA3) was generated.
  • the gene for Myc tag was inserted into pcDNA3 (Invitrogen) using Hind III and BamH I sites.
  • the cftr gene or its mutant (A508F ) was inserted into Myc-pcDNA3 using Not I and Xho I sites (Myc-CFTR- pcDNA3 or Myc-CFTR A508F -pcDNA3).
  • the gene for the amino acids (human bag3 gene amino acid 419-536: indicated by (S) in the constructs) was inserted into Myc-pcDNA3 with two restriction sites (BamH I and EcoR I), then the cftr gene was inserted into it using Not I and Xho I sites (Myc-(S)-CFTR-pcDNA3 or Myc-(S)-CFTR D508F -pcDNA3).
  • the plasmids for Myc- CFTR, Myc-(S)-CFTR, Myc-CFTR A508F, and Myc-(S)-CFTR A508F were transfected to HEK293 cells with pEGFP-N2 plasmid (Clontech), and cultured for two days. Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail and PMSF.
  • RIPA buffer 50 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS
  • Example 3 Intracellular localization of CFTR in human cells
  • the plasmid for Myc-CFTR-pcDNA3 or Myc-(S)-CFTR-pcDNA3 was transfected into HEK293 as described above.
  • cells were washed with PBS, and fixed with 4% paraformaldehyde in PBS for 5 minutes. After three 5-minute washes with PBS, the fixed samples were incubated in 0.2% Triton X-100 in PBS for 3 minutes and blocked with 2% BSA in PBS for 1 hour. The antibody against Myc tag was incubated with the samples for 1 hour at room temperature.
  • HEK293 cells were cultured onto 96-well plate. The cultured cells were transfected with the plasmid for Myc -CFTR-pcDN A3 or Myc-(S)-CFTR-pcDNA3, and incubated for an additional two days. Cells were loaded with SPQ (6-methoxy-iV-(3-sulfopropyl)
  • Soluble IL13Ra2 may be used to inhibit inflammation (e.g., in connection with asthma), or to reduce fibrosis (pulmonary fibrosis, hepatic fibrosis etc).
  • Generating recombinant proteins in mammalian cells offers several advantages over other expression systems. Most of recombinant proteins require complicated post- translational modifications such as glycosylation for their function, which cannot be attained or are not accurate in other host cells, such as E. coli or yeast. Despite their slower growth rates and requirements of complex nutrition, mammalian cells have grown as a major host cell for generating complex recombinant proteins 25. However, mammalian cells have strict intracellular quality control systems for protein production, in which misfolded or unfolded proteins are rapidly degraded in cells 26 . This system could lead to extremely low efficiency of recombinant protein generation. Therefore, attempting enhancement of protein folding has been one of the major issues in this field.
  • IL13 Interleukin 13
  • IL13Ra2 its decoy receptor, IL13Ra2 protein is used in the treatment of asthma.
  • the production of soluble IL-13Ra2 using human cells is inefficient because of its instability in cells.
  • the present invention provides efficient methods and compositions for the production of IL-13Ra2.
  • the gene sequence for Flag tag was made by annealing two single strand nucleotides, and inserted into pcDNA3 with two restriction enzyme sites (Xba I and Apa I; pcDNA3- Flag).
  • cDNA of Jurkat cells immortalized cell line of human T lymphocyte
  • the gene for IL13Ra2 was inserted into pcDNA3-Flag with two restriction sites (Kpn I and Xho I).
  • the cDNA for the FOLDING TAG was inserted between Xho I and Xba I sites.
  • IL13Ra2-Flag-pcDNA3 or IL13Ra2-(S)-Flag-pcDNA3 was transfected to HEK293 cells with pEGFP-N2 plasmid (Clontech), and cultured cells for two days. Cells were lysed in
  • RIPA buffer 50 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% NP-40, 0.5% sodium
  • IL13Ra2 protein was detected with anti-Flag antibody ( Figure 10). GFP expression was used for the transfection efficiency, and actin as a loading control.
  • IL13Ra2 protein The activity of IL13Ra2 protein is measured by ELISA, which is commercially available.
  • the accumulation of unfolded proteins in the ER elicits the unfolded protein response.
  • the typical response has three different part, (i) suppression of translation, (ii) enhanced expression of molecular chaperones, and (iii) degradation of unfolded proteins .
  • Accumulation of unfolded protein in ER induces cell death unless the unfolded proteins are cleared . This explains why enhancing the efficiency of secretion is as important as enhancing the expression level.
  • al anti-trypsin is secreted from liver, and circulates in blood vessels.
  • the function of al anti-trypsin is to protect tissues, particularly lung tissue, from excess proteases.
  • the lung is damaged by proteases in a disease called a 1 anti-trypsin deficiency.
  • Subjects affected by a 1 anti-trypsin deficiency may develop emphysema, asthma and/or chronic obstructive pulmonary disease (COPD) 30.
  • COPD chronic obstructive pulmonary disease
  • a 1 anti-trypsin human serum
  • This treatment is expensive and subjects that receive the human serum are at risk of contracting pathogens present in the human serum 31. Therefore, the present invention provides recombinant protein technology useful in generating a 1 anti-trypsin, which is not subject to the expense and risks
  • al anti-trypsin expressing plasmid The construction of an al anti-trypsin expressing plasmid is similar to that described above (refer "Construction of IL13Ra2 expressing plasmid").
  • cDNA of al anti-trypsin was generated by PCR using cDNA of Jurkat cells (immortalized cell line of human T
  • lymphocyte as a template.
  • the generated cDNA for al anti-trypsin was inserted into pcDNA3-Flag with two restriction sites (EcoR I and Xho I), and cDNA for FOLDING TAG was inserted into Xho I and Xba I sites.
  • the plasmid for al anti-trypsin was transfected to HEK293 cells with pEGFP-N2 plasmid (Clontech), and cultured cells for two days.
  • Cell medium was harvested to monitor the secretion of al anti-trypsin, and cells were lysed in RIPA buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail and PMSF.
  • Cell lysate was briefly sonicated, and the sample was boiled in SDS-sample buffer, and applied onto SDS-PAGE. After transfer to PVDF membrane, al anti-trypsin was detected with anti-Flag antibody (Figure 11).
  • GFP expression was used for the transfection efficiency, al anti-trypsin activity is carried out using a commercially available kit.
  • the invention provides compositions and methods for generating BAG3 fusion polypeptides comprising the proteins delineated in the following tables, as well as vectors encoding these BAG3 fusion polypeptides.
  • STN1 Survival motor neuron- 1
  • DMD Dystrophin Muscular dystrophy
  • DMD Utrophin Muscular dystrophy
  • HIV1 gag HIV vaccine 40 HIV1 gag HIV vaccine 40
  • GAD65 glutamic acid Parkinson's disease 42
  • CD86(B7-2) Malignant melanoma 54
  • Interferon-beta Relapsing multiple sclerosis 65 glycodelin Immunosuppression 66 sIL13 receptor alpha 2 Asthma, Pulmonary fibrosis 6/
  • Fogolin MB Wagner R, Etcheverrigaray M, Kratje R. Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells. J Biotechnol. Apr 8 2004;109(1-2): 179-191.
  • Metabolic acetate therapy improves phenotype in the tremor rat model of Canavan disease. Inherit Metab Dis.
  • Carbohydrate -remodelled acid alpha-glucosidase with higher affinity for the cation- independent mannose 6-phosphate receptor demonstrates improved delivery to muscles of Pompe mice. Biochem J. Aug 1 2005;389(Pt 3):619-628.
  • TNF tumor necrosis factor

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Pulmonology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des compositions et des procédés qui favorisent le repliement d'une protéine, comprenant un domaine BAG.
PCT/US2011/065563 2010-12-22 2011-12-16 Compositions et procédés pour favoriser le repliement d'une protéine Ceased WO2012087835A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201061426326P 2010-12-22 2010-12-22
US61/426,326 2010-12-22

Publications (2)

Publication Number Publication Date
WO2012087835A2 true WO2012087835A2 (fr) 2012-06-28
WO2012087835A3 WO2012087835A3 (fr) 2012-11-01

Family

ID=46314784

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/065563 Ceased WO2012087835A2 (fr) 2010-12-22 2011-12-16 Compositions et procédés pour favoriser le repliement d'une protéine

Country Status (1)

Country Link
WO (1) WO2012087835A2 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014089375A1 (fr) 2012-12-05 2014-06-12 Boston Strategics Corporation Polypeptides améliorant l'expression protéique
WO2015191892A3 (fr) * 2014-06-11 2016-05-06 Beth Israel Deaconess Medical Center, Inc. Compositions d'α1-antitrypsine et méthodes de traitement de maladies auto-immunes
US10364292B2 (en) 2014-01-06 2019-07-30 Hoffmann-La Roche Inc. Monovalent blood brain barrier shuttle modules
US10370692B2 (en) 2013-12-20 2019-08-06 Hoffmann-La Roche Inc. Recombinant polypeptide production methods
US10941205B2 (en) 2015-10-02 2021-03-09 Hoffmann-La Roche Inc. Bispecific anti-human A-beta/human transferrin receptor antibodies and methods of use
EP3812473A1 (fr) * 2014-01-31 2021-04-28 Temple University Of The Commonwealth System Of Higher Education Bag3 comme cible pour le traitement de l'insuffisance cardiaque
US11390899B2 (en) 2016-09-26 2022-07-19 SOLA Biosciences, LLC Cell-associated secretion-enhancing fusion proteins
US11584793B2 (en) 2015-06-24 2023-02-21 Hoffmann-La Roche Inc. Anti-transferrin receptor antibodies with tailored affinity
US11603411B2 (en) 2015-10-02 2023-03-14 Hoffmann-La Roche Inc. Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054868A1 (fr) * 2003-12-05 2005-06-16 Multimmune Gmbh Compositions et procedes pour traiter et diagnostiquer des maladies neoplasiques et infectieuses
JP2005229874A (ja) * 2004-02-18 2005-09-02 Institute Of Physical & Chemical Research Ｂａｇ３ドメインポリペプチド及びその用途

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014089375A1 (fr) 2012-12-05 2014-06-12 Boston Strategics Corporation Polypeptides améliorant l'expression protéique
US9758807B2 (en) 2012-12-05 2017-09-12 SOLA Biosciences, LLC Protein expression enhancing polypeptides
US10370692B2 (en) 2013-12-20 2019-08-06 Hoffmann-La Roche Inc. Recombinant polypeptide production methods
US10364292B2 (en) 2014-01-06 2019-07-30 Hoffmann-La Roche Inc. Monovalent blood brain barrier shuttle modules
US11236389B2 (en) * 2014-01-31 2022-02-01 Temple University Of The Commonwealth System Of Higher Education BAG3 as a target for therapy of heart failure
EP3812473A1 (fr) * 2014-01-31 2021-04-28 Temple University Of The Commonwealth System Of Higher Education Bag3 comme cible pour le traitement de l'insuffisance cardiaque
US11542555B2 (en) 2014-01-31 2023-01-03 Temple University Of The Commonwealth System Of Higher Education BAG3 as a target for therapy of heart failure
US10781248B2 (en) 2014-06-11 2020-09-22 Beth Israel Deaconess Medical Center, Inc. α1-antitrypsin compositions and methods of treating autoimmune diseases
WO2015191892A3 (fr) * 2014-06-11 2016-05-06 Beth Israel Deaconess Medical Center, Inc. Compositions d'α1-antitrypsine et méthodes de traitement de maladies auto-immunes
US11584793B2 (en) 2015-06-24 2023-02-21 Hoffmann-La Roche Inc. Anti-transferrin receptor antibodies with tailored affinity
US12252533B2 (en) 2015-06-24 2025-03-18 Hoffmann-La Roche Inc. Anti-transferrin receptor antibodies with tailored affinity
US10941205B2 (en) 2015-10-02 2021-03-09 Hoffmann-La Roche Inc. Bispecific anti-human A-beta/human transferrin receptor antibodies and methods of use
US11603411B2 (en) 2015-10-02 2023-03-14 Hoffmann-La Roche Inc. Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use
US12030952B2 (en) 2015-10-02 2024-07-09 Hoffmann-La Roche Inc. Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use
US12358997B1 (en) 2015-10-02 2025-07-15 Hoffmann-La Roche Inc. Bispecific anti-human A-beta/anti-human transferrin receptor antibodies
US11390899B2 (en) 2016-09-26 2022-07-19 SOLA Biosciences, LLC Cell-associated secretion-enhancing fusion proteins

Also Published As

Publication number Publication date
WO2012087835A3 (fr) 2012-11-01

Similar Documents

Publication Publication Date Title
WO2012087835A2 (fr) Compositions et procédés pour favoriser le repliement d'une protéine
Chatzifrangkeskou et al. Cofilin-1 phosphorylation catalyzed by ERK1/2 alters cardiac actin dynamics in dilated cardiomyopathy caused by lamin A/C gene mutation
Csortos et al. High complexity in the expression of the B′ subunit of protein phosphatase 2A0: Evidence for the existence of at least seven novel isoforms
US9746475B2 (en) Antibody and antibody mimetic for visualization and ablation of endogenous proteins
EP3098241B1 (fr) Protéine de fusion inhibant l'angiogenèse ou la croissance et son utilisation
CN111787940B (zh) 使用工程化的去泛素化酶探测泛素依赖性细胞过程的组合物和方法
EP2910632B1 (fr) Enzyme hautement fonctionnelle ayant une spécificité de substrat béta-hexosaminidase b humaine modifiée, et présentant une résistance à la protéase
AU2014345501A1 (en) New methods to produce active TERT
JP2010120881A (ja) ヒト癌タンパク質ＭＤＭ２とヒト癌抑制タンパク質ｐ５３との相互作用阻害ペプチド及びその使用
AU2012306826B2 (en) Endostatin mutants with mutations at ATP binding sites
CN107022557B (zh) 新的死亡受体5抗体融合蛋白及其应用
EP2502991A1 (fr) Transformé qui produit du collagène dans lequel un résidu de lysine et un résidu de proline sont hydroxylés
US20170204157A1 (en) Human fgfr2c extracellular protein as well as coding gene and application thereof
Lee et al. Phosphocode-dependent glutamyl-prolyl-tRNA synthetase 1 signaling in immunity, metabolism, and disease
Zhang et al. MLKL-USP7-UBA52 signaling is indispensable for autophagy in brain through maintaining ubiquitin homeostasis
US7666641B2 (en) Canine COX-2 proteins and uses thereof
CN106754817B (zh) 一种重组自分泌运动因子的表达方法
CN112794895A (zh) 外源性atg10s蛋白在制备抗病毒药物中的应用
CN103319605B (zh) 一种肝靶向肽与血管生成抑制因子融合蛋白及其制备方法与应用
EP3808842A1 (fr) Composition pharmaceutique pour le traitement d'une immunodéficience complexe sévère comprenant une protéine de fusion d'un peptide de pénétration cellulaire et de l'adénosine désaminase
EP2841449A2 (fr) Archéase en tant que membre d'un complexe d'arn ligase
CN115607675B (zh) Nav1.9互作蛋白PRMT7及其下调剂在制备镇痛药物中的用途
AU2015258537B2 (en) Platelet-derived growth factor B mutant, preparation method therefor, and use thereof
JP4615230B2 (ja) ガラクトシルセラミド発現因子−１のｃ領域によるがん細胞転移抑制剤
CN115521360B (zh) 特异性结合Sestrin2蛋白的多肽及其在消化道癌治疗中的应用

Legal Events

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

Ref document number: 11851423

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct app. not ent. europ. phase

Ref document number: 11851423

Country of ref document: EP

Kind code of ref document: A2