Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/93—Ligases (6)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
  • C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain

Definitions

• the field of the currently claimed embodiments of the present invention relate to inteins, split inteins, compositions comprising inteins and methods for use of the like for protein engineering.
• Protein splicing is a posttranslational auto-processing event in which an intervening protein domain called an intein excises itself from a host protein in a traceless manner such that the flanking polypeptide sequences (exteins) are ligated together via a normal peptide bond.
• an intervening protein domain called an intein excises itself from a host protein in a traceless manner such that the flanking polypeptide sequences (exteins) are ligated together via a normal peptide bond.
• intein flanking polypeptide sequences
• Embodiments of the invention include a split intein N-fragment including an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a split intein N-fragment including an amino acid sequence, wherein said amino acid sequence comprises an amino acid sequence of at least 80%>, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a split intein C-fragment including an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• Embodiments of the invention include a split intein C-fragment including an amino acid sequence, wherein said amino acid sequence of said C-fragment comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a split intein C-fragment including an amino acid sequence, wherein said amino acid sequence of said C-fragment comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• VKIISRKSLGTQNVYDIGVGEPHNFLLK GLVASN (SEQ ID NO: 389).
• Embodiments of the invention include a composition including a split intein N- fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• split intein C-fragment comprising an amino acid sequence of at least 80%o, 85%, 90%>, 95%, 98%, 99%, or 100% sequence identity
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 4
• VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN SEQ ID NO: 389.
• Embodiments of the invention include a nucleotide plasmid including a nucleotide sequence encoding for a split intein N-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a nucleotide plasmid comprising a nucleotide sequence encoding for a split intein C-fragment including an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN SEQ ID NO: 389.
• Embodiments of the invention include a method for splicing two complexes including the following: contacting a first complex comprising a first compound and a split intein N-fragment with a second complex comprising a second compound and a split intein C-fragment, with contacting performed under conditions that permit binding of the split intein N-fragment to the split intein C- fragment to form an intein intermediate; and reacting the intein intermediate to form a conjugate of the first compound with the second compound.
• the split intein N-fragment includes an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• the split intein C-fragment comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%>, 99%), or 100%) sequence identity to
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• Embodiments of the invention include a method including the following: contacting a first complex comprising a first compound and a split intein N-fragment with a second complex comprising a second compound and a split intein C-fragment, with the contacting performed under conditions that permit binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and reacting the intein intermediate with a nucleophile to form a conjugate of the first compound with the nucleophile.
• the split intein N-fragment includes an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCL EDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP SEQ ID NO: 2
• the split intein C-fragment includes an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN SEQ ID NO: 389.
• the compound, first compound, or second compound is or includes a peptide or a polypeptide. In some embodiments, the compound, first compound, or second compound is or includes an antibody, antibody chain, or antibody heavy chain. In some embodiments, the compound, first compound, or second compound is or includes a peptide, oligonucleotide, drug, or cytotoxic molecule.
• Embodiments of the invention include an intein comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a kit for splicing two complexes together including the following: a split intein N-fragment including an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• CLSYDTEILTVEYGFLPIGKTVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP SEQ ID NO: 1
• CLSYDTEILTVEYGFLPIGKTVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP SEQ ID NO: 2
• split intein C-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%), 99%, or 100% sequence identity to
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• V IISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN (SEQ ID NO: 389);
• reagent(s) for permitting the binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate for permitting the binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and a nucleophilic agent.
• Embodiments of the invention include a method for generating a synthetic consensus intein peptide sequence including the following: generating a population of a plurality of homologous intein peptide sequences; identifying amino acids associated with fast splicing within the population of a plurality of homologous intein peptide sequences; generating a subpopulation of a second plurality of homologous intein peptide sequences, with the second plurality of homologous intein peptide sequences including amino acids associated with fast splicing; creating an alignment of at least three peptide sequences of the subpopulation; determining a most frequently occurring amino acid residue at each position of the at least three peptide sequences; and generating a synthetic consensus intein peptide sequence based on the most frequently occurring amino acid residue at each position of the at least three peptide sequences.
• Embodiments of the invention include a method including the following: fusing a first nucleotide sequence encoding an amino acid sequence of a first intein fragment (split intein N- fragment) including with a second nucleotide sequence encoding an amino acid sequence of a second intein fragment (split intein C-fragment), so that the fusion of the first nucleotide sequence and the second nucleotide sequence codes for a contiguous intein.
• the split intein N-fragment includes an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 1) or
• CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCL EDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP SEQ ID NO: 2
• the split intein C-fragment includes an amino acid sequence of at least 80%>, 85%>, 90%>, 95%, 98%, 99%, or 100% sequence identity to
• V IISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3), MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 4), or
• VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN SEQ ID NO: 389.
• Embodiments of the invention include a method including the following: fusing a first nucleotide sequence encoding an amino acid sequence of a first intein fragment (split intein N- fragment) including
• CLSYDTEILTVEYGFLPIGKWEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 1) with a second nucleotide sequence encoding an amino acid sequence of a second intein fragment (split intein C-fragment) including VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3), so that the fusion of the first nucleotide sequence and the second nucleotide sequence codes for a contiguous intein.
• Embodiments of the invention include a gene fusion including the following: a first nucleotide sequence encoding an amino acid sequence of a first intein fragment (split intein N- fragment) with a second nucleotide sequence encoding an amino acid sequence of a second intein fragment (split intein C-fragment).
• the split intein N-fragment includes an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCL EDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP SEQ ID NO: 2
• the split intein C-fragment includes an amino acid sequence of at least 80%o, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN SEQ ID NO: 389.
• Embodiments of the invention include a gene fusion including the following: a first nucleotide sequence encoding an amino acid sequence of a first intein fragment (split intein N- fragment) including
• Embodiments of the invention include a complex (e.g., a fusion protein) comprising a split intein N-fragment and a compound.
• the compound can be or include a peptide, a polypeptide or an antibody chain, such as an antibody heavy chain.
• the compound can include a peptide, oligonucleotide, drug, or cytotoxic molecule.
• the compound can be a 1,2-amino thiol or a 1,2-amino alcohol bonded to a peptide, oligonucleotide, drug, or cytotoxic molecule.
• the split intein N-fragment includes an amino acid sequence of at least 80%, 85%, 90%>, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a complex (e.g., a fusion protein) comprising a split intein C-fragment and a compound.
• the compound can be or include a dendrimer, peptide or polypeptide.
• the compound can include a peptide, an oligonucleotide, a drug, or a cytotoxic molecule.
• the compound can be a 1,2-amino thiol or a 1,2-amino alcohol bonded to a peptide, oligonucleotide, drug, or cytotoxic molecule.
• the split intein C-fragment includes an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3), an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to MVKIISRKS LGTQNV YDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 4), or an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN (SEQ ID NO: 389).
• the dendrimer can be a compound having the structure
• Rl, R2, R3, and R4 are each (independently) hydrogen (H) or a cargo molecule (the cargo molecules on Rl, R2, R3, and R4 can be different from each other).
• Rl, R2, R3, and R4 can each be a dye molecule.
• Rl , R2, R3, and R4 can each be a fluorescein derivative havin the structure
• Embodiments of the invention include a complex of the structure dendrimer - - cargo
• Embodiments of the invention include a complex of the structure
• Embodiments of the invention include a complex of the structure
• the split intein C-fragment comprises an amino acid sequence of at least 80%, 85%>, 90%>, 95%>, 98%, 99%>, or 100% sequence identity to VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3), an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%>, 99%, or 100% sequence identity to MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 4), or an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN (SEQ ID NO: 389).
• Embodiments of the invention include a contiguous intein that can be used, for example, in traditional semi-synthesis applications such as Expressed Protein ligation.
• Fig. 1 shows an alignment and a computer-generated model of the design of the Cfa split intein according to an embodiment of the invention
• Fig. 2 shows graphs showing the characterization of the Cfa intein according to an embodiment of the invention
• FIG. 3 shows expression and modification of a mouse monoclonal antibody using the
• FIG. 4 shows the identification of second shell 'accelerator' residues important for rapid protein trans-splicing according to an embodiment of the invention
• Fig. 5 shows kinetic analysis of Batch 2 mutations and computer generated models according to an embodiment of the invention
• Fig. 6 shows an analysis of Batch 1 mutations and computer generated models according to an embodiment of the invention
• Fig. 7A and Fig. 7B show an alignment and refinement of the DnaE intein family according to an embodiment of the invention
• Fig. 8 is an image of an SDS-PAGE analysis of test expression of His6-SUMO-Npu N and His 6 -SUMO-Cfa N according to an embodiment of the invention
• Fig. 9 shows a schematic and graph showing increased promiscuity of CfaGEP according to an embodiment of the invention.
• Fig. 10 shows graphs and schematics showing cyclization of eGFP in E. coli with variable residues according to an embodiment of the invention.
• Fig. 11 shows a table illustrating several complexes and compounds according to an embodiment of the invention.
• Embodiments of the invention include a split intein N-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a split intein N-fragment comprising an amino acid sequence, wherein said amino acid sequence comprises an amino acid sequence of at least 80%o, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP (SEQ ID NO: 2).
• Embodiments of the invention include a split intein C-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• Embodiments of the invention include a split intein C-fragment comprising an amino acid sequence, wherein said amino acid sequence of said C-fragment comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a composition comprising the following: a split intein N-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLED GSIIRATKDHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 1); and a split intein C-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3).
• Embodiments of the invention include a nucleotide plasmid comprising a nucleotide sequence encoding for a split intein N-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a nucleotide plasmid comprising a nucleotide sequence encoding for a split intein C-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• Embodiments of the invention include a method for splicing two complexes comprising: contacting a first complex comprising a first compound and a split intein N-fragment and a second complex comprising a second compound and a split intein C-fragment, wherein contacting is performed under conditions that permit binding of the split intein N-fragment to the split intein C- fragment to form an intein intermediate; and reacting the intein intermediate to form a conjugate of the first compound with the second compound, wherein said split intein N-fragment comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• reacting the intein intermediate comprises contacting the intein intermediate with a nucleophile.
• said first compound is a polypeptide.
• said first compound is an antibody.
• Embodiments of the invention include an intein comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• Embodiments of the invention include a kit for splicing two complexes together comprising the following: a split intein N-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to
• CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDK GFVYTQPIAQWHNRGEQEVFEYCLED GSIIRAT DHKFMTTDGQMLPIDEIFERGL (SEQ ID NO: 1); a split intein C-fragment comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 3); reagents for permitting the binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and a nucleophilic agent.
• Embodiments of the invention include a method for generating a synthetic consensus intein peptide sequence comprising: generating a population of a plurality of homologous intein peptide sequences; identifying amino acids associated with fast splicing within said population of a plurality of homologous intein peptide sequences; generating a subpopulation of a second plurality of homologous intein peptide sequences, wherein said second plurality of homologous intein peptide sequences comprise amino acids associated with fast splicing; creating an alignment of at least three peptide sequences of said subpopulation; determining a most frequently occurring amino acid residue at each position of said at least three peptide sequences; and generating a synthetic consensus intein peptide sequence based on said most frequently occurring amino acid residue at each position of said at least three peptide sequences.
• Embodiments of the invention include a method comprising: fusing a first nucleotide sequence encoding an amino acid sequence of a first intein fragment comprising
• Embodiments of the invention include a gene fusion comprising a first nucleotide sequence encoding an amino acid sequence of a first intein fragment comprising
• VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN SEQ ID NO: 3
• Embodiments of the invention include a contiguous intein that can be used, for example, in traditional semi-synthesis applications such as Expressed Protein ligation.
• the various intein fragments described are linked, fused, chemically bonded, complexed or coupled by conventional methods known in the art to polymers, peptides, polypeptides, oligopeptides, small molecules, nucleotides, polynucleotides, oligonucleotides, drugs, cytotoxic molecules or combinations thereof.
• Npu and Ssp the basis of rapid protein splicing through a comparative study of the first two characterized split inteins, Npu and Ssp was investigated.
• the substantial difference in splicing rate between these two proteins is especially puzzling given their highly similar sequences (63% identity) and near superimposable active site structures.
• Previous mutagenesis studies on Npu and Ssp suggest that the difference in activity between the two is likely due to the combined effects of several residues, rather than a single site. 6 ' 8
• Fig. 4 shows the identification of second shell 'accelerator' residues important for rapid protein trans-splicing according to an embodiment of the invention.
• Panels A and B design of second shell batch mutants on chimera 1 (Ssp N -Npu c ) and chimera 2 (Npu N -Ssp c ) is shown.
• Catalytic residues are shown in black (rendered as sticks).
• Fig. 5 shows kinetic analysis of Batch 2 mutations and computer generated models according to an embodiment of the invention.
• Fig. 6 shows an analysis of Batch 1 mutations and computer generated models according to an embodiment of the invention.
• Consensus protein engineering is a tool applied to a homologous set of proteins in order to create athermostable variant derived from the parent family. 13 ' 14
• a multiple sequence alignment is first generated from homologues of a particular protein, from which the most statistically frequent residue at each position is chosen as the representative in the consensus sequence.
• DnaE inteins 105 sequences were identified through a BLAST 15 search of the JGI 16 and NCBI 17 databases (Fig. 7A).
• the alignment was filtered to only contain sequences bearing the second shell indicators of fast splicing: K70, M75, M81, and SI 36.
• the 73 theoretically fast inteins left in the MSA (Fig. 7B) were then used to generate a consensus fast DnaE intein sequence (Cfa) (Fig. 1).
• the various sequences disclosed in Figs. 7 A and 7B are presented below:
• VKIISRKSLGIQPVYDIGVERDHKFVLKNGLVASN SEQ ID NO:8
• VKVVTRKYIGKENVYDIGVERDHNFVIRNGLVASN SEQ ID NO: 16
• VKILSRKSLGIQSVYDIGVEKDHNFLLANGLVASN SEQ ID NO: 24
• VKIISRQLAGNQTVYDLGVEKDHNFLLANGLIASN SEQ ID NO: 28
• VFEYDLEDGSVIRATKDHKFMTSEGQMLAIDEIFERGLELKQVKRSQP SEQ ID NO: 41
• VKIVRRKSLGIQTVYDIGVERDHNFLLANGLVASN SEQ ID NO: 42
• VKIIGRQSLGVQKVYDIGVEKEHNFLLHNGLIASN SEQ ID NO: 50
• VKIVS YRSLGKQFVYDIGVAQDHNFLLANGSIASN (SEQ ID NO: 52)
• VKIINRQSLGKQTVYDIGVEKDHNFILGNGLVASN SEQ ID NO:70
• VKIIMRSYVGRENVYDIGVERDHNFVAKNGLIAAN (SEQ ID NO:72)
• VKVIGRRSLGVQRIFDIGLPQDHNFLLANGAIAAN (SEQ ID NO:74)
• VKWRRRSLGMHRVFDIGLAQDHNFLLANGAIAAN (SEQ ID NO:78)
• VFEYTLENGQTIQATKDHKFMTNDGEMLAIDTIFERGLDLKSSDFS (SEQ ID NO: 79) [00179] VKIISRQSLGRKPVYDIGVEKDHNFLLGNGLIASN (SEQ ID NO:80)
• VKIISRQSLGKQSVFDIGVAKDHNFLLANGLVASN SEQ ID NO:136
• VKIISRTYVGQANVYDIGVENDHNFVIKNGFIAAN (SEQ ID NO:138)
• VKIVSRRYLGKADVYDIGVAKDHNFIIKNGLVASN SEQ ID NO: 140
• VKIIERRSLGKQNVYDIGVEKDHNFLLSNNLIASN SEQ ID NO: 142
• VKIISRQSLGKQSVYDIGVAKDHNFLLANGMVASN SEQ ID NO: 1436
• VKIIKRTSLGVRPVYDIGVIQDHNFLLENGLVASN SEQ ID NO: 152
• VKIISRQFLGRKPVYDIGVEKDHNFLLGNGLIASN SEQ ID NO: 1544
• VKITQRRSLGLQ S V YDIGL AQDHNFVIANGWVAAN (SEQ ID NO: 160)
• VKILKRRSISSQQVYDIGVEKDHNFLLANGLVASN SEQ ID NO: 162
• VKIISRQSLGVQPVYDIGVARDHNFLLADGQVASN SEQ ID NO: 164
• VKIIAKKSLGTQNVYDIGVERDHNFVIKNGLVASN SEQ ID NO: 168
• VKILARKSLGTQKVYDIGVNDDHNFALSNSFIASN SEQ ID NO: 170
• VKIIRRNLIGEAATYDIGLGKDHNFLLGQGLIASN SEQ ID NO:178
• VKIIRRKFIGHAPTYDIGLSQDHNFLLGQGLIAAN SEQ ID NO: 180
• V IIRRKFVGHAPTYDIGLSQDHNFLLGQGLIAAN (SEQ ID NO: 182)
• VKIVSYRSLGKQFVYDIGVAQDHNFLLANGSIASN SEQ ID NO: 184.
• VKIIGRQSLGVQKVYDIGVEKEHNFLLHNGLIASN SEQ ID NO: 190
• VKIISRKSLGTQPVYDIGVKDDHNFILANGMVASN SEQ ID NO: 192
• VKIIRRKFIGHAPTYDIGLSQDHNFLLGQGLIAAN SEQ ID NO: 1936
• VKIIQRRSLGLQSVYDIGLAQDHNFVMANGWVAAN SEQ ID NO: 198
• VKIISRQSLGRKPVYDIGVEKDHNFLLGNGLIASN SEQ ID NO:200
• VKIISRTYVGQANVYDIGVENDHNFVIKNGFVAAN SEQ ID NO:202
• VKIISRKSLGTQPVYDIGVQEDHNFVLNNGLVASN SEQ ID NO:208
• VKIISRKSLGTQPVYDIGVQEDHNFLLNNGLVASN SEQ ID NO:210
• VKIISRKSLGTQPVYDIGVQEDHNFVLNNGLVASN SEQ ID NO:214
• VKIISRQLAGNQTVYDLGVEKDHNFLLANGLIASN SEQ ID NO:2378
• VKIIGRQSLGVQKVYDIGVEKEHNFLLHNGLIASN SEQ ID NO:2578
• VKIVSYRSLGKQFVYDIGVAQDHNFLLANGSIASN SEQ ID NO:260
• VKIVSCKPLRVQTVYDIGVEKDHNFILDNGLVASN SEQ ID NO:276
• VKIISRKSLGTQPVYDIGVQEDHNFVLNNGLVASN SEQ ID NO:280
• V IISRKSLGTQPVYDIGVQEDHNFVL NGLVASN SEQ ID NO:284.
• VKIVSYRSLGKQFVYDIGVAQDHNFLLANGSIASN SEQ ID NO:286
• VKIIGRQSLGVQKVYDIGVEKEHNFLLHNGLIASN SEQ ID NO:290
• VKIISRKSLGTQPVYDIGVKDDHNFILANGMVASN SEQ ID NO:292
• VKIISRQSLGRKPVYDIGVEKDHNFLLGNGLIASN SEQ ID NO:296
• VKIIAKKSLGTQNVYDIGVERDHNFVIKNGLVASN SEQ ID NO:300
• VKILARKSLGTQKVYDIGVNDDHNFALSNSFIASN (SEQ ID NO:302)
• VKILKRRSIS S QQ V YDIGVEKDHNFLL ANGLV ASN (SEQ ID NO:304)
• VKIVSRKSLGKQPVYDLGVAKDHNFLLANGTVASN SEQ ID NO:310
• VKISTKKSLGKQKVYDIGVVRDHNFIIK GFVASN (SEQ ID NO.330)
• VKIISRRSVGVQSVYDIGVKQDHNFFLRNGLIASN SEQ ID NO:340
• VFEYRLENGSVIRATKDHKMMTADGQMLPIDEIFKQNLDLKQLN SEQ ID NO:345
• VKIISRQSLGKQSVFDIGVAKDHNFLLANGLVASN SEQ ID NO:3436
• VKP SRRYLGKADVYDIGVAKDHNFIIKNGLVASN SEQ ID NO:348)
• VKIIERRSLGKQNVYDIGVEKDHNFLLSNNLIASN SEQ ID NO:350
• VKIISRQSLGKQSVYDIGVAKDHNFLLANGMVASN SEQ ID NO:354
• VKIIGRQSLGRKPVYDIGVEKDHNFLLGNGLIASN SEQ ID NO:360
• FIG. 1 shows an alignment and a computer-generated model of the design of the Cfa split intein according to an embodiment of the invention.
• Panel A shows a sequence alignment of Npu DnaE and Cfa DnaE. The sequences share 82% identity with the differences (underlined, cyan) evenly distributed through the primary sequence. Catalytic residues and second shell 'accelerator' residues are shown in caret, orange and asterisk, green, respectively.
• the Cfa intein has high sequence similarity to Npu (82%), and the non-identical residues are spread throughout the 3D structure of the protein.
• Cfa intein fragments fused to model exteins were generate and their PTS activity was measured using the aforementioned in vitro assay (Fig. 2). This revealed that the Cfa intein splices 2.5 fold faster at 30°C than Npu (ti/ 2 20s vs. 50s), a notable enhancement in activity since the latter is the fastest characterized DnaE split intein (Fig. 2A). This accelerated rate manifests itself both in branch formation (3 -fold increase) and branch resolution (2-fold increase). In line with parent DnaE inteins, Cfa retains the preference for a bulky hydrophobic residue at the +2 position of the C-extein.
• Cfa shows an increased splicing rate as a function of temperature and is consistently faster than Npu (Fig. 2 A).
• the Cfa intein even maintains activity at 80°C, albeit with reduced yield of splice products, while Npu is inactive at this temperature.
• Fig. 2 shows graphs showing the characterization of the Cfa intein according to an embodiment of the invention.
• Panel A splicing rates for Cfa and Npu as a function of temperature are shown.
• Panels B and C splicing rates for Cfa and Npu as a function of added chaotrope are shown.
• Npu is inactive in 3M GuHCl or 8M Urea.
• Cfa N fusions would also exhibit increased protein expression levels in mammalian cells.
• intein fusions to the heavy chain (HC) of monoclonal antibodies (mAbs) have emerged as a powerful tool for site-specific conjugation of synthetic cargoes. 19"21
• the expression levels in HEK293 cells of a mAb (aDec205) as a function of the N-intein fused to its HC was explored. Consistent with the bacterial expression results, production of the HC-Cfa N fusion was significantly higher than for the other inteins examined; for example, the secreted levels of the mAb-Cfa construct were ⁇ 10-fold higher than for the corresponding Npu fusion (Fig. 3A and 3B).
• Fig. 8 is an SDS-PAGE analysis of test expression of His 6 -SUMO-Npu N and His 6 - SUMO-Cfa N .
• Lanes correspond to (P) the inclusion body pellet, (FT) flow through of batch bound Ni-NTA solution, (Wl) a 5CV wash with 5 mM imidazole, (W2) a 5CV wash of 25 mM imidazole, (E1-E4) and four 1.5CV elutions of 250 mM imidazole.
• FIG. 3 shows expression and modification of a mouse monoclonal antibody using the Cfa intein according to an embodiment of the invention.
• Panel A shows test expression in HEK293T cells of various Int N homologues (Npu, Mcht, Ava and Cfa) fused to the C-terminus of the heavy chain of a mouse aDec205 monoclonal antibody.
• Top Western blot analysis (aMouse IgG) of antibody levels present in the medium following the 96 hour expression.
• Bottom a-actin western blot of cell lysate as a loading control.
• Panel C shows the structure of the Cfa c -dendrimer construct used in PTS reactions with the ocDEC205 HC-Int N fusion. For simplicity, the Cfa c peptide sequence is depicted symbolically in green (as a rectangle with a triangular cut-out on the left).
• Panel D is a schematic of the in situ PTS approach used to modify the HC of a mAb with a multivalent cargo.
• Panel E is an SDS-PAGE analysis of PTS reaction. Lane 1 : Wild type mouse aDEC205 mAB.
• Lane 2 Mouse aDEC205-Cfa N mAB fusion.
• Lane 3 addition of the Cfa c - dendrimer to the media containing the aDEC205-Cfa N mAB.
• the splicing reaction was analyzed by fluorescence (bottom) and western blot (top, aMouse IgG).
• Oligonucleotides and synthetic genes were purchased from Integrated DNA
• Hotsart fusion polymerase were purchased from Agilent (La Jolla, CA). All restriction enzymes and
• DH5a competent cells purchased from Invitrogen (Carlsbad, CA). Dulbecco's Modified Eagle Medium (DMEM), Lipofectamine 2000, and low IgG fetal bovine serum were purchased from Invitrogen as well. DNA purification kits were purchased from Qiagen (Valencia, CA). All plasmids were sequenced by GENEWIZ (South
• N,N-diisopropylethylamine DIPEA
• Luria Bertani LB
• buffering salts were purchased from Fisher Scientific (Pittsburgh, PA).
• Dimethylformamide DMF
• dichloromethane DCM
• Coomassie brilliant blue triisopropylsilane (TIS)
• ⁇ -Mercaptoethanol DAIPEA
• DIPEA Dimethylformamide
• DCM dichloromethane
• TIS triisopropylsilane
• BME DL-dithiothreitol
• DTT sodium 2-mercaptoethanesulfonate
• SSNa sodium 2-mercaptoethanesulfonate
• Pd(PPh 3 ) 4 tetrakis(triphenylphosphine)palladium(0)
• 5(6)-carboxyfluorescein purchased from Sigma-Aldrich (Milwaukee, WI) and used without further purification.
• Tris(2- carboxyethyl)phosphine hydrochloride (TCEP) and isopropyl- -D-thiogalaetopyranoside (IPTG) were purchased from Gold Biotechnology (St. Louis, MO).
• the protease inhibitor used was the Roche
• Nickel-nitrilotriacetic acid (Ni-NTA) resin was purchased from Thermo scientific (Rockford, IL). Fmoc amino acids were purchased from
• HBTU hexafluorophosphate
• Rink Amide-ChemMatrix resin was purchased from Biotage (Charlotte, NC).
• Trifluoroacetic acid was purchased from Halocarbon (North Augusta, SC). Immun-blot PVDF membrane (0.2 ⁇ ) and Criterion XT Bis-Tris gels (12% polyacrylamide) were purchased from Bio-
• MES-SDS running buffer was purchased from Boston Bioproducts (Ashland, MA).
• Anti-Mouse IgG secondary antibody (Licor mouse 800) and Mouse aActin primary antibody were purchased from Li-COR biotechnology (Lincoln, NE).
• Analytical RP-HPLC was performed on Hewlett-Packard 1100 and 1200 series instruments equipped with a C 18 Vydac column (5 ⁇ , 4.6 x 150 mm) at a flow rate of 1 mL/min.
• Preparative RP-HPLC was performed on a Waters prep LC system comprised of a Waters 2545 Binary Gradient Module and a Waters 2489 UV detector. Purifications were carried out on a Ci 8 Vydac 218TP1022 column (10 ⁇ ; 22 x 250 mm) at a flow rate of 18 mL/min.
• Coomassie-stained gels and western blots were imaged using a LI-COR Odyssey Infrared Imager. Fluorescent gels were imaged using a GE ImageQuant LAS 4000 Imager. The splicing-dependent E. coli growth assay was performed on a VersaMax tunable microplate reader from Molecular Devices. Cell lysis was carried out using a S- 450D Branson Digital Sonifier.
• WT Ssp N pet30- His 6 -SUMO-AEY- Ssp N
• Plasmid 2 [00622] WT Npu N : pet30- His 6 -SUMO-AEY-Npu N
• ESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTA NHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVP GLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNG FVSHAHHHHHH (SEQ ID NO:364)
• Batch 1 Pet30- His 6 -SUMQ-AEY-Ssp N (R73K. L75M, Y79G, L81M
• Ssp N R73K Pet30- His 6 -SUMO-AEY- Ssp N (R73K [00636] MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVS
• Batch 2 Pet30- His 6 -SUMO-AEY- Ssp N (L56F, S70K, A83P. E85D
• Ssp N S70K A83P Pet30- His 6 -SU O-AEY- Ssp N (S70K, A83P)
• Ssp N L56, S70K, A83P Pet30- His 6 -SUMO-AEY- Ssp N (L56F. S70K, A83P)
• Batch 3 Pet30- His6-SUMO-AEY- Ssp N (S23E. E24K, E25R, N27E
• Batch 4 Pet30- His 6 -SUMO-AEY- Ssp N (P35N. E36N, R38N, V39I)
• LLDAGTIK (SEQ ID NO:374)
• the four batch mutants (Batches 5-8) and A136S point mutant on the Ssp c intein were cloned by inverse PCR using Pfu Ultra II HS Polymerase (Agilent) using plasmid 3 as a template and code the protein sequences shown below:
• VSHAHHHHHH SEQ ID NO:375
• VSHAHHHHHH SEQ ID NO:377)
• Batch 8 pTXBl- Ssp c -MxeGyrA-His 6 (X128A. A130K. A133F)
• VSHAHHHHHH SEQ ID NO:378)
• Plasmid 19 [00675] Ssp c A136S: pTXBl- Ssp c -MxeGyrA-His 6 (A136S
• the gene for the fused Consensus DnaE sequence was codon-optimized for E. coli expression through IDT DNA and purchased as a gBlock.
• the DNA gBlock sequence is shown below:
• Cfa plasmids used to screen the dependency of splicing at the +2 position of the C- extein were generating using restriction cloning into a previously generated plasmid 2 containing a dual expression system of the split aminoglycoside phosphotransferase (Kan R ) gene.
• the Cfa dual expression construct is shown below:
• the +2 position of the C-extein is underlined, and is either phenylalanine, glycine, arginine, or glutamate.
• pCMV Plasmids containing the aDEC205 antibody light chain (LC), heavy chain (HC), and HC-intein fusions (HC-Npu N , HC-Mcht N , HC-Ava N ) were obtained as previously described.
• LC antibody light chain
• HC heavy chain
• HC-intein fusions HC-Npu N , HC-Mcht N , HC-Ava N
• a codon-optimized Cfa DnaE sequence for mammalian cell expression was generated using JCAT 4 and purchased as a gBlock through IDT DNA. The sequence is shown below: [00699] TGCCTGAGCTACGACACCGAGATCCTGACCGTGGAGTACGGCTTCCTGC
• HC-Cfa N pCMV-HC-Cfa N
• Cfa c -link pTXB 1 -H6-Cfa c -CFNSGG-MxeGyrA-H6
• the inclusion body pellet was resuspended in 30 mL of lysis buffer containing 6M Urea, and the suspension was incubated overnight at 4 ° C to extract and resolubilize the protein. This mixture was then centrifuged at 35,000 rcf for 30 minutes.
• the supernatant was then mixed with 4 mL of Ni-NTA resin (for affinity purification using the His 6 tag) and incubated at 4 ° C for 30 minutes to batch bind the protein.
• This mixture was loaded on a fritted column, the flow through was collected, and the column was washed with 5 column volumes (CV) of lysis buffer with 6M Urea and 5 CV of lysis buffer with 25 mM imidazole and 6M urea.
• the protein was then eluted in four 1.5 CV fractions of lysis buffer with 250 mM imidazole and
• the first two elution fractions were generally found by SDS-PAGE (12% Bis-Tris gel, run for 50 minutes at 170V) to contain the expressed protein and were combined for refolding.
• N-inteins were refolded by stepwise dialysis into lysis buffer with 0.5 mM DTT at 4°C. This refolded protein was then treated with 10 mM TCEP and Ulpl protease (overnight, RT) to cleave the His 6 -SUMO expression tag. The solution was then mixed with 4 mL Ni-NTA resin and incubated for 30 minutes at 4°C. The slurry was applied to a fritted column and the flow through was collected together with a 3 CV wash with lysis buffer.
• the protein was then treated with 10 mM TCEP, concentrated to 10 mL, and further purified by size exclusion chromatography using an S75 16/60 gel filtration column employing degassed splicing buffer (100 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.2) as the mobile phase. Fractions were analyzed by SDS-PAGE, analytical RP- HPLC, and ESI-MS. Pure protein was stored by flash-freezing in liquid N 2 following the addition of glycerol (20 % v/v). Note: during the refolding step, significant protein precipitation was observed for Batch 3, suggesting it is prone to aggregation.
• the cell pellet (from expression of plasmid 20) was first resuspended in 30 mL of lysis buffer (50 mM phosphate, 300 mM NaCl, 5 mM imidazole, pH 8.0) containing the Roche Complete protease inhibitor cocktail. The cells were then lysed by sonication (35% amplitude, 8 x 20 second pulses on / 30 seconds off), and the lysate was pelleted by centrifugation (35,000 rcf, 30 rnin). The supernatant was incubated with 4 mL of Ni-NTA resin for 30 minutes at 4°C to enrich for the soluble Cfa N protein.
• lysis buffer 50 mM phosphate, 300 mM NaCl, 5 mM imidazole, pH 8.0
• the cells were then lysed by sonication (35% amplitude, 8 x 20 second pulses on / 30 seconds off), and the lysate was pelleted by centrifugation (3
• the slurry was then loaded onto a fritted column, and the column was washed with 20 mL of wash buffer 1 (lysis buffer) followed by 20 mL of wash buffer 2 (lysis buffer with 25 mM imidazole). Finally, the protein was eluted from the column with 4 x 1.5 CV of elution buffer (lysis buffer +250 mM imidazole).
• the desired protein which was present in elution fractions 1 and 2 as determined by SDS-PAGE (12% bis-tris gel run in MES-SDS running buffer at 170V for 50 minutes), was then dialyzed into lysis buffer for 4 hours at 4°C. Following dialysis, the protein was treated with 10 mM TCEP and Ulpl protease overnight at room temperature to cleave the His 6 -SUMO expression tag. The solution was then incubated with 4 mL Ni-NTA resin for 30 minutes at 4°C. The slurry was applied to a fritted column and the flow through was collected together with a 3 CV wash with lysis buffer.
• the protein was then treated with 10 mM TCEP, concentrated to 10 mL, and purified over an S75 16/60 gel filtration column employing degassed splicing buffer (100 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.2) as the mobile phase. Fractions were analyzed by SDS-PAGE (12% bis- tris gel run in MES-SDS running buffer at 170V for 60 minutes), analytical RP-HPLC, and ESI-MS. Pure Protein was stored in glycerol (20% v/v) and flash-frozen in liquid N 2 .
• Cell pellets were harvested by centrifugation (10,500 rcf, 30 min), resuspended in lysis buffer, and lysed by sonication on ice (35% amplitude, 10 x 20 second pulses on / 30 seconds off).
• the protein in the soluble fraction was isolated by centrifugation (35,000 rcf, 30 min) and then enriched by Ni-NTA purification (4 mL beads, carried out as described for N-intein constructs). Following elution in lysis buffer with 250 mM imidazole, the imidazole was removed by dialysis into fresh lysis buffer.
• the ligation was then carried out overnight at room temperature with the addition of 10 mM TCEP, the Roche Complete protease inhibitor cocktail, 100 mM MESNa, 5 mM EDTA, and 5 mM CFN-NEb (pH 7.0).
• N- and C-inteins (15 ⁇ Int N , 10 ⁇ Int c ) were individually preincubated in splicing buffer
• [P] is the normalized intensity of product
• k is the rate constant (s "1 ).
• the mean and standard deviation (n 3) are reported.
• Figs. 7 A and 7B show an alignment and refinement of the DnaE intein family.
• Fig. 7A shows the multiple sequence alignment (MSA) of the 105 members of the DnaE intein family found from a BLAST search of the JGI and NCBI sequences databases. The locations of the 'accelerator' residues used to filter the alignment are indicated with black arrows.
• Fig. 7B shows MSA of the 73 DnaE inteins predicted to demonstrate fast splicing kinetics due to the presence of all four accelerator residues.
• coli growth was measured at various concentrations of kanamycin (2.5, 10, 25, 50, 100, 250, 1000 ⁇ g/mL kanamycin with 100 ⁇ g/mL ampicillin).
• the cell optical density at 650 nm (OD 65 o) at the 24-hour end point was fit to a dose response curve with variable slope.
• E. coli inclusion bodies containing His 6 -Sumo-Cfa N expression (plasmid 20) were resuspended and extracted overnight at 4°C in lysis buffer containing 6M urea. Following centrifugation (35,000 rcf, 30 min), the supernatant was removed and the protein enriched by Ni-NTA under denaturing conditions (as described above). However, instead of refolding the protein, trans- splicing was directly initiated by the addition of Cfa c -CFN ( ⁇ Cfa c , 2mM TCEP, 2mM EDTA, 2hrs, RT). Reaction progress was monitored by SDS-PAGE.
• Expression yield was measured as the amount of HC-Int N in the media as determined by densitometry. To account for varying cell growth and survival, the yield was normalized using an a-actin blot of the HEK293T cell lysate (5s sonication, 35% amplitude, in lx loading dye) and then represented relative to the expression of HC-Cfa N . Four replicates of this test expression were carried out, and the mean was calculated with error represented as the standard deviation.
• Compound 2 (dendrimer thioester). This compound was synthesized on the solid phase using the route outlined in Supplemental Scheme 1 on a scale of 400 mg of Rink Amide resin (substitution: 0.47 mmol/g, 188 ⁇ ). General procedures are given first, followed by any specific methods for this peptide. The Fmoc group was removed with 3 mL of 20% piperidine in DMF and performed twice (one deprotection for 30 sec followed by an additional deprotection for 15 min). After each deprotection step, as well as all subsequent synthetic steps, flow washes were used (3 5 sec. with ⁇ 5 mL of DMF each). Coupling was performed using 4 eq. of monomer, 4 eq.
• Trityl protecting group was selectively removed using 1 % TFA, 5% TIS in DCM using a total of 30 mL ( 1 Ox 3 mL) of deprotection cocktail. Thorough washing of the resin with DCM both during and after these cycles ensured the removal of any liberated Trityl species.
• the resin was also neutralized with 5% DIPEA in DMF before the next coupling was undertaken.
• the Alloc group was deprotected using 0.1 eq of tetrakis(triphenylphosphine) palladium(O), 20 eq of phenylsilane in DCM for 3x 45 min each. Thorough washing of the resin with DCM during and after these cycles was used, as well as a 5% DIPEA in DMF wash before the next coupling.
• the glutaric anhydride monomer was used as a preactivated dicarboxylic acid to allow the formation of the thioesters (i.e.
• Compound 3 was synthesized by native chemical ligation (scheme 2).
• Compound 2 was dissolved in ligation buffer and mixed with five eq. of Cys-Gly-Lys(Fluorescein) (1 mM 2, 5 mM peptide, 4M Guanidine, 100 mM phosphate, 150 mMNaCl, lOOmM MPAA, 20 mM TCEP, pH 7.0) and allowed to react overnight at room temperature. Deprotection of the thiazolidine was then accomplished by the addition of 0.1 M methoxyamine (final concentration) and decreasing the pH of the ligation buffer to 4.0 (overnight, RT).
• Compound 1 was synthesized by expressed protein ligation.
• Compound 3 was dissolved in ligation buffer and mixed with 1.5 eq of the Cfa c -MESNa thioester (100 ⁇ 3, 150 ⁇ Cfa c -MESNa, 4M Guanidine, 100 mM phosphate, 150 mM NaCl, 20 mM TCEP, 100 mM MPAA). The reaction was allowed to proceed overnight at room temperature. The ligated product was then purified by semi-preparative RP-HPLC. Desired fractions were pooled and lyophilized. Expected mass: 9860.8 Da. Found: 9860.3 Da.
• the aDec205 mAb with Cfa N fused to its C-terminus was expressed as described above. Following the 96 hr expression, the media was concentrated 10-fold in an Amicon 30K concentrator (0.5 mL). Compound 1 was dissolved in splicing buffer (100 mM phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.2) and then mixed with the concentrated media (2 ⁇ compound 1, 2 mM TCEP, 1 mM EDTA) and the reaction allowed to proceed for 2 hrs at room temperature.
• splicing buffer 100 mM phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.2
• the splicing mixture was then analyzed by SDS-PAGE (12% Bis-Tris run in MES-SDS running buffer at 170V for 50 minutes) and imaged on a fluorescence imager. This was followed by transfer to a PVDF membrane and western blot analysis (aMouse IgG).
• the invention allows for the formation of various complexes between a split intein fragment and a compound.
• IntC is a split intein fragment, for example, a split intein C-fragment.
• the dendrimer can have the form of Compound 2, Compound 3, or portions of these.
• the cargo can be a dye (e.g., fluorescein), another marker molecule, a drug (e.g., a cytotoxic molecule, such as used in the treatment of cancer), or a nucleotide.
• the polypeptide can be a wholly or partially synthetic or a naturally occurring polypeptide or portion thereof.
• a dendrimer can be a molecule having a branched chemical structure onto which one or more "cargo" molecules can be “loaded".
• a "cargo” molecule can be a synthetic of naturally occurring molecule.
• the cargo molecule can be structured to have no free 1,2-amino thiols or 1,2-amino alcohols.
• the intein is bonded through an amino thiol or amino alcohol to a polypeptide, as shown in row 3 of the table of Fig. 11, the complex formed can be considered to be a recombinant fusion protein.
• a major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site, hi addition to a mandatory catalytic Cys, Ser, or Thr residue at position +1 (i.e., the first residue within the C-extein), there is a bias for residues resembling the proximal N- and C-extein sequence found in the native insertion site. Deviation from this preferred sequence context leads to a marked reduction in splicing activity, limiting the applicability of PTS-based methods. 23 ' 24 Accordingly, there is a need for split inteins whose activities are minimally affected by local sequence environment. For DnaE inteins, extein sequence preferences are largely confined to the catalytic cysteine at the +1 position and large hydrophobic residues that are preferred at the +2 position. 25
• VKIISRKSLGTQNVYDIGVGEPHNFLLKNGLVASN (SEQ ID NO: 389).
• Fig. 9 shows a schematic and a table showing the increased promiscuity of CfaGEP.
• Panel A shows a schematic depicting the PTS-dependent E. coli selection system with the Cfa split intein.
• the kanamycin resistance protein, KanR is split and fused to N- and C-intein fragments (Cfa N and Cfa c ).
• the +2 C-extein residue (red X) is varied in the system.
• FIG. 10 shows schematics and graphs showing eGFP Cyclization with the CfaoEP split intein.
• Panel A is a schematic depicting cyclization of eGFP in E. coli with variable residues at the +2 C-extein position (red X).
• Panel C is a schematic depicting the cyclization of eGFP in E.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Medicinal Chemistry (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Biophysics (AREA)
• Microbiology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Immunology (AREA)
• Gastroenterology & Hepatology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)

Priority Applications (13)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (2)

Family

ID=59398921

Family Applications (1)

Country Status (14)

Cited By (50)

Families Citing this family (11)

Family Cites Families (4)

• 2017
  • 2017-01-27 CA CA3051195A patent/CA3051195A1/en active Pending
  • 2017-01-27 HU HUE17745022A patent/HUE062276T2/hu unknown
  • 2017-01-27 SI SI201731373T patent/SI3408292T1/sl unknown
  • 2017-01-27 PT PT177450228T patent/PT3408292T/pt unknown
  • 2017-01-27 JP JP2018539361A patent/JP7290305B2/ja active Active
  • 2017-01-27 EP EP23168286.5A patent/EP4234689A3/en active Pending
  • 2017-01-27 AU AU2017211395A patent/AU2017211395B2/en active Active
  • 2017-01-27 FI FIEP17745022.8T patent/FI3408292T3/fi active
  • 2017-01-27 EP EP17745022.8A patent/EP3408292B1/en active Active
  • 2017-01-27 DK DK17745022.8T patent/DK3408292T3/da active
  • 2017-01-27 CN CN201780021267.0A patent/CN108884154A/zh active Pending
  • 2017-01-27 ES ES17745022T patent/ES2949163T3/es active Active
  • 2017-01-27 WO PCT/US2017/015455 patent/WO2017132580A2/en not_active Ceased
  • 2017-01-27 US US16/073,602 patent/US11142550B2/en active Active
  • 2017-01-27 PL PL17745022.8T patent/PL3408292T3/pl unknown
• 2021
  • 2021-06-30 US US17/363,698 patent/US12297472B2/en active Active

Non-Patent Citations (21)

Also Published As

Similar Documents

Legal Events