US20150344549A1 - Split inteins, conjugates and uses thereof - Google Patents

Split inteins, conjugates and uses thereof Download PDF

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US20150344549A1
US20150344549A1 US14/411,702 US201314411702A US2015344549A1 US 20150344549 A1 US20150344549 A1 US 20150344549A1 US 201314411702 A US201314411702 A US 201314411702A US 2015344549 A1 US2015344549 A1 US 2015344549A1
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fragment
intein
split
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Tom W. Muir
Miquel Vila-Perello
Zhihua Liu
Neel H. Shah
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Princeton University
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    • 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
    • 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
    • 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
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • 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

  • Protein splicing is a post-translational process catalyzed by a family of proteins known as inteins.(1) During this process, an intein domain catalyzes its own excision from a larger precursor protein and simultaneously ligates the two flanking polypeptide sequences (exteins) together. While most inteins catalyze splicing in cis, a small subset of these proteins exist as naturally fragmented domains that are separately expressed but rapidly associate and catalyze splicing in trans. Given their capacity to make and break polypeptide bonds (inteins can be considered protein ligases), both cis and trans-splicing inteins have found widespread use as chemical biological tools.(2)
  • inteins Despite the growing use of inteins in chemical biology, their practical utility has been constrained by two common characteristics of the family, namely (i) slow kinetics and (ii) context dependent efficiency with respect to the immediate flanking extein sequences.(3,4) Recently, a split intein from the cyanobacterium Nostoc punctiforme (Npu) was shown to catalyze protein trans-splicing on the order of a minute, rather than hours like most cis- or trans-splicing inteins.(5) Furthermore, this intein was slightly more tolerant of sequence variation at the critical +2 C-extein residue than other inteins.(6)
  • split intein N- and C-fragments Disclosed are split intein N- and C-fragments, variants thereof, and methods of using these split inteins in polypeptide purification and modification.
  • fusion proteins of a polypeptide and a split intein N-fragment, or variant thereof as described below in greater detail.
  • complexes of the fusion protein and a split intein C-fragment or variant thereof as described in detail below can be via a covalent interaction between the fusion protein and C-fragment or variant or via a noncovalent interaction (e.g., ionic, H-bonding, and/or van der Waals interaction).
  • split intein C-fragments or variants thereof further comprises a linker, such as a peptide linker, or other linkers as described below in detail.
  • a linker such as a peptide linker, or other linkers as described below in detail.
  • a specific peptide linker contemplated is -SGGC (SEQ ID NO: 705) attached to any of the split intein C-fragments described below.
  • the linker can be tailored so as to allow for attachment of a split intein C-fragment of interest to a support, e.g., a bead, a resin, a slide, a particle.
  • the split intein C-fragment, or variant thereof is bound to a support.
  • the support is a bead, a resin, a particle or a slide.
  • selection of the N-fragment and C-fragment can be from the same wild type split intein (e.g., both from Npu, or a variant of either the N- or C-fragment as discussed in great detail below), or alternatively can be selected from different wild type split inteins or the consensus split intein sequences discussed below, as it has been discovered that the affinity of a N-fragment for a different C-fragment (e.g., Npu N-fragment or variant thereof with Ssp C-fragment or variant thereof) still maintains sufficient binding affinity for use in the disclosed methods.
  • the fusion protein can be in a whole cell lysate or secreted from a cell (e.g., a mammalian cell) and in a cell supernatant.
  • the polypeptide of the fusion protein is an antibody, e.g., an IgG antibody.
  • the N-fragment is fused to one or both of the heavy chains of the antibody.
  • the N-fragment is fused to one or both of the light chains of the antibody.
  • the methods disclosed herein can further comprise washing the intein intermediate (prior to contact with the nucleophile) to remove the cell lysate or cell supernatant, for example.
  • the methods disclosed herein can further comprise isolating the resulting conjugate of the polypeptide and nucleophile.
  • the methods disclosed herein can be useful as an efficient purification for polypeptides prepared by recombinant protein methods.
  • the nucleophile can be a thiol to form a conjugate that is an ⁇ -thioester of the polypeptide.
  • the resulting ⁇ -thioester can be further modified by contacting with a second nucleophile, employing the well known ⁇ -thioester chemistry for protein modification.
  • the methods disclosed herein can provide conjugates of the polypeptide, which in some cases is an antibody (e.g., an IgG antibody), and a nucleophile (e.g., a drug, a polymer, an oligonucleotide).
  • FIG. 1 shows trans-splicing of split DnaE inteins.
  • FIG. 2 shows in vitro half-lives of trans-splicing reactions.
  • Indicated split intein pairs fused to model exteins Ub or SUMO Ub-Int N and Int C -SUMO
  • Ub-Int N and Int C -SUMO were mixed at either 30° C. or 37° C., and the formation of products was monitored over time by gel electrophoresis.
  • Representative coomassie-stained SDS-PAGE gels showing (b) fast Ava splicing at 37° C. and (c) inefficient Ssp splicing at 37° C.
  • FIG. 3 shows sequence-activity relationships in split DnaE inteins.
  • (c) In vivo analysis of the C120G mutation in the Aha intein ( ⁇ SD, n 3).
  • (e) In vivo analysis of Ssp-to-Npu point mutations that improve Ssp activity ( ⁇ SD, n 4). Note that all residue numberings correspond to the relevant positions on Npu as defined by the NMR structure (PDB: 2KEQ).(21)
  • FIG. 4 shows engineered versions of ultrafast DnaE inteins support efficient expressed protein ligation.
  • FIG. 5 shows sequence alignments of split DnaE inteins. Numbering follows that of Npu as assigned for the NMR structure (PDB 2KEQ). Critical catalytic residues are marked with an asterisk.
  • FIG. 6 shows sequence logos for high- and low-activity inteins.
  • Inteins are ranked based on in vivo activity with a “CFN” C-extein sequence.
  • the high and low activity inteins are distinguished based on a cut-off IC 50 value of 350 ⁇ g/mL of kanamycin, and the Aha intein is included in the high-activity set, given that the C 120 G mutation dramatically restores high activity.
  • FIG. 7 shows purification of C-terminal ⁇ -thioesters using split-inteins.
  • FIG. 8 shows Purification of soluble protein ⁇ -thioesters using the Npu C -AA affinity column.
  • FIG. 9 shows RP-HPLC and MS analysis of Ub and MBP ⁇ -thioesters.
  • FIG. 10 shows the effect of the ⁇ 1 residue on the efficiency of the on-resin thiolysis.
  • 20 different Ub-Npu N proteins were expressed containing each of the 20 proteinogenic amino acids at the C-terminus of Ub ( ⁇ 1 residue) and purified over Npu C -AA columns.
  • Cleavage yields from the Npu C -AA column were estimated by gel electrophoresis and amounts of thioester versus side reactions (mainly hydrolysis) were determined by RP-HPLC and MS analysis.
  • FIG. 11 shows purification of H2B(1-116)- ⁇ -thioester under denaturing conditions.
  • FIG. 12 shows purification of ⁇ DEC thioesters expressed in 293T cells using the split-intein column.
  • EPL Expressed Protein Ligation
  • FIG. 13 shows EPL directly using Int C -column eluted thioesters.
  • RP-HPLC (30-73% B gradient, 214 nm and 440 nm detection) and MS analysis of the reactions between the H-CGK(F1)-NH 2 peptide and MBP (A) and PHPT1 (B) MES thioesters, purified from E. coli using the Int C -column.
  • FIG. 14 shows a one-pot purification/ligation experiment of ubiquitin to the H-CGK(Fluorescein)-NH 2 peptide (CGK(F1)).
  • Ub-Npu N from E. coli cell lysates was bound to the Int C -column, and after removal of contaminants through extensive washes, intein cleavage and ligation were triggered by addition of 200 mM MES and 1 mM CGK(F1) peptide.
  • FIG. 15 shows the semi-synthesis of H2B-K120Ac under denaturing conditions.
  • A) Coomassie stained SDS-PAGE analysis of H2B(1-116) ⁇ -thioester generation in the presence of 2 M urea (sup: cell lysate supernatant, trit: 1% triton wash of the inclusion bodies, inp: solubilized inclusion bodies used as input for the Int C -column).
  • E1-E6 were pooled, concentrated to 150 ⁇ M and ligated to the peptide H-CVTK(Ac)YTSAK-OH at 1 mM for 3 h at r.t.
  • FIG. 16 shows the characterization of ⁇ DEC205 ligated to the H-CGK(Fluorescein)-NH2 peptide (CGK(F1)).
  • Elution fractions from the Npu C -column containing ⁇ DEC205-MES thioester were concentrated to 20 ⁇ M and ligated to the CGK(F1) fluorescent peptide at 1 mM for 48 h at r.t.
  • Expected mass for ligation product 50221.2 Da.
  • Free HC 49575.0 Da.
  • FIG. 17 shows purification of ⁇ DEC thioesters expressed in CHO cells using a split-intein column. Top) Coomassie stained SDSPAGE gel of the purification of ⁇ DEC-MES thioester from CHO cells using a Npu C -column. Bottom) Western blot analysis of the same purification.
  • FIG. 18 shows purification of ⁇ DEC thioesters using an Ava C split-intein column and Western blot analysis of the purification of ⁇ DEC thioesters from mammalian cell supernatants using an Ava C -column.
  • FIG. 19 shows expression tests of ⁇ DEC205 antibody fused to Ava N split intein through the C-terminus of the antibody light chain and western blot analysis of CHO cell supernatants expressing the ⁇ DEC205-AvaN fusion at different timepoints.
  • inteins Of the roughly 600 inteins currently catalogued, (7) less than 5% are split inteins, mostly from a family known as the cyanobacterial split DnaE inteins (8). Surprisingly, only six of these, including Npu, have been experimentally analyzed to any extent, (6,9,10) and only Npu and its widely-studied, low-efficiency ortholog from Synechocystis species PCC6803 (Ssp) have been rigorously characterized in vitro.(5,11)
  • This assay can be carried out in the background of varying local C-extein sequences without significantly perturbing the dynamic range. Since all DnaE inteins splice the same local extein sequences in their endogenous context, this screen was originally carried out in a wild-type C-extein background (CFN) within the KanR enzyme. As expected, bacteria expressing the Npu intein had a high relative IC 50 , whereas clones expressing Ssp showed poor resistance to kanamycin. Remarkably, more than half of the DnaE inteins showed splicing efficiency comparable to Npu in vivo at 30° C. ( FIG. 1 b ).
  • Npu, Cra(CS505), and Cwa inteins were analyzed in vitro in the presence of a +2 glycine. All three of these reactions were characterized by rapid accumulation of thioester intermediates, which slowly resolved over tens of minutes into the spliced product and the N-extein cleavage product.
  • intein folds their locations on the intein fold (14) may provide some insights into their function.
  • an aromatic residue is preferred in the high-activity inteins. This position is adjacent to the conserved catalytic TXXH motif (positions 69-72), and an aromatic residue may facilitate packing interactions to stabilize those residues.
  • a glutamate is preferred at position 122, proximal to catalytic histidine 125. The glutamate at position 89 is involved in an intimate ion cluster that was previously shown to be important for stabilizing the split intein complex.(13)
  • E23 is distant from the catalytic site and has no obvious structural role. This position is conceivably important for fold stability or dynamics as has previously been observed for activating point mutations in other inteins.(15,16)
  • the fragments of the different fast-splicing split inteins can be mixed as non-cognate pairs and still retain highly efficient splicing activity, further expanding the options available for any trans-splicing application.
  • the N-fragment split intein of Npu or variant thereof can bind to the C-fragment of Npu or variant thereof or any of the other split intein C-fragments or variants discussed below.
  • the N-fragment of Ssp or variant thereof can bind to the C-fragment of Ssp or variant thereof or any of the other split intein C-fragments or variants thereof discussed below;
  • the N-fragment of Aha or variant thereof can bind to the C-fragment of Aha or variant thereof or any of the other split intein C-fragments or variants thereof discussed below;
  • the N-fragment of Aov or variant thereof can bind to the C-fragment of Aov or variant thereof or any of the other split intein C-fragments or variants thereof discussed below;
  • the N-fragment of Asp or variant thereof can bind to the C-fragment of Asp or variant thereof or any of the other split intein C-fragments or variants thereof discussed below;
  • the N-fragment of Ava or variant thereof can bind to the C-fragment of Ava or variant thereof or any of the other split intein C-fragments or variants thereof discussed below;
  • any split intein can be artificially fused and then utilized as a cis-splicing intein in this application ( 1 in FIG. 4 a ).
  • Ultrafast split inteins are especially attractive in this regard due to their speed and efficiency.
  • artificially fused variants of Npu, Ava, and Mcht with an N-terminal ubiquitin domain were generated.
  • premature C-terminal cleavage or undesired high levels of competing hydrolysis residues Asn137 and Cys+1 were mutated to Ala.
  • This thioester intermediate is generally thought to be transiently populated in protein splicing, and to our knowledge, it has never been directly observed.(1) Surprisingly, when analyzing the ubiquitin-DnaE intein fusions by reverse phase HPLC, two major peaks and a third minor peak were often observed, all bearing the same mass. The relative abundance of these species could be modulated by unfolding the proteins or by changes in pH, and the two major species were almost equally populated from pH 4-6 ( FIG. 4 d ). The major peaks most likely correspond to the precursor amide, 1, and the linear thioester, 2, and the minor peak as the tetrahedral oxythiazolidine intermediate.
  • polypeptide refers to any amino acid based polymer, interchangeable referred to as a “protein” throughout, and can include glycoproteins and lipoproteins.
  • the polypeptide is a polypeptide excreted from a cell (e.g., a mammalian cell).
  • the polypeptide is an antibody or a fragment thereof.
  • the polypeptide can be any naturally occurring or synthetic polypeptide of interest, including polypeptides having one or more amino acid residues other than the 20 naturally occurring amino acids.
  • the polypeptide has a molecular weight of 45 kDa or greater, 50 kDa or greater, 60 kDa or greater, 75 kDa or greater, 100 kDa or greater, 120 kDa or greater, or 150 kDa or greater.
  • the polypeptide can be, e.g., an antibody or a fragment thereof.
  • the split intein N-fragment can be fused to one or both of the heavy chains, and/or to one or both of the light chains.
  • the polypeptide is a protein secreted from a cell, e.g., a mammalian cell.
  • the split intein N-fragment comprises a sequence as shown in FIG. 5 , e.g., Npu (SEQ ID NO: 1), Ssp (SEQ ID NO: 2), Aha (SEQ ID NO: 3), Aov (SEQ ID NO: 4), Asp (SEQ ID NO: 5), Ava (SEQ ID NO: 6), Cra(CS505) (SEQ ID NO: 7), Csp(CCY0110) (SEQ ID NO: 8), Csp(PCC8801) (SEQ ID NO: 9), Cwa (SEQ ID NO: 10), Maer (NIES843) (SEQ ID NO: 11), Mcht(PCC7420) (SEQ ID NO: 12), Oli (SEQ ID NO: 13), Sel(PC7942) (SEQ ID NO: 14), Ssp(PCC7002) (SEQ ID NO: 15), Tel (SEQ ID NO: 16), Ter (SEQ ID NO: 17), Tvu (SEQ ID NO: 18), or a variant thereof.
  • the spilt intein N-fragment sequence comprises a sequence other than Npu (SEQ ID NO: 1) or Ssp (SEQ ID NO: 2), and in other cases, comprises a sequence other than Npu (SEQ ID NO: 1), Ssp (SEQ ID NO: 2), or Aha (SEQ ID NO: 3).
  • the split intein N-fragment sequence comprises a sequence of Ava (SEQ ID NO: 6), Cra (SEQ ID NO: 7), Csp(PCC8801) (SEQ ID NO: 9), Cwa (SEQ ID NO: 10), Mcht (PCC7420) (SEQ ID NO: 12), Oli (SEQ ID NO: 13), Ter (SEQ ID NO: 17) and Tvu (SEQ ID NO: 18).
  • the split intein N-fragment has a sequence comprising a consensus sequence of SEQ ID NO: 19: (CLSYDTEILTVEYGAVPIGKIVEENIECTVYSVDENGFVYTQPIAQWHDRGEQEVFE YCLEDGSTIRATKDHKFMTEDGEMLPIDEIFEQGLDLKQVKGLPD).
  • a variant of a split intein N-fragment is a mutated split intein N-fragment as disclosed herein that maintains the activity of the split intein N-fragment (e.g., its ability to bind to a split intein C-fragment and/or catalyze nucleophilic attack of the polypeptide fused to it).
  • Contemplated variants of a split intein N-fragments disclosed herein include mutation of one or more C residues, except for Cysl, to an aliphatic residue, such as an A, I, L, or F, or to a S residue.
  • One such variant contemplated is a mutant Npu with Cys28 and Cys59 mutated to Ser, SEQ ID NO: 20 (CLSYETEILTVEYGLLPIGKIVEKRIESTVYSVDNNGNIYTQPVAQWHDRGEQEVFEY SLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN).
  • the split intein C-fragments disclosed herein are mutated from the naturally occurring sequences to mutate the N137 and C+1 residues to a residue other than Asn or Gln for N137 and a residue other than Cys for C+1 (SEQ ID NOs: 129-146).
  • the mutations at these two positions are to a hydrophobic residue, e.g., not containing a free SH thiol (Cys), a carboxylic acid (Asp, Glu), or a base (Arg, His, Lys) or other undesired group (e.g., Asn, Gln) on the side chain.
  • the two mutated aliphatic residues can be the same or different and can be A, V, I, S, M, H, L, F, Y, G, or W or can be a unnatural (e.g., not encoded by genetic code) aliphatic amino acid residue such as norleucine, 2-aminobutyric acid, nor-valine, 2-aminopentoic acid, or 2-aminohexaanoic acid (SEQ ID NOs: 219-236).
  • mutations where both residues are selected from A, I, V, L, Y, G and F (SEQ ID NOs: 309-326).
  • at least one of the two mutated residues is A.
  • a mutated split intein C-fragment comprising a mutation at N137 and Cys+1 of Npu (SEQ ID NOs: 129, 147, 165, 183, 201, 219, 237, 255, 273, 291, 309, 327, 345, 363, 381 and 399), Ssp (SEQ ID NOs: 130, 148, 166, 184, 202, 220, 238, 256, 274, 292, 310, 328, 346, 364, 382 and 400), Aha (SEQ ID NOs: 131, 149, 167, 185, 203, 221, 239, 257, 275, 293, 311, 329, 347, 365, 383 and 401), Aov (SEQ ID NOs: 132, 150, 168, 186, 204, 222, 240, 258, 276, 294, 312, 330, 348, 366, 384 and 402), Asp (SEQ ID NOs: 129, 147,
  • At least one of the mutations is A (SEQ ID NOs: 183-200, 255-272, 327-344, and 345-416) and in more specific cases, both mutations are A (SEQ IS NOs: 399-416).
  • the solid support is a polymer or substance that allows for linkage of the split intein C-fragment, optionally via a linker.
  • the linker can be further amino acid residues engineered to the C-terminus of the split intein C-fragment or can be other known linkers for attachment of a peptide to a support.
  • One contemplated linker is a small peptide -SGGC (SEQ ID NO: 705), where the thiol of the C-terminal Cys can be used to attach the split intein C-fragment to the support.
  • SEQ ID NO: 705 small peptide -SGGC
  • sequences noted above having a -SGGC peptide linker (SEQ ID NO: 705) (e.g., specifying the residues starting at the N137 position: AAFN-SGGC) (SEQ ID NO:706).
  • the length of a pepide linker can be modified to provide varying lengths and flexibility in any individual sitation (e.g., more than 2 Gly residues).
  • the C-terminus residue of a peptide linker can be modified to introduce an appropriately reactive functional group to attach the split intein C-fragment to a surface of choice (e.g., Lys to react via an amine, Cys to react via a thiol, or Asp or Glu to react via a carboxylic acid).
  • peptide linker to provide other functional group moieties to allow for different attachment chemistry of the C-fragment to a support of interest (e.g., azide, alkynes, carbonyls, amino-oxy, cyano-benzothiazoles, tetrazoles, alkenes, alkyl-halides).
  • the linker can alternatively be a polymeric linker.
  • a consensus sequence for the split intein C-fragment is derived: SEQ ID NO: 707 (VKIISRQSLGKQNVYDIGVEKDHNFLLANGLIASN), as well as a mutated version where the N137 is mutated to other than Asn or Gln (SEQ ID NO:708), or more specifically, N137 is mutant to a naturally occurring or unnaturally occurring hydrophobic residue, such as A, V, I, M, H, L, F, Y, G, S, H, or W or can be a unnatural (e.g., not encoded by genetic code) aliphatic amino acid residue such as norleucine, 2-aminobutyric acid, nor-valine, 2-aminopentanoic acid, or 2-aminohexanoic acid (SEQ ID NO: 709).
  • a naturally occurring or unnaturally occurring hydrophobic residue such as A, V, I, M, H, L, F, Y, G, S, H, or W
  • +1 position can be a naturally occurring or unnaturally occurring hydrophobic residue such as A, V, I, M, H, L, F, Y, G, S, H, or W or can be a unnatural (e.g., not encoded by genetic code) aliphatic amino acid residue such as norleucine, 2-aminobutyric acid, nor-valine, 2-aminopentanoic acid, or 2-aminohexanoic acid (SEQ ID NOs: 713, 717, 721, and 725).
  • mutation at +1 position is selected from A, I, V, L, Y, G, and F (SEQ ID NOs: 714, 718, 722, and 726).
  • at least one of the mutated residues of the consensus sequence is A (SEQ ID NOs: 715, 719, and 723).
  • the consensus C-fragment sequence has both mutations as Ala (SEQ ID NO:727).
  • a consensus sequence comprising FN at the +2 and +3 positions (SEQ ID NO:728-743.
  • a consensus sequence comprising a peptide linker for attachment to a solid support, and one embodiment is -SGGC at positions +4-+7 (SEQ ID NO: 744-759).
  • the split intein C-fragment or variant thereof as disclosed herein can be attached to a solid support via a linker.
  • the linker is a polymer, including but not limited to a water-soluble polymer, a nucleic acid, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, or combinations thereof. It is not critical what the linker's chemical structure is, since it serves primarily as a linker. The linker should be chosen so as not to interfere with the activity of the C-fragment.
  • the linker can be made up of amino acids linked together by peptide bonds.
  • the linker comprises Y n , wherein Y is a naturally occurring amino acid or a steroisomer thereof and “n” is any one of 1 through 20.
  • the linker is therefore can be made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally-occurring amino acids.
  • the 1 to 20 amino acids are selected from Gly, Ala, Ser, Cys.
  • the linker is made up of a majority of amino acids that are sterically un-hindered, such as Gly.
  • Non-peptide linkers are also possible.
  • These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C 1 -C 6 ), halogen (e.g., Cl, Br), CN, NH 2 , phenyl, etc.
  • non-peptide linker is a polyethylene glycol group, such as: —HN—(CH 2 ) 2 —(O-CH 2 —CH 2 ) n —O—CH 2 —CO, wherein n is such that the overall molecular weight of the linker ranges from approximately 101 to 5000, preferably 101 to 500.
  • the linker has a length of about 0-14 sub-units (e.g., amino acids).
  • the length of the linker in various embodiments is at least about 10 nucleotides, 10-30 nucleotides, or even greater than 30 nucleotides.
  • the bases of the polynucleotide linker are all adenines, all thymines, all cytidines, all guanines, all uracils, or all some other modified base.
  • a non-nucleotide linker of the invention comprises a basic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds.
  • Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res.
  • non-nucleotide further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.
  • linkers contemplated include linear polymers (e.g., polyethylene glycol, polylysine, dextran, etc.), branched chain polymers (see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); lipids; cholesterol groups (such as a steroid); or carbohydrates or oligosaccharides.
  • Other linkers include one or more water soluble polymer attachments such as polyoxyethylene glycol, or polypropylene glycol as described U.S. Pat. Nos.
  • polystyrene resin 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337.
  • Other useful polymers as linkers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers.
  • polystylated polyols e.g., glycerol
  • oligonucleotide such as poly-A or hydrophilic or amphiphilic polymers are contemplated as linkers, including, for example, amphiphiles (including oligonucletoides).
  • Contemplated solid supports include resins, particles, and beads. More specific solid supports include polyhydroxy polymers, e.g. based on polysaccharides, such as agarose, dextran, cellulose, starch, pullulan, or the like, and synthetic polymers, such as polyacrylic amide, polymethacrylic amide, poly(hydroxyalkylvinyl ethers), poly(hydroxyalkylacrylates) and polymethacrylates (e.g. polyglycidylmethacrylate), polyvinyl alcohols and polymers based on styrenes and divinylbenzenes, and copolymers in which two or more of the monomers corresponding to the above-mentioned polymers are included.
  • polysaccharides such as agarose, dextran, cellulose, starch, pullulan, or the like
  • synthetic polymers such as polyacrylic amide, polymethacrylic amide, poly(hydroxyalkylvinyl ethers), poly(hydroxyalkylacryl
  • solid supports contemplated include agarose, sepharose, cellulose, polystyrene, polyethylene glycol, derivatized agarose, acrylamide, sephadex, sepharose, polyethyleneglycol (PEG)-acrylamide, and polystyrene-PEG based supports.
  • the solid support can be a resin such as p-methylbenzhydrylamine (pMBHA) resin (Peptides International, Louisville, Ky.), polystyrenes (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), including chloromethylpolystyrene, hydroxymethylpolystyrene and aminomethylpolystyrene, poly (dimethylacrylamide)-grafted styrene co-divinyl-benzene (e.g., POLYHIPE resin, obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (e.g., TENTAGEL or ARGOGEL, Bayer, Tubingen, Germany) polydimethylacrylamide resin (obtained from Milligen/Biosearch, California), or Sepharose (Pharmacia, Sweden).
  • pMBHA p-methylbenzhydrylamine
  • EPL Expressed Protein Ligation
  • EPL was first described in 1998 (21), as an expansion to recombinant proteins of Native Chemical Ligation (NCL) (19,22), and it consists on the reaction between a C-terminal recombinant protein ⁇ -thioester with a synthetic peptide containing a Cys at its N-terminus through the formation of a new native peptide bond between the two fragments.
  • NCL Native Chemical Ligation
  • Inteins such as GyrA or VMA, have been successfully harnessed to prepare a wide variety of protein thioesters.
  • inteins are usually fused to affinity tags such as the chitin bidning domain or the hexa-His tag.
  • affinity tags such as the chitin bidning domain or the hexa-His tag.
  • the reaction conditions required for efficient thiolysis affect the performance of such tags and subsequent additional purification steps are often required to obtain the desired pure product for ligation (24-26).
  • significant levels of in vivo premature cleavage can occur, significantly reducing the final product yield.
  • the Npu split-intein was chosen, which is one of the fastest DnaE split-inteins previously known [10]. Initially the ability of split Npu to generate protein thioesters in solution was tested by mixing the model protein ubiquitin fused to NpuN with a mutant NpuC (Asn137 and Cys+1 to Ala) in the presence (and absence) of the thiol MESNa. SDSPAGE, HPLC and MS analysis of the reactions showed the formation of the desired ubiqutin C-terminal ⁇ -thioester in a few hours.
  • NpuC-AA SEQ. ID NO: 777
  • NpuC-AA affinity resin protein C-terminal ⁇ -thioesters (Ubiquitin, MBP, PHPT1) could be easily produced and purified out of cell lysates ( FIG. 8 ).
  • HPLC and MS analysis confirmed the formation of the desired protein thioesters with very low levels of undesired hydrolysis ( FIG. 9 ).
  • split-inteins and also inteins
  • the N-terminal junction is regarded as more tolerant towards deviations from the native N-extein residues it was important to evaluate the effect that the C-terminal amino acid of the protein of interest ( ⁇ 1 residue according to intein numbering conventions) would have on the yields of thioester formation.
  • a complete library of Ub-X-NpuN fusion proteins was constructed where the C-terminal Ub residue (X) was varied from its native Gly to all other 19 proteinogenic amino acids. Proteins were expressed in E. coli and cell lysates, applied to the NpuC-AA affinity resin and purified.
  • Protein yields were estimated from the SDSPAGE analysis for each purification and hydrolysis levels from RP-HPLC and MS analysis of the elution fractions ( FIG. 10 ). Results show similar trends to those known for non-split inteins, such as GyrA (29), and most amino acids display high yields of cleavage after overnight incubation with MESNa, the exceptions being Pro and Glu, for which recovery were 49 and 50%, respectively. As expected, the Asn ⁇ -thioester could not be isolated due to the well-known reaction of its side-chain with the adjacent ⁇ -thioester to form a succinimide.
  • the system was next tested for the purification from inclusion bodies of a fragment of the histone H2B.
  • Preparation of site-specifically modified histones using EPL is a topic of major interest due to their crucial role in the understanding of epigenetic regulation.
  • histone fragments are remarkably poorly behaved and the preparation of their recombinant C-terminal ⁇ -thioesters particularly challenging.
  • a H2B(1-116) fragment fused to NpuN was expressed in E. coli and the inclusion bodies extracted with 6 M urea.
  • the H2B-NpuN fusion was subsequently diluted into a 3 M urea buffer and the corresponding C-terminal ⁇ -thioester generated concomitant with purification over the NpuC-AA affinity resin ( FIG. 11 ).
  • H2B(1-116)C-terminal thioester was obtained in excellent purity (>90% by RP-HPLC) and isolated yield ( ⁇ 20 mg per L of culture). This represents a significant improvement over previous protocols which afford less protein (4 mg per L of culture) and require the use of multiple chromatographic purification steps including RP-HPLC.
  • the H2B(1-116)-MES thioester obtained from the IntC-column can be directly used in EPL reactions without further purification. Accordingly, the protein was successfully ligated to a synthetic H2B(117-125) peptide containing an acetylated Lys at position 120 to yield semi-synthetic H2B-K120Ac ( FIG. 15 ).
  • the modification of antibodies is a field of intense research, specially focused on the development of therapeutic antibody-drug-conjugates (30).
  • the identity of the N-intein could have a significant effect on the expression levels of its fusion to a given protein of interest.
  • the N-fragment of several of the fastest split DnaE inteins cross-reacted with NpuC, allowing one to use any of them with the same NpuC based affinity column. Accordingly, the expression levels of a model antibody ( ⁇ DEC, antibody against the DEC205 receptor) were tested and found the highest levels of expression were obtained when ⁇ DEC was fused to the AvaN intein ( FIG. 12A ).
  • ⁇ DEC-AvaN fusions were transfected into 293T cells and after 4 days of culture the supernatants were collected and purified over the NpuC-AA-column.
  • the presence of a C-terminal thioester in the purified ⁇ DEC was confirmed by reacting it with a short fluorescent peptide with an N-terminal Cys residue ( FIGS. 12B and C).
  • MS of the deglycosylated and reduced ⁇ DEC-fluorophore conjugate was used to confirm its identity and SEC-MALS demonstrate the product was monodisperse and of the expected size for an IgG antibody.
  • Split-inteins can be engineered for the preparation of protein ⁇ -thioesters and that the strong affinity between the two split-intein fragments provides a powerful handle for their purification.
  • the generality of the approach is demonstrated by using it to generate highly pure thioesters of both soluble (ubiqutin, MBP, PHPT) and insoluble proteins (H2B fragment) as well as monoclonal antibodies ( ⁇ DEC).
  • ubiqutin, MBP, PHPT soluble proteins
  • H2B fragment insoluble proteins
  • ⁇ DEC monoclonal antibodies
  • several N-inteins can be tested for optimal expression levels of the protein of interest and used with one single NpuC-column.
  • split inteins disclosed herein can be used to purify and modify a polypeptide of interest.
  • a polypeptide of interest is provided in a fusion protein with a split intein N-fragment, e.g., via well-known recombinant protein methods.
  • the fusion protein is then contacted with a corresponding split intein C-fragment under conditions that allow binding of the N-fragment and C-fragment to form an intein intermediate.
  • the split intein C-fragment can be bound to a support (e.g., a solid support such as a resin) or can subsequently (e.g., after binding to the split intein N-fragment to form the intein intermediate) be bound to a support.
  • Washes can include detergents, denaturing agents and salt solutions (e.g., NaCl).
  • the intein intermediate can be reacted with a nucleophile to release the polypeptide of interest from the bound N- and C-fragment inteins wherein the C-terminus of the polypeptide is modified by the nucleophile added.
  • the nucleophile can be a thiol to directed yield the polypeptide as an ⁇ -thioester, which in turn can be further modified, e.g., with a different nucleophile (e.g., a drug, a polymer, another polypeptide, a oligonucleotide), or any other moiety using the well-known ⁇ -thioester chemistry for protein modification at the C-terminus.
  • a different nucleophile e.g., a drug, a polymer, another polypeptide, a oligonucleotide
  • One advantage of this chemistry is that only the C-terminus is modified with a thioester for further modification, thus allowing for selective modification only at the C
  • the nucleophile that is used in the methods disclosed herein either with the intein intermediate or as a subsequent nucleophile reacting with, e.g., a ⁇ -thioester can be any compound or material having a suitable nucleophilic moiety.
  • a thiol moiety is contemplated as the nucleophile.
  • the thiol is a 1,2-aminothiol, or a 1,2-aminoselenol.
  • An ⁇ -selenothioester can be formed by using a selenothiol (R-SeH).
  • Alternative nucleophiles contemplated include amines (i.e.
  • nucleophile can be a functional group within a compound of interest for conjugation to the polypeptide of interest (e.g., a drug to form a protein-drug conjugate) or could alternatively bear an additional functional group for subsequent known bioorthogonal reactions such as an azide or an alkyne (for a click chemistry reaction between the two function groups to form a triazole), a tetrazole, an ⁇ -ketoacid, an aldehyde or ketone, or a cyanobenzothiazole.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • DIPEA N,N-diisopropylethylamine
  • Kanamycin sulfate Kan
  • BME ⁇ -Mercaptoethanol
  • DTT sodium 2-mercaptoethanesulfonate
  • EDT ethanedithiol
  • Coomassie brilliant blue N,N-dimethylformamide (DMF)
  • Tetrakis(triphenylphosphine)palladium(0) Pd(PPh 3 ) 4
  • phenylsilane triisopropylsilane (TIS) sodium diethyldithiocarbamate trihydrate
  • 5(6)-carboxyfluorescein were purchased from Sigma-Aldrich (St. Louis, Mo.).
  • Tris(2-carboxyethyl)phosphine hydrochloride was purchased from Thermo Scientific (Rockford, Ill.). Fmoc-Gly-OH, Fmoc-Lys(Alloc)-OH, and Boc-Cys(Trt)-OH were purchasd from Novabiochem (Laufelfingen, Switzerland). Piperidine was purchased from Alfa Aesar (Ward Hill, Mass.). Dichloromethane (DCM) and rink amide resin were purchased from EMD Chemicals (Billerica, Mass.). 1-Hydroxybenzotriazole hydrate (HOBt) was purchased from AnaSpec (Fremont, Ca).
  • Trifluoroacetic acid was purchased from Halocarbon (North Augusta, S.C.). Complete protease inhibitor tablets were purchased from Roche Diagnostics (Mannheim, Germany). Nickel-nitrilotriacetic acid (Ni-NTA) resin was from Novagen (Gibbstown, N.J.). The QuikChange XL II site directed mutagenesis kit was from Agilent (La Jolla, Calif.). DpnI and the Phusion High-Fidelity PCR kit were from New England Biolabs (Ipswich, Mass.).
  • DNA purification kits QIAprep spin minikit, QIAquick gel extraction kit, QIAquick PCR purification kit
  • Qiagen Valencia, Calif.
  • Sub-cloning efficiency DH5 competent cells and One Shot BL21(DE3) chemically competent E. coli were purchased from Invitrogen (Carlsbad, Calif.) and used to generate “in-house” high-competency cell lines.
  • Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, Iowa). The new intein genes were generated synthetically and purchased from GENEWIZ (South Plainfield, N.J.). All plasmids used in this study were sequenced by GENEWIZ.
  • Criterion XT Bis-Tris gels (12%), Immun-blot PVDF membrane (0.2 ⁇ m), and Bradford reagent dye concentrate were purchased from Bio-Rad (Hercules, Calif.). 20 ⁇ MES-SDS running buffer was purchased from Boston Bioproducts (Ashland, Mass.). Mouse anti-myc monoclonal antibody ( ⁇ -myc) was purchased from Invitrogen (Carlsbad, Calif.). Anti-His Tag, clone HIS.H8 mouse monoclonal antibody ( ⁇ -His6) was purchased from Millipore (Billerica, Mass.). Mouse HA.11 monoclonal antibody ( ⁇ -HA) was purchased from Covance (Princeton, N.J.). IRDye 800CW goat anti-Mouse IgG secondary antibody (Licor mouse 800) and Licor Blocking Buffer were purchased from LI-COR Biotechnology (Lincoln, Nebr.).
  • Size-exclusion chromatography was carried out on an ⁇ KTA FPLC system from GE Healthcare. Both preparative and analytical FPLC were carried out on a Superdex 75 10/300 or S200 10/300 column. For all runs, proteins were eluted over 1.35 column volumes of buffer (flow rate: 0.5 mL/min).
  • Analytical RP-HPLC was performed on Hewlett-Packard 1100 and 1200 series instruments equipped with a C18 Vydac column (5 ⁇ m, 4.6 ⁇ 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.
  • Protein sequences of the split DnaE inteins were obtained from the NEB InBase1. This list consisted of 23 entries as of May 2011. Of these entries, two were discarded from the study as they did not have a C-intein sequence: Csp(PCC7822) and Nosp(CCY9414). Two pairs of inteins had identical sequences: Nsp(PCC7120) with Asp (these are most likely the same organism with two different names) and Sel(PCC6301) with Sel(PC7942). Thus, Nsp(PCC7120) and Sel(PCC6301) were removed from the library.
  • the Mcht(PCC7420) and Oli C-intein sequences were identical, but both inteins were kept in the library as their N-intein sequences were different.
  • the Aov intein had an “X” at position 87 in place of an absolutely conserved isoleucine (I), so 187 was utilized at this position.
  • the plasmid for the kanamycin resistance assays bearing the Csp(PCC7424) intein proved to be unstable and yielded highly variable results; thus this intein was excluded from the analyses.
  • the final library contained 18 inteins, Table 1.
  • N- and C-inteins were manually aligned using the multiple alignment software Jalview2. All N-intein sequences were “left-justified” to align the first cysteine residue, and the variable N-intein tail region was not aligned. All C-intein sequences were “right-justified” to align the C-terminal asparagine.
  • the residue numbering used in this study is based on the numbering for the NMR structure of a fused Npu intein (PDB code 2KEQ).
  • PDB code 2KEQ fused Npu intein
  • the C-intein numbering starts at 103, except for the Tel and Tvu inteins, which have a gap at this position and start at 104.
  • sequence logos FIG. 6
  • the high activity sequence logos were comprised of Cwa, Cra(CS505), Csp(PCC8801), Ava, Npu, Csp(CCY0110), Mcht(PCC7420), Maer(NIES843), Asp, Oli, and Aha (which was included based on the high activity of the C120G mutant).
  • the low activity sequence logos were comprised of Aov, Ter, Ssp(PCC7002), Tvu, Tel, Ssp, and Sel(PC7942).
  • the sequence logos were generated using WebLogo.
  • Heat maps were generated using the statistical computing and graphics program “R”.
  • KanR promoter [KanR promoter]-[RBS]-[myc-KanRN]-[IntN]-[iRBS]-[IntC]-[CFN-KanRC]
  • RBS is a common E. coli ribosomal binding site
  • iRBS is an intervening ribosomal binding site preceded by a linker
  • myc encodes for a c-myc epitope tag (EQKLISEEDL) (SEQ ID NO: 760)
  • KanRN and KanRC are fragments of the KanR protein
  • IntN and IntC are split intein fragments.
  • Ssp plasmid was also constructed as previously described (36,37). These plasmids are referred to as myc-KanR-NpuDnaE-Split and myc-KanR-SspDnaE-Split.
  • synthetic genes were designed and purchased from GENEWIZ containing the following architecture:
  • the entire synthetic gene was amplified with Phusion High-Fidelity Polymerase using primers annealing to the 5′ and 3′ overhangs.
  • the resulting megaprimer was inserted into the myc-KanR plasmid in place of Npu by overlap-extension PCR with Phusion polymerase (39).
  • the plasmids are named as: myc-KanR-XyzDnaE-Split (where Xyz indicates the intein name as given in Table 1). Specific point mutations were made to various inteins using a QuikChange Site-Directed Mutagenesis kit with the standard recommended protocol.
  • Intein activity-coupled kanamycin resistance (KanR) assays were conducted in 96-well plate format as previously described (36,37). Typically, plasmids were transformed into 15 ⁇ L of sub-cloning efficiency DH5 ⁇ cells by heat shock, and the transformed cells were grown for 18 hours at 37° C. in 3 mL of Luria-Bertani (LB) media with 100 ⁇ g/mL of ampicillin (LB/amp). The over-night cultures were diluted 250-fold into LB/amp solutions containing 8 different kanamycin concentrations (150 ⁇ L per culture). The cells were grown at 30° C.
  • KanR Intein activity-coupled kanamycin resistance
  • OD Obs OD Min + ( OD Max - OD Min ) 1 + 10 [ ( log ⁇ ⁇ IC 50 - log ⁇ [ Kan ] ) ⁇ HillSlope ]
  • DH5 ⁇ cells were transformed with the assay plasmids identically as for the 96-well plate setup and grown for 18 hours at 37° C. while shaking. The overnight cultures were used to inoculate 3 mL of fresh LB/amp at a 1:300 dilution, and the cells were incubated at 30° C. for 24 hours. The ODs of the 30° C. cultures were measured at 650 nm to assess relative bacterial levels, then 150 ⁇ L of each culture was transferred to an Eppendorf tube and centrifuged at 17,000 rcf for 2 minutes.
  • the supernatant was aspirated off, and the cell pellets were resuspended/lysed in ⁇ 200 ⁇ L of 2 ⁇ SDS gel loading dye containing 4% BME (the resuspension volumes were varied slightly to normalize for differences in OD).
  • the samples were boiled for 10 minutes, then centrifuged at 17,000 rcf for 1 minute.
  • Each sample (5 ⁇ L) was loaded onto a 12% Bis-Tris gel and run in MES-SDS running buffer.
  • the proteins were transferred to PVDF membrane in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 15% methanol) at 100V for 90 minutes.
  • Membranes were blocked with 4% milk in TBST, then the primary antibody ( ⁇ -myc, 1:5000) and secondary antibody (Licor mouse 800, 1:15,000) were sequentially applied in 4% milk in TBST. The blots were imaged using the Licor Odyssey scanner.
  • N-intein expression plasmids were derived from a previously described NpuN plasmid, pMR-Ub-NpuN(WT) (36,37). This plasmid encoded for the following protein sequence:
  • IntN plasmids were cloned using overlap-extension PCR to generate Ub-IntN fusion genes in homologous plasmids in a traceless manner (39).
  • N-intein genes were amplified by Phusion polymerase from the synthetic gene plasmids using primers with overhangs that anneal to the plasmid sequences surrounding NpuN in pMR-Ub-NpuN- ⁇ Gly 4 .
  • the resulting megaprimer was then used to insert the new N-intein gene in place of NpuN to generate a new plasmid called pMR-Ub-IntN that was identical to the NpuN plasmid except for the N-intein gene.
  • the C-intein plasmids were all derived from a previously described NpuC plasmid, pET-NpuC(WT)-SUMO (37). This plasmid encoded for the following protein sequence:
  • IntC plasmids were cloned using overlap-extension PCR to generate IntC-SUMO fusion genes in homologous plasmids in a traceless manner (39).
  • C-intein genes were amplified by Phusion polymerase from the synthetic gene plasmids using primers with overhangs that anneal to the plasmid sequences surrounding NpuC in pET-NpuC(WT)-SUMO.
  • the resulting megaprimer was then used to insert the new C-intein gene in place of NpuC to generate a new plasmid called pET-IntC-SUMO that was identical to the NpuC plasmid except for the C-intein gene.
  • lysis buffer 50 mM phosphate, 300 mM NaCl, 5 mM imidazole, 2 mM BME, pH 8.0
  • the cell pellets were resuspended by adding an additional 15 mL of lysis buffer supplemented with Complete protease inhibitor cocktail. Cells were lysed by sonication (35% amplitude, 8 ⁇ 20 second pulses separated by 30 seconds on ice). The soluble fraction was recovered by centrifugation (35,000 rcf, 30 min). The soluble fraction was mixed with 2 mL of Ni-NTA resin and incubated at 4° C. for 30 minutes. After incubation, the slurry was loaded onto a fritted column.
  • the column was washed with 5 column volumes (CV) of lysis buffer, 5 CV of wash buffer 1 (lysis buffer with 20 mM imidazole), and 3 CV of wash buffer 2 (lysis buffer with 50 mM imidazole).
  • the protein was eluted with elution buffer (lysis buffer with 250 mM imidazole) in four 1.5 CV elution fractions.
  • the wash and elution fractions were analyzed by SDS-PAGE.
  • the proteins were purified by gel filtration.
  • the wash and elution fractions were all treated with 50 mM DTT for 30 minutes on ice.
  • the first elution fraction was then directly injected on an S75 10/300 gel filtration column (3 ⁇ 1 mL injections) and eluted over 1.35 CV in freshly prepared, degassed splicing buffer (100 mM phosphates, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.2).
  • the concentration of pure proteins were determined by UV A280 nm and by the Bradford assay.
  • the Ub-CwaN protein did not express well in the soluble fraction, and all of the enriched protein was aggregated, as observed by gel filtration analysis.
  • the protein was extracted from the insoluble fraction of the lysate as follows. First, the lysate pellet was resuspended in 20 mL of Triton wash buffer (lysis buffer with 0.1% Triton X-100) and incubated at room temperature for 30 minutes. The Triton wash was centrifuged at 35,000 rcf for 30 minutes, and the supernatant was discarded.
  • Triton wash buffer lysis buffer with 0.1% Triton X-100
  • the pellet was resuspended in 20 mL of lysis buffer containing 6 M urea, and the mixture was incubated overnight at 4° C. The mixture was centrifuged at 35,000 rcf for 30 minutes, and then the supernatant was mixed with 2 mL of NiNTA resin.
  • the Ni column was run identically as for the native purifications described above, except that every buffer had a background of 6 M urea. Following enrichment over a Ni-NTA column, the 50 mM imidazole wash and the first two elution fractions were pooled and diluted to 0.2 mg/mL.
  • the diluted protein was refolded into lysis buffer (without urea) by step-wise dialysis removal of the urea at 4° C.
  • the protein was concentrated four-fold to 3 mL and immediately purified by gel filtration as indicated for the native purifications above.
  • the pure protein was analyzed by analytical gel filtration, analytical RP-HPLC, and mass spectrometry. Note that this construct was highly susceptible to aggregation. When re-folded at 2 mg/mL rather than 0.2 mg/mL, less than 10% of the obtained protein was monomeric, whereas more dilute refolding yielded roughly 50% monomeric protein.
  • the obtained protein was 80% monomeric, and the monomer to aggregate ratio did not change after 24 hours of storage at 4° C.
  • the concentration of pure protein was determined by the Bradford assay.
  • the AvaC-SUMO and Csp(PCC8801)C-SUMO proteins did not express well at 37° C., so the proteins were re-expressed by induction at 18° C. for 16 hours.
  • the 50 mM imidazole wash and the first two elution fractions were pooled and dialyzed into TEV cleavage buffer (50 mM phosphate, 300 mM NaCl, 5 mM imidazole, 0.5 mM EDTA, 0.5 mM DTT, pH 8.0) then treated with 40 ⁇ g of His-tagged TEV protease overnight at room temperature.
  • TEV cleavage buffer 50 mM phosphate, 300 mM NaCl, 5 mM imidazole, 0.5 mM EDTA, 0.5 mM DTT, pH 8.0
  • the cleavage was confirmed by RP-HPLC/MS, after which the reaction solution was incubated with Ni-NTA resin at room temperature for 30 min. The flow-through and two 1.5 CV washes with wash buffer 1 were collected and pooled.
  • the protein was then concentrated to 3-4 mL, injected onto the S75 10/300 gel filtration column (3 ⁇ 1 mL injections), and eluted over 1.35 CV in freshly prepared, degassed splicing buffer (100 mM phosphates, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.2).
  • degassed splicing buffer 100 mM phosphates, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.2.
  • FPLC fractions were analyzed by SDS-PAGE, and the purest fractions were pooled and analyzed by analytical gel filtration, analytical RP-HPLC, and mass spectrometry.
  • the concentration of pure protein was determined by UV A 280 nm and by the Bradford assay.
  • a typical reaction volume was 300 ⁇ L and was carried out in an Eppendorf tube on a heat block. During the reaction, 20 ⁇ L aliquots of the reaction solution were removed at the desired time points and quenched in 20 ⁇ L of 2 ⁇ concentrated SDS gel loading dye on ice to afford a final quenched solution with 40 mM Tris ( ⁇ pH 7.0), 10% (v/v) glycerol, 1% (w/v) SDS, 0.02% (w/v) bromophenol blue, and 2% (v/v) BME. For each reaction, an artificial zero time point was taken by mixing equivalent amounts of starting materials directly into the quencher solution. Samples were boiled for 10 minutes then centrifuged at 17,000 rcf for 1 minute. Aliquots of starting materials and time points (15 ⁇ L) were loaded onto Bis-Tris gels and run in MES-SDS running buffer. The gels were Coomassie-stained then imaged using the Licor Odyssey scanner.
  • each lane of a gel was analyzed using the Licor Odyssey quantification function or ImageJ. Given the close proximity of the starting material bands, these bands were typically integrated together. To normalize for loading error, the integrated intensity of each band in a lane was expressed as a fraction intensity of the total band intensity in that lane (which remained relatively constant between lanes). These normalized intensities were plotted as a function of time, and data from three independent reactions were collectively fit to first-order rate equations using the GraphPad Prism software:
  • Y is the fractional intensity of a species
  • t is time in minutes
  • S is a scaling factor for reactant depletion (allowed to vary)
  • Z indicates the fraction of reactant remaining at the reaction endpoint (allowed to vary)
  • Y max is a scaling factor for product formation
  • k obs is the observed first-order rate constant for the splicing reaction (allowed to vary).
  • reaction solution 90 ⁇ L aliquots of the reaction solution were removed at the desired time points and quenched in 30 ⁇ L of a quenching solution (6 M guanidine hydrochloride with 4% trifluoroacetic acid). 100 ⁇ L of each quenched time point were injected onto an analytical C18 RP-HPLC column and eluted over a 25-73% buffer B gradient in 30 minutes, preceded by a two minute isocratic phase in 25% buffer B (see Equipment section for column and running buffer specifications). At different time points, various HPLC peaks were collected and their identities were confirmed by mass spectrometry. The IntC-(Ub)SUMO species were identified by MS, verifying branched intermediate formation and depletion.
  • Fmoc-based solid phase peptide synthesis was used to produce a peptide with the sequence H-Cys-Gly-Lys(Fluorescein)-NH 2 .
  • the peptide was synthesized on Rink amide resin at a 0.2 mmol scale as follows: 20% piperidine in DMF was used for Fmoc deprotection using a one minute equilibration of the resin followed by a 20 minute incubation. After Fmoc deprotection, amino acids were coupled using DIC/HOBt as activating agents.
  • the amino acid (1.1 mmol) was dissolved in 50:50 DCM:DMF (2 mL) and was activated with DIC (1.0 mmol) and HOBt (1.2 mmol) at 0° C. for 15 minutes. The mixture was added to the N-terminally deprotected resin and coupled for 10 minutes at room temperature.
  • the lysine side chain was deprotected by treatment with Pd(Ph 3 ) 4 (0.1 eq.) and phenylsilane (25 eq.) in dry DCM for 30 minutes.
  • the peptidyl resin was washed with DCM (2 ⁇ 5 mL) and DMF (2 ⁇ 5 mL) followed by two washes with 0.5% DIPEA in DMF (v/v) and two washes with 0.5% sodium diethyldithiocarbamate trihydrate in DMF (w/v) to remove any remaining traces of the Pd catalyst.
  • 5(6)-Carboxyfluorescein was then coupled to the lysine side chain using the DIC/HOBt activation method overnight at room temperature.
  • the peptide was cleaved off the resin using 94% TFA, 1% TIS, 2.5% EDT, and 2.5% H 2 O (6.5 mL) for one hour. After cleavage, roughly half of the TFA was evaporated under a stream of nitrogen. The crude peptide was precipitated with cold ether and washed with cold ether twice. Finally, the peptide was purified by RP-HPLC on C18 prep column over a 15-80% buffer B gradient in 40 minutes. The purified peptide was analyzed by analytical RP-HPLC and ESI-MS to confirm its identity. Note that no attempt was made to separately isolate the 5-carboxyfluorescein and 6-carboxyfluorescein conjugates, thus the peptide is a mixture of these two isomers.
  • This plasmid was modified to replace the MxeGyrA intein with a fused Npu intein.
  • the myc-KanR-NpuDnaE-Split plasmid was modified by QuikChange to remove the iRBS sequence separating the NpuN and NpuC genes.
  • the resulting plasmid, myc-KanR-NpuDnaE-Fused, was then used as a template to amplify megaprimers bearing the fused Npu intein with overhangs homologous to the sequences surrounding MxeGyrA in the modified pTXB1 vector.
  • Npu gene with the N137A mutation was inserted in place of MxeGyrA using overlap-extension PCR with the Phusion polymerase. (39) Importantly, this construct was modified to include the native C-extein residues of Npu (CFN) instead of those for MxeGyrA (TEA).
  • CFN C-extein residues of Npu
  • TEA MxeGyrA
  • proteins were fused to AvaDnaE or MchtDnaE to test the sequence dependence on thiolysis from these inteins.
  • the proteins utilized were the N-terminal SH3 domain of human Grb2 (AAs 1-55+/ ⁇ an exogenous C-terminal Gly), the SH2 domain of human Abl kinase (AAs 122-217), eGFP, and the catalytic domain of human PARP1 (AAs 657-1015). All plasmids were cloned using the aforementioned methods to yield plasmids encoding the following proteins:
  • the cell pellets were transferred to 50 mL conical tubes with 5 mL of lysis buffer (50 mM phosphate, 300 mM NaCl, 5 mM imidazole, No BME, pH 8.0) and stored at ⁇ 80° C. The cell pellets were resuspended by adding an additional 15 mL of lysis buffer supplemented with Complete protein inhibitor cocktail. Cells were lysed by sonication (35% amplitude, 8 ⁇ 20 second pulses separated by 30 seconds on ice). The soluble fraction was recovered by centrifugation (35,000 rcf, 30 min).
  • the soluble fraction was mixed with 2 mL of Ni-NTA resin and incubated at 4° C. for 30 minutes. After incubation, the slurry was loaded onto a fritted column. After discarding the flow-through, the column was washed with 5 column volumes (CV) of lysis buffer, 5 CV of wash buffer 1 (lysis buffer with 20 mM imidazole), and 3 CV of wash buffer 2 (lysis buffer with 50 mM imidazole).
  • the protein was eluted with elution buffer (lysis buffer with 250 mM imidazole) in four 1.5 CV elution fractions. The wash and elution fractions were analyzed by SDS-PAGE with loading dye containing no thiols.
  • the cleanest fractions were pooled and treated with 10 mM TCEP for 20 minutes on ice. Then, the solution was injected on an S75 or 5200 10/300 gel filtration column (2 ⁇ 1 mL injections), and eluted over 1.35 CV in thiolysis buffer (100 mM phosphates, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, pH 7.2).
  • thiolysis buffer 100 mM phosphates, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP, pH 7.2.
  • the FPLC fractions were analyzed by SDS-PAGE with loading dye containing no thiols, and the purest fractions were pooled and analyzed by analytical RP-HPLC and mass spectrometry. The concentration of pure protein was determined by UV A 280 nm .
  • reaction solution 5 ⁇ L were removed and quenched in 30 ⁇ L 2 ⁇ SDS loading dye containing no thiols. As time points were collected, they were stored at ⁇ 20° C. until the end of the reaction. After the reaction, the 35 ⁇ L quenched time points were thawed, treated with 1 ⁇ L of a 1 M TCEP stock solution, boiled for 10 minutes, and centrifuged at 17,000 rcf for 1 minute. Time points (5 ⁇ L) were loaded onto 12% Bis-Tris gels and run in MES-SDS running buffer. The gels were first imaged on a fluorescence imager to visualize the Ub-CGK-Fluorescein ligation product.
  • the gels were coomassie-stained and imaged using the Licor Odyssey scanner.
  • the reaction endpoints were quenched by 20-fold dilution in H 2 O with 0.1% TFA and injected on an analytical C18 RP-HPLC column. The mixture was separated over a 2 minute isocratic phase in 0% B followed by a 0-73% B linear gradient in 30 minutes. The major peaks were collected and analyzed by MS.
  • the 45 ⁇ L quenched time points were thawed, treated with 1 ⁇ L of a 1 M TCEP stock solution, boiled for 10 minutes, and centrifuged at 17,000 rcf for 1 minute.
  • Time points (15 ⁇ L) were loaded onto 12% Bis-Tris gels and run in MES-SDS running buffer. Then the gels were coomassie-stained and imaged using the Licor Odyssey scanner.
  • the reaction endpoints were quenched by 4-fold dilution in H 2 O with 0.1% TFA and injected on an analytical C18 RP-HPLC column. The mixture was separated over a 2 minute isocratic phase in 0% B followed by a 0-73% B linear gradient in 30 minutes. The product peaks were collected and analyzed by MS.
  • this species appears to accumulate during protein expression resulting in a mixture of “trapped” (dehydrated) and “free” (native, hydrated) fusion protein.
  • the “free” protein Upon addition of MESNa at neutral pH, the “free” protein rapidly undergoes thiolysis to yield the desired product, and the “trapped” protein slowly rehydrates and is also thiolyzed to yield the same desired product.
  • a “burst” phase was observed followed by a slower phase.
  • the accumulation of dehydrated fusion protein could be reduced by expression at lower temperatures (18° C. instead of 37° C.), and these reactions could be driven faster and closer to completion by increasing the MESNa concentration from 100 mM to 200 mM MESNa.

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