WO2013188635A2 - Nouveaux procédés d'identification de protéines par étiquetage n-terminal et extraction sélective - Google Patents

Nouveaux procédés d'identification de protéines par étiquetage n-terminal et extraction sélective Download PDF

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WO2013188635A2
WO2013188635A2 PCT/US2013/045594 US2013045594W WO2013188635A2 WO 2013188635 A2 WO2013188635 A2 WO 2013188635A2 US 2013045594 W US2013045594 W US 2013045594W WO 2013188635 A2 WO2013188635 A2 WO 2013188635A2
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protein
transferase
azido
amino acid
derivative
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PCT/US2013/045594
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WO2013188635A3 (fr
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E. James PETERSSON
Christopher J. Noren
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The Trustees Of The University Of Pennsylvania
New England Biolabs, Inc.
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Publication of WO2013188635A2 publication Critical patent/WO2013188635A2/fr
Publication of WO2013188635A3 publication Critical patent/WO2013188635A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • Post-translational modification of a protein by proteases may regulate the activity of the protein, such as in the case of zymogens (e.g., angiotensinogen, trypsinogen, chymotrypsinogen, pepsinogen, most proteins in the coagulation system, pro-caspases, pro-elastase, pro-lipase and pro-carboxypo f ypeptidases), and may be an important disease marker (caspases are themselves proteases that regulate cell death, and aberrant caspase activity is common in cancer).
  • N-terminal modification can regulate protein activity through degradation by the Clp machinery in prokaryotes or the 26S proteosome in eukaryotes. Thus, identifying the substrates of proteases and N-terminal modifying enzymes may be important to understand protein stability and its role in regulating enzyme activity.
  • conjugation examples include protein microarrays to investigate protein-protein interactions, and proteins labeled with near-IR quantum dots for imaging in deep tissue.
  • Protein modification can also significantly improve protein properties so that they may be used as therapeutic and diagnostic agents: thermostability, resistance to degradation (both enzymatic and non-enzymatic in nature), increase in solubility, and improved formulation characteristics.
  • Degradation-resistant modifications have been achieved by PEG modification of proteins and diagnostic antibodies. In most cases, the formation of well-defined conjugates is valuable, if not essential, for product development.
  • N-termini can be ligated to synthetic peptides to produce semi-synthetic proteins (Hackenberger et al., 2008, Angew. Chem. Int. Ed. 47:10030-10074).
  • Reverse proteolysis methods can be used to modify the N-terminus under conditions that do not require protein unfolding, but the reaction can be difficult to drive to completion in the absence of high protein
  • Aminoacyl tRNA transferases are members of a gro wing class of enzymes that use aminoacyl tRNAs in secondary metabolism (Lahoud et al, 2010, Nat. Chem. Biol. 6:795-796).
  • AaT catalyzes the transfer of Leu, Phe, or Met from an aminoacyl tRNA to a protein bearing an ' N-terminal Arg or Lys (Kaji et al., 1963, Biochem. Biophys. Res. Comm. 10:406-409; Suto et al., 2006, EMBO J.
  • BpT catalyzes the transfer of Leu from an aminoacyl tRNA to a protein bearing an -terminal Asp or Glu (Graciet et al, 2006, Proc. Natl. Acad. Sci. USA 103:3078-3083).
  • Leu or Phe targets that protein for degradation by ClpA as part of the N-end rule pathway (Mogk et al., 2007, Trends Cell Biol. 17: 165-172; Tobias et al, 1991, Science 254: 1374-1377; Varshavsky, 2008, Nat. Struct. Mol. Biol. 15:1238-1240;
  • Soffer and Leibowitz subsequently reconstituted the purified AaT enzyme and characterized its specificity for both the RNA and amino acid components of the donor molecule, demonstrating its use in transferring a non-natural amino acid, jp-fluorophenylalanine (Leibowitz et al., 1969, Biochem.
  • AaT has been used to modify proteins in vitro with a variety of non-natural amino acids charged onto tRNAs, either by chemical semisynthesis or the use of a mutant aminoacyl tRNA synthetase (aaRS)
  • aaRS aminoacyl tRNA synthetase
  • AaT could bind phenylalanyl adenosine and transfer Phe to peptides in trace amounts (Watanabe et al, 2007, Nature 449:867-871).
  • adenosine mononucleoside as a donor substrate for AaT has not been further explored. No such exploration of donor scope has occurred for BpT.
  • the present invention provides a method of determining whether a protein of interest is present in a sample, the method comprising contacting the sample with a transferase and a derivative of an azido-containing amino acid or a salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the N- terminus of the protein of interest, thereby yielding a first system.
  • the method also optionally comprises isolating a protein-containing fraction from the first system.
  • the method further comprises contacting the first system or protein-containing fraction thereof with a dibenzocyclooctyne-containing biotin derivative, under conditions whereby the azido and dibenzocyclooctyne groups react to form a triazole group, thereby yielding a second system.
  • the method optionally comprises isolating a protein- containing fraction from the second system.
  • the method further comprises contacting the second system or protein-containing fraction thereof with a solid surface comprising an avidin-containing molecule immobilized thereon, under conditions whereby biotin and the avidin-containing molecule bind to each other.
  • the method further comprises sequencing any protein bound to the avidin-containing molecule immobilized on the solid surface, thereby determining whether the protein of interest is present in the sample.
  • the contacting of the sample with the transferase and the derivative of an azido-containing amino acid or a salt thereof is carried out under conditions that do not significantly denature the protein of interest.
  • the first system is substantially free of a synthetase.
  • the synthetase comprises aminoacy! tR A synthetase.
  • the derivative of the azido-containing amino acid is the adenosine ester of the azido-containing amino acid.
  • the derivative of the azido-containing amino acid comprises Azf-A or a salt thereof.
  • the transferase is an aminoacyl tRNA transferase
  • the transferase comprises E. coli AaT, V. vulnificus BpT, a mutant thereof and any combinations thereof.
  • the N-terminus of the protein of interest comprises Arg or Lys if the transferase comprises E. coli AaT, and the N-terminus of the protein of interest comprises Asp or Glu if the transferase comprises V, vulnificus BpT.
  • the protein of interest is produced by proteolysis of a larger protein.
  • the sequencing of any protein bound to the avidin- containing molecule immobilized on the solid surface comprises trypsinizing the protein before it is sequenced.
  • the present invention also provides a method of determining whether a protein comprising an internal cleavage site is present in a sample, the method comprising contacting the sample with a transferase and a derivative of an azido- containing amino acid or salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the N-terminus of the protein, thereby yielding a first system, wherein the first system is substantially free of a synthetase.
  • the method optionally comprises isolating a protein-containing fraction from the first system.
  • the method further comprises contacting the first system or protein-containing fraction thereof with a dibenzocyclooctyne-containing biotin derivative, under conditions whereby the azido and dibenzocyclooctyne groups react to form a triazole group, thereby yielding a second system.
  • the method optionally comprises isolating a protein- containing fraction from the second system.
  • the method further comprises submitting the second system or protein-containing fraction thereof to reagents capable of cleaving the internal cleavage site, thereby yielding a third system.
  • the method optionally comprises isolating a protein-containing fraction from the third system.
  • the method further comprises contacting the third system or protein-containing fraction thereof with a solid surface comprising an avidin-containing molecule immobilized thereon, under conditions whereby biotin and the avidin-containing molecule bind to each other, thereby yielding a fourth system.
  • the method further comprises determining whether any protein remains in the supernatant of the fourth system, wherein, if any protein remains in the supernatant of the fourth system, a protein comprising the internal cleavage site is present in the sample.
  • the contacting of the sample with the transferase and the derivative of an azido-containing amino acid or a salt thereof is carried out under conditions that do not significantly denature the protein comprising the internal cleavage site.
  • the synthetase comprises aminoacyi tRNA synthetase.
  • derivative of the azido-containing amino acid is the adenosine ester of the azido-containing amino acid.
  • the derivative of the azido-containing amino acid comprises Azf-A or a salt thereof.
  • the transferase is an aminoacyi tRNA transferase (AaT) or mutants thereof.
  • the transferase comprises E. coli AaT, V. vulnificus BpT, a mutant thereof and any combinations thereof.
  • the N-termi is of the protein of interest comprises Arg or Lys if the transferase comprises E, coli AaT, and the N-terminus of the protein of interest comprises Asp or Giu if the transferase comprises V. vulnificus BpT.
  • determining whether a protein remains in the supernatant of the fourth system comprises sequencing any protein in the supernatant.
  • the present invention also provides a kit comprising a transferase, a derivative of an azido-containing amino acid or a salt thereof, a dibenzocyclooctyne- containing biotin derivative, and an instructional material for use thereof.
  • instructional material comprises instructions for a method of determining whether a protein of interest is present in a sample, and the method comprises contacting the sample with the transferase and the derivative of an azido-containing amino acid or a salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the N-terminus of the protein of interest, thereby yielding a first system.
  • the method optionally comprises isolating a protein-containing fraction from the first system.
  • the method further comprises contacting the first system or protein-containing fraction thereof with the dibenzocyclooctyne-containing biotin derivative, under conditions whereby the azido and dibenzocyclooctyne groups react to form a triazole group, thereby yielding a second system.
  • the method optionally comprises isolating a protein- containing fraction from the second system.
  • the method further comprises contacting the second system or protein-containing fraction thereof with a solid surface comprising an avidin-containing molecule immobilized thereon, under conditions whereby biotin and the avidin-containing molecule bind to each other.
  • the method further comprises identifying any proteinaceous molecule that may be bound to the avidin-containing molecule immobilized on the solid surface, thereby determining whether the protein of interest is present in the sample.
  • the present invention also provides a kit comprising a transferase, a derivative of an azido-containing amino acid or a salt thereof, a dibenzocyclooctyne- containing biotin derivative, and an instructional material for use thereof.
  • the instructional material comprises instructions for a method of determining whether a protein comprising an internal cleavage site is present in a sample, and the method comprises contacting the sample with the transferase and the derivative of an azido- containing amino acid or salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the N-terminus of the protein, thereby yielding a first system, wherein the first system is substantially free of a synthetase.
  • the method optionally comprises isolating a protein-containing fraction from the first system.
  • the method further comprises contacting the first system or protein-containing fraction thereof with a dibenzocyclooctyne-containing biotin derivative, under conditions whereby the azido and dibenzocyclooctyne groups react to form a triazole group, thereby yielding a second system.
  • the method optionally comprises isolating a protein- containing fraction from the second system.
  • the method further comprises submitting the second system or protein-containing fraction thereof to reagents capable of cleaving the internal cleavage site, thereby yielding a third system.
  • the method optionally comprises isolating a protein-containing fraction from the third system.
  • the method comprises contacting the third system or protein-containing fraction thereof with a solid surface comprising an avidin-containing molecule immobilized thereon, under conditions whereby biotin and the avidin-containing molecule bind to each other.
  • the method optionally comprises determining whether a protein remains in the supernatant, wherein, if a protein remains in the supernatant, a protein comprising the internal cleavage site is present in the sample.
  • the present invention also provides a method of determining one or more cleavage sites for a protein-cleaving reagent, the method comprising constructing a random peptide library, wherein peptides contained therein comprise a fixed N-terminal residue followed by at least one randomized residue, wherein the peptides in the library are physically linked to an amplifiable genetic moiety and the identity of each peptide in the library is specified by the sequence of a corresponding nucleic acid contained within the amplifiable genetic moiety.
  • the method further comprises contacting the library with a transferase and a derivative of an azido-containing amino acid or salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the fixed N-terminal residue of the peptides in the library, thereby yielding a first system.
  • the method optionally comprises isolating a protein-containing fraction from the first system.
  • the method further comprises contacting the first system, or the isolated protein- containing fraction thereof, with an alkyne-containing biotin derivative under conditions whereby the azido and alkyne groups react to form a triazole group, thereby yielding a second system.
  • the method optionally comprises isolating a protein-containing fraction from the second system.
  • the method further comprises subjecting the second system or the isolated protein-containing fraction thereof, to a protein-cleaving reagent capable of cleaving an internal cleavage site, thereby yielding a third system.
  • the method optionally comprises isolating a protein-containing fraction from the third system.
  • the method further comprises contacting the third system or the isolated protein-containing fraction thereof, with a solid surface comprising a biotin-binding molecule immobilized thereon, under conditions whereby biotin and the biotin-binding molecule bind to each other, thereby yielding a fourth system.
  • the method further comprises sequencing the nucleic acids contained within the amplifiable genetic moiety in the fourth system to identify individual peptide molecules which remain in the supernatant of the fourth system, wherein any sequences that remain in the supernatant of the fourth system comprise an internal cleavage site for the protein cleavage reagent.
  • the transferase comprises E. coli AaT
  • the fixed N-terminal residue of the peptide library comprises Arg or Lys.
  • the N-terminal residue of the peptide library comprises Asp or Glu.
  • the amplifiable genetic moiety comprises a phage virion, a living cell, mRNA, a plasmid, or a paused ribosome.
  • the alkyne in the alkyne-containing biotin derivative comprises a dibenzocyclooctyne moiety.
  • the biotin-binding molecule comprises avidin, streptavidin, an anti-biotin antibody, or derivatives thereof.
  • the protein-cleavage reagent comprises a proteolytic enzyme.
  • the protein-cleavage reagent comprises a chelated metal or other small molecule.
  • the present invention also provides a method of determining one or more cleavage sites for a protein-cleaving reagent, the method comprising constructing a random peptide library, wherein peptides contained therein comprise a fixed N-terminal residue followed by at least one randomized residue, wherein the peptides in the library are physically linked to an amplifiable genetic moiety and the identity of each peptide in the library is specified by the sequence of a corresponding nucleic acid contained within the amplifiable genetic moiety.
  • the method further comprises contacting the library with a transferase and a derivative of an azido-containing amino acid or salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the fixed N-terminal residue of the peptides in the library, thereby yielding a first system.
  • the method optionally comprises isolating a protein-containing fraction from the first system.
  • the method further comprises contacting the first system, or the isolated protein- containing fraction thereof, with an alkyne-containing biotin derivative under conditions whereby the azido and alkyne groups react to form a triazole group, thereby yielding a second system.
  • the method optionally comprises isolating a protein-containing fraction from the second system.
  • the method further comprises subjecting the second system or the isolated protein-containing fraction thereof, to a protein-cleaving reagent capable of cleaving an internal cleavage site, thereby yielding a third system.
  • the method further comprises sequencing the nucleic acids contained within the amplifiable genetic moiety in the third system to identify individual peptide molecules which remain in the supernatant of the third system, wherein any sequences that remain in the supernatant of the third system comprise an internal cleavage site for the protein cleavage reagent.
  • the transferase comprises E. coli AaT
  • the fixed N-terminal residue of the peptide library comprises Arg or Lys.
  • the N-terminal residue of the peptide library comprises Asp or Glu.
  • the amplifiable genetic moiety comprises a phage virion, a living cell, mRNA, a plasmid, or a paused ribosome.
  • the alkyne in the alkyne-containing biotin derivative comprises a dibenzocyclooctyne moiety.
  • the biotin-binding molecule comprises avidin, streptavidin, an anti-biotin antibody, or derivatives thereof.
  • the protein-cleavage reagent comprises a proteolytic enzyme.
  • the protein-cleavage reagent comprises a chelated metal or other small molecule.
  • Figure 1 is a series of schemes depicting transferase-mediated N-terminal protein modification.
  • Figure 1 A illustrates that fully enzymatic methods use aminoacyl tRNA synthetase (aaRS), tRNA, aminoacyltransferase (AaT), aminoacid, and ATP.
  • Figure IB illustrates that chemoenzymatic methods use only a synthetic nucleic acid donor and AaT.
  • an aminoacyl mononucleoside (R n H) was used.
  • Figure 2 is a series of schemes illustrating expressed protein ligation strategies.
  • FIG. 2A Strategy 1 - A protein fragment is expressed as a fusion to a "stalled" intein, and thioester exchange produces the protein as a thioester capable of undergoing ligation to a synthetic peptide with an N-terminal Cys, selenocysteine (Sec) or equivalent.
  • the ligated product places the synthetic peptide at the C-terminus.
  • Sec can be converted to Ala by treatement with TCEP (tris(carboxyethyl)phosphine) under mild conditions.
  • Strategy 2 - A protein fragment is expressed with a tag that can be proteolyzed to reveal an N-terminal Cys, which is then ligated to a synthetic peptide thioester.
  • Traceless Ligation - A protein fragment is expressed with an N-terminal Lys, Arg, Asp, or Glu (generated by proteolysis, action of methionine aminopeptidase, or any other means known to those skilled in the art).
  • a transferase (AaT recognizes Lys or Arg, BpT recognizes Asp or Glu) attaches a hemiselenide-protected amino acid such as C*sp (a protected Sec derivative).
  • C*sp a protected Sec derivative
  • FIG. 2B Top - Protein Modification for Traceless Protein Ligation Reactions.
  • Compounds 4j and 4k can be synthesized from Boc-protected amino acids as described herein, and then used to transfer a masked Cys or Sec to the protein N-terminus using Arg or Lys as the recognition element for AaT and Asp or Glu as the recognition element for BpT. Sec can then be used in a ligation with a thioester and then deselenized using tris(carboxyethyl) phosphine (TCEP) to form Ala, leaving any Cys in the protein intact.
  • TCEP tris(carboxyethyl) phosphine
  • the right portion of the panel illustrates Sec transfer to N-terminus as part of a strategy to synthesize large semi-synthetic proteins.
  • the left portion of the panel illustrates modification of proteins with N-terminal Arg or Lys by aminoacyl adenosine donors.
  • the right portion of the panel illustrates labeling of antibodies with arbitrary N-terminal sequences using mutant AaTs with alternate recognition sequences.
  • Figure 3 is a series of reversed-phase HPLC traces for the analysis of AaT transferase reactions.
  • Figures 3A-3C depict AaT-mediated transfer of Phe from adenosyl donor 4a to LysAlaAcm reporter peptide.
  • HPLC chromatograms obtained after 0 h ( Figure 3A) or after 4 h ( Figure 3B) show conversion of LysAlaAcm (1 1.8 min retention time) to PheLysAlaAcm (13.0 min retention time). Conversion after one addition of donor 4a was 92% ( Figure 3B).
  • a second addition of 1 mM 4a drives the reaction to completion with a small amount of double Phe addition as depicted in Figure 3C.
  • Figure 3D depicts the results of HPLC analysis of transfer of Nap from adenosyl donor 4d to LysAlaAcm after 4 h shows conversion to NapLysAlaAcm (14.1 min retention time). Product identities were confirmed by observation of indicated masses by MALDI MS.
  • Figure 4 is a series of images illustrating the results of kinetic analysis of AaT reactions.
  • Figure 4A depicts real time monitoring of Nap transfer to LysAlaAcm from 4d by quenching of Acm fluorescence. No significant inhibition was observed in the presence of 1 mM adenosine.
  • Figure 4B depicts saturation curve used to determine Michaelis-Menten kinetic parameters for LysAlaAcm
  • Figure 5 is a series of images illustrating
  • FIG. 5A shows an a-casein modification scheme.
  • Figure 5B is an image depicting PAGE gel analysis of ⁇ -casein modification. Lanes (left to right): (1) molecular weight (MW) markers (Masses in kDa: 17, 25, 30, 46, 58, 80, 175); (2) a-casein; (3) ⁇ -casein mixed with fluorescein alkyne (FlAlk); (4) ⁇ -casein mixed with FlAlk, CuS0 4 , THPTA, and sodium ascorbate; (5) ⁇ -casein mixed with Azf-A (4c) and AaT; (6) ⁇ -casein mixed with 4c and AaT, then FlAlk; (7) ⁇ -casein mixed with 4c and AaT, then propargylamine (Alk), CuS0 4 , tris-(3-hydroxypropyltriazolylmethyl) amine
  • Figure 5C depicts the results of MALDI MS analysis of trypsinized N-terminal fragment of a-casein with or without modification by Azf using AaT and 4c (double addition).
  • Figure 6 is a series of images depicting the results of reversed-phase HPLC analysis of AaT transferase reactions.
  • Figures 6A-6C depict AaT -mediated transfer of Cys and Hcs from adenosyl donor 4k, 41, or 4n to the LysAlaAcm reporter peptide.
  • HPLC chromatograms obtained after 4 h show conversion of LysAlaAcm (11.3 min retention time) to CsmLysAlaAcm ( Figure 6A, 12.3 min retention time), CspLysAlaAcm ( Figure 6B, 13.0 min retention time), or
  • Figure 6C depicts the results of HPLC analysis of transfer of Hem from adenosyl donor 4n to LysAlaAcm, deprotection, and ligation to the Ac-MetAspValPhe thioester peptide to form Ac- MetAspValPheHcsLysAlaAcm (17.7 min retention time). Product identities were confirmed by observation of indicated masses by MALDI MS.
  • Figure 7 is an image depicting 1H and 13 C NMR characterization of Boc- Leu-(DMT)-A 3b.
  • Figure 8 is an image depicting 1H and 13 C NMR characterization of Boc-Azf-(DMT)-A 3c.
  • Figure 9 is an image depicting 1H and 13 C NMR characterization of Boc-Nap-(DMT)-A 3d.
  • Figure 10 is an image depicting 1H and 13 C NMR characterization of
  • Figure 11 is an image depicting 1H and 13 C NMR characterization of Boc-Acf-(DMT)-A 3f.
  • Figure 12 is an image depicting 1H and 13 C NMR characterization of Boc-N 3 f-(DMT)-A 3g.
  • Figure 13 is an image depicting 1H and 13 C NMR characterization of Boc-Mcm-(DMT)-A 3h.
  • Figure 14 is an image depicting 1H and 13 C NMR characterization of Boc-Bzf-(DMT)-A 3i.
  • Figure 15 is an image depicting the results of HPLC analysis of donor 4a purity after TFA deprotection.
  • Figure 16 is an image depicting the results of HPLC analysis of donor 4a hydrolysis in mock transferase reactions. Slight differences in retention time were observed, and compound identities were confirmed by MALDI MS.
  • Figure 17 is an image depicting PAGE gel analysis of AaT expression and purification. Lanes (left to right): 1) Molecular weight markers (Masses in kDa: 16, 25, 32, 47, 80, 100, 210); 2) Pre-induction; 3) Post-induction; 4) Crude cell lysate; 5) Supernatant after centrifugation; 6) Ni-NTA column flow-through; 7) Column wash 1 ; 8) Column wash 3; 9) Elution fraction 1 ; 10) Combined elution fractions 3-5 after overnight dialysis.
  • Lanes (left to right): 1) Molecular weight markers (Masses in kDa: 16, 25, 32, 47, 80, 100, 210); 2) Pre-induction; 3) Post-induction; 4) Crude cell lysate; 5) Supernatant after centrifugation; 6) Ni-NTA column flow-through; 7) Column wash 1 ; 8) Column wash 3; 9) Elution fraction 1 ; 10) Combined elution
  • Figure 18, is a series of images demonstrating fluorescence emission of NapLysAlaAcm solutions.
  • Figure 18A depicts fluorescence emission of mixtures of LysAlaAcm and NapLysAlaAcm (5d);
  • Xsd 0.00, 0.10, 0.20, 0.25, 0.33, 0.50, 1.00.
  • Figure 18B depicts linear fit of normalized
  • Figure 19 is a series of images illustrating Phe transfer reaction kinetics.
  • Figure 20 is a series of images depicting the results of Edman degradation analysis of a-casein modification.
  • Figure 20A depicts the results of unmodified a-casein.
  • R peak area was 196 counts total, F peak area was 44 counts.
  • Figure 20B depicts the results of Phe-modified a-casein.
  • F peak area was 97 counts, R peak area was 53 counts.
  • Figure 21 is a series of images demonstrating the results of PAGE gel analysis of ⁇ -casein modification in cleared E. coli lysate.
  • Figure 21A depicts the results of Coomassie-stained gel.
  • Figure 21B is a fluorescence image from 302 nm excitation.
  • Figure 22 is an image illustrating click chemistry reagents.
  • Figure 23 illustrates a direct pull-down experiment contemplated within the invention.
  • specific N-terminal amino acids in a protein are detected by transferase labeling.
  • the transferase recognition site (the N-terminus amino acid, wherein X is its side chain) is generated either by proteolysis or by modification by a ligating enzyme.
  • the N-terminus amino acid is recognized by a transferase (AaT recognizes and is capable of transferring an amino acid to N-terminus R or K; BpT recognizes and is capable of transferring an amino acid to N-terminus D or E); in this case, the transferase attaches Azf to the N-terminus, utilizing Azf-A as the donor.
  • the Azf-modified protein is labeled with biotin via reaction with a dibenzocyclooctyne (DBCO) biotin molecule.
  • Biotin pull-down is used to capture the protein, which may be trypsinized and sequenced.
  • Figure 24 illustrates the identification of proteolyzed proteins by selective elimination of unproteolyzed proteins from solution.
  • the transferase recognition site (X) at the N-terminus allows tagging of substrate library with Azf and DBCO biotin.
  • the N-terminal residue of the protein or peptide is recognized by a transferase (AaT recognizes and is capable of transferring an amino acid to N-terminus R or K; BpT recognizes and is capable of transferring an amino acid to N-terminus D or E); in this case, the transferase attaches Azf to the N-terminus, utilizing Azf-A as the donor.
  • the Azf-modified protein is labeled with biotin via reaction with a DBCO biotin molecule.
  • Proteolysis is then performed under conditions wherein the Z internal site is not cleaved but the Z 2 internal site is cleaved. Under such conditions, the protein comprising the internal Z 2 site has its biotin label cleaved, but the protein comprising the internal Zi site retains its biotin label.
  • An immobilized biotin affinity reagent may be used to remove uncleaved or unproteolyzed protein, and remaining cleaved or
  • proteolyzed protein may be identified by MS/MS sequencing, for example.
  • Figure 25 illustrates non-limiting examples of dibenzocyclooctyne (DBCO)-containing biotin derivatives contemplated within the invention.
  • DBCO dibenzocyclooctyne
  • the present invention includes a method of identifying a proteolyzed or modified protein.
  • the N-terminus of the proteolyzed or modified protein comprises an amino acid reside that is recognized by an enzyme.
  • the enzyme is an aminoacyl tRNA transferase.
  • the aminoacyl tRNA transferase is selected from the group consisting of E. coli (AaT), V. vulnificus (BpT), mutants thereof, and any combination thereof.
  • the transferase is capable of transferring a non-natural amino acid to the N-terminus of the proteolyzed or modified protein, using a derivative of the non-natural amino acid as the amino acid donor.
  • the donor of the non-natural amino acid is the adenosine ester of the non-natural amino acid.
  • the non-natural amino acid comprises an azido group.
  • the proteolyzed or modified protein comprising the azide-containing amino acid on its N-terminus may then be reacted with an alkynyl-containing compound, such as a dibenzocyclootyne (DBCO)- containing compound or a derivative thereof, whereby a triazole-containing adduct is formed.
  • DBCO dibenzocyclootyne
  • the alkynyl-containing compound further comprises a label, such as but not limited to biotin or a derivative thereof.
  • Biotin may bind tightly to avidin, streptoavidin or any avidin-related molecule known to those skilled in the art.
  • the N-terminus modification method described herein does not require prior protein manipulation, and may be performed under conditions that maintain the protein's natural fold and activity.
  • Various non-natural amino acids may be transferred to the N-terminus of a protein with high efficiency at relatively low protein concentrations using the methods of the invention.
  • the amino acids are provided within the methods of the invention as easily synthesized adenosine esters thereof.
  • the synthetic molecule is a detectable label that allows for the identification and quantification of the protein.
  • an element means one element or more than one element.
  • “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • MALDI MS matrix-assisted laser desorption/ionization mass spectrometry
  • ligation refers to the process to creating covalent chemical bonds among the two or more molecules, as to form at least one molecule that incorporates at least a portion of each of the two or more molecules.
  • abnormal when used in the context of organisms, tissues, cells or components thereof refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal” (expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.
  • amino acid refers to any natural or non-natural compound having a carboxyl group and an amino group in a molecule.
  • amino acid as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids.
  • Natural amino acid means any of the twenty L-amino acids commonly found in naturally occurring peptides.
  • Non- natural amino acid residues means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source.
  • synthetic amino acid encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or
  • a disulfide linkage may be present or absent in the peptides.
  • natural amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated below:
  • labeled amino acid refers to an amino acid that is bound to a label compound.
  • amino acid skeleton used herein includes a carboxyl group, an amino group, and a portion connecting them in an amino acid.
  • aromatic ring used herein generally refers to every type of unsaturated cyclic compound. Accordingly, it includes a 5- or 6-membered
  • a particularly preferable aromatic ring is a benzene ring.
  • naturally-occurring amino acids phenylalanine, tryptophan, and tyrosine are naturally-occurring aromatic amino acids comprising aromatic rings on their side chains.
  • a preferable example of the labeled amino acid of the present invention is a labeled compound in which a label compound is bound to the aromatic ring.
  • an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression that can be used to
  • the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal.
  • the instructional material of the kit of the invention can, for example, be affixed to a container that contains the identified compound, composition, vector, or delivery system of the invention or be shipped together with a container which contains the identified compound, composition, vector, or delivery system.
  • the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
  • salt embraces addition salts of free acids or free bases that are compounds useful within the invention.
  • Suitable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, phosphoric acids, perchloric and tetrafluoroboronic acids.
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, ⁇ -hydroxybutyric, sal
  • Suitable base addition salts of compounds useful within the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, lithium, calcium, magnesium, potassium, sodium and zinc salts.
  • Acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl- glucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding free base compound by reacting, for example, the appropriate acid or base with the corresponding free base.
  • label or “detectable label” or “tag” is a composition detectable by mass spectrometric, spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive isotopes (e.g., 3 H, 35 S,
  • a label as used in the context of the present invention is any entity that may be used to detect or isolate the product of interest.
  • any entity that is capable of binding to another entity may be used in the practice of this invention, including without limitation, epitopes for antibodies, ligands for receptors, and nucleic acids, which may interact with a second entity through means such as complementary base pair hybridization.
  • the term "organic group” is used for the purpose of this disclosure to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups).
  • polypeptide as used herein is defined as a chain of amino acid residues, usually having a defined sequence.
  • polypeptide is mutually inclusive of the terms “peptide” and "protein”.
  • Polypeptide also refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
  • protein typically refers to large polypeptides.
  • peptide typically refers to short polypeptides.
  • a peptide ester can carry a detectable label and a site for proteolysis or another form of chemical cleavage (e.g., through introduction of photolabile, acid-labile, or base-labile functional groups).
  • polypeptide sequences the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
  • proteases generally refer to a class of enzymes that cleave peptide bonds between amino acids of proteins. Because proteases use a molecule of water to effect hydrolysis of peptide bonds, these enzymes can also be classified as hydrolases.
  • Six classes of proteases are presently known: serine proteases, threonine proteases, cysteine proteases, aspartic acid proteases, metalloproteases, and glutamic acid proteases (see, e.g., Barrett A. J. et al, The Handbook of Proteolytic Enzymes, 2 nd ed. Academic Press, 2003).
  • proteases are involved in a multitude of physiological reactions from simple digestion of food proteins to highly regulated cascades (e.g., the cell cycle, the blood clotting cascade, the complement system, and apoptosis pathways). It is well known to the skilled artisan that proteases can break either specific peptide bonds, depending on the amino acid sequence of a protein, or break down a polypeptide to constituent amino acids.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject or individual is a human.
  • an “effective amount” or “therapeutically effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
  • treating a disease or disorder means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient.
  • Disease and disorder are used interchangeably herein.
  • terapéuticaally effective amount refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition described herein, including alleviating symptoms of such diseases.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the present invention includes a chemoenzymatic method of modifying a protein or peptide at the N-terminus, thus allowing for the labeling and/or introduction of at least one additional amino acid residue at the N-terminus of the protein or peptide.
  • the method utilizes a nucleoside-based amino acid donor in
  • the transferase transfers the non-natural or natural amino acid from the donor to the N-terminus of the protein, yielding a N-terminus modified protein.
  • the N-terminus modification of the protein may be further manipulated to further comprise a detectable label.
  • the present invention relates to the discovery that natural or non-natural amino acids may be transferred from adenosine donors to the N-terminus of proteins (or peptides) with high yields at relatively low protein concentrations.
  • the methods of the invention afford high yields under non-denaturing conditions.
  • the transferase useful within the method requires only a single amino acid on the protein for specific recognition. In one embodiment, the single amino acid recognized by the transferase is charged.
  • the methods of the invention are also useful in the context of protein ligation.
  • the chemoenzymatic method of the invention may be used to introduce a specific residue (such as cysteine, homocysteine, selenocysteine, or a derivative thereof) on the N-terminus of a protein.
  • This specific residue may then be used in a protein ligation reaction, thus resulting in a larger product protein.
  • the homocysteine residue incorporated in the product protein is methylated to yield a methionine residue.
  • the selenocysteine residue incorporated in the product protein is converted to alanine.
  • This invention includes methods for synthetically manipulating protein structures.
  • AaT can efficiently use a minimal adenosine substrate, which can be synthesized in one to two steps, as the source of the natural or non-natural aminoacid.
  • the invention includes adenosyl donors and the use of these donors in AaT-catalyzed protein modifications.
  • the use of adenosyl amino acid donors offers an advantage over the prior art by removing the substrate limitations associated with synthetases, and avoiding the complex synthesis of an oligonucleotide donor.
  • the use of AaT donors within the invention increases the potential substrate scope and reaction scale for N-terminus protein modification, without resorting to conditions that promote protein unfolding.
  • Escherichia coli (AaT) and V. vulnificus (BpT) can modify the N-terminus of a protein with an amino acid from a tRNA or a synthetic oligonucleotide donor.
  • AaT can efficiently use a minimal adenosine substrate, which can be synthesized in one to two steps from readily available starting materials.
  • the enzymatic activity of AaT with aminoacyl adenosyl donors has been characterized and it has been found that the reaction products do not inhibit AaT.
  • adenosyl donors removes the substrate limitations imposed by the use of synthetases for tRNA charging and avoids the complex synthesis of an oligonucleotide donor.
  • the present AaT donors increase the potential substrate scope and reaction scale for N- terminal protein modification under conditions that maintain folding.
  • results presented herein demonstrate that AaT can efficiently use an aminoacyl adenosine substrate to transfer disulfide-protected Cys analogs, which can be used in subsequent peptide ligation reactions. These Cys analogs can then be converted to other amino acids, making the ligation "traceless.” For example, Hcs-based analogs can be converted to Met by alkylation. The scope of molecules transferred thus far indicates that a variety of analogs including Sec derivatives should be transferable, allowing the ligation site to be masked as other amino acids, including Ala, Leu, Phe, and He. This effectively removes the requirement of Cys at the peptide ligation site of an expressed protein fragment. Compositions
  • the invention includes a composition comprising at least one compound selected from the group consisting of la-lz, 2a, 3a-3z, 4a-4z, a salt thereof, and any combinations thereof.
  • the structures of compounds la-lz, 2a, 3a-3z, and 4a-4z are depicted in Scheme 1 disclosed elsewhere herein.
  • the invention also includes a composition comprising at least one azido- containing amino acid, a salt thereof, and any combinations thereof.
  • the azido-containing amino acid is 4-azido-phenylalanine (Azf). Azf is depicted in Scheme 1 disclosed elsewhere herein.
  • the invention also includes a composition comprising at least one dibenzocyclooctyne (DBCO)-containing biotin derivative, a salt thereof, and any combinations thereof.
  • DBCO dibenzocyclooctyne
  • biotin derivatives include, but are not limited to, the molecules exemplified in Figure 26.
  • the invention includes a method of determining whether a protein of interest is present in a sample.
  • the method comprises contacting the sample with a transferase and a derivative of an azido-containing amino acid or a salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the N- terminus of the protein of interest, thereby yielding a first system.
  • the method optionally comprises isolating a protein-containing fraction from the first system
  • the method further comprises contacting the first system or protein-containing fraction thereof with a dibenzocyclooctyne-containing biotin derivative, under conditions whereby the azido and dibenzocyclooctyne groups react to form a triazole group, thereby yielding a second system
  • the method optionally comprises isolating a protein-containing fraction from the second system.
  • the method further comprises contacting the second system or protein- containing fraction thereof with a solid surface comprising an avidin-containing molecule immobilized thereon, under conditions whereby biotin and the avidin-containing molecule bind to each other.
  • the method further comprises sequencing any protein bound to the avidin-containing molecule immobilized on the solid surface, thereby determining whether the protein of interest is present in the sample.
  • the contacting of the sample with the transferase and the derivative of an azido-containing amino acid or a salt thereof is carried out under conditions that do not significantly denature the protein of interest.
  • the first system is substantially free of a synthetase.
  • the synthetase comprises aminoacyl tRNA synthetase.
  • the derivative of the azido-containing amino acid is the adenosine ester of the azido-containing amino acid.
  • the derivative of the azido- containing amino acid comprises Azf-A or a salt thereof.
  • the transferase is an aminoacyl tRNA transferase (AaT) or mutants thereof.
  • the transferase comprises E. coli AaT, V. vulnificus BpT, a mutant thereof and any combinations thereof
  • the N-terminus of the protein of interest comprises Arg or Lys if the transferase comprises E. coli AaT
  • the N- terminus of the protein of interest comprises Asp or Giu if the transferase comprises V. vulnificus BpT.
  • the protein of interest is produced by proteolysis of a larger protein.
  • the sequencing of any protein bound to the avidin-containing molecule immobilized on the solid surface comprises trypsinizing the protein before it is sequenced.
  • the invention further includes a method of determining whether a protein comprising an internal cleavage site is present in a sample.
  • the method comprises contacting the sample with a transferase and a derivative of an azido-containing amino acid or salt thereof, under conditions whereby the transferase transfers the azido- containing amino acid to the N-terminus of the protein, thereby yielding a first system, wherein the first system is substantially free of a synthetase.
  • the method optionally comprises isolating a protein-containing fraction from the first system.
  • the method further comprises contacting the first system or protein-containing fraction thereof with a dibenzocyclooctyne-containing biotin derivative, under conditions whereby the azido and dibenzocyclooctyne groups react to form a triazole group, thereby yielding a second system.
  • the method optionally comprises isolating a protein-containing fraction from the second system.
  • the method further comprises submitting the second system or protein- containing fraction thereof to reagents capable of cleaving the internal cleavage site, thereby yielding a third system.
  • the method optionally comprises isolating a protein- containing fraction from the third system.
  • the method further comprises contacting the third system or protein-containing fraction thereof with a solid surface comprising an avidin-containing molecule immobilized thereon, under conditions whereby biotin and the avidin-containing molecule bind to each other, thereby yielding a fourth system.
  • the method further comprises determining whether any protein remains in the supernatant of the fourth system, wherein, if any protein remains in the supernatant of the fourth system, a protein comprising the internal cleavage site is present in the sample.
  • contacting of the sample with the transferase and the derivative of an azido-containing amino acid or a salt thereof is carried out under conditions that do not significantly denature the protein comprising the internal cleavage site.
  • the synthetase comprises aminoacyl tRNA synthetase.
  • the derivative of the azido-containing amino acid is the adenosine ester of the azido-containing amino acid.
  • the derivative of the azido-containing amino acid comprises Azf-A or a salt thereof.
  • the transferase is an aminoacyl tRNA transferase (AaT) or mutants thereof.
  • the transferase comprises E. coli AaT, V. vulnificus BpT, a mutant thereof and any combinations thereof.
  • the N- terminus of the protein of interest comprises Arg or Lys if the transferase comprises E.
  • determining whether a protein remains in the supernatant of the fourth system comprises sequencing any protein in the supernatant.
  • Also included in the invention is a method of determining one or more cleavage sites for a protein-cleaving reagent.
  • the method comprises the following steps.
  • a random peptide library is constructed wherein peptides contained therein comprise a fixed N-terminal residue followed by at least one randomized residue.
  • the peptides in the library are physically linked to an amplifiable genetic moiety, and the identity of each peptide in the library is specified by the sequence of a corresponding nucleic acid contained within the amplifiable genetic moiety.
  • the library is contacted with a transferase and a derivative of an azido-containing amino acid or salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the fixed N-terminal residue of the peptides in the library, thereby yielding a first system.
  • a protein-containing fraction is isolated from the first system.
  • the first system, or the isolated protein-containing fraction thereof is contacted with an alkyne- containing biotin derivative, under conditions whereby the azido and alkyne groups react to form a triazole group, thereby yielding a second system.
  • a protein- containing fraction is isolated from the second system.
  • the second system, or isolated protein-containing fraction thereof is subjected to a protein-cleaving reagent capable of cleaving an internal cleavage site, thereby yielding a third system.
  • a protein- containing fraction is isolated from the third system.
  • the third system or the isolated protein-containing fraction thereof, is contacted with a solid surface comprising a biotin- binding molecule immobilized thereon, under conditions whereby biotin and the biotin- binding molecule bind to each other, thereby yielding a fourth system.
  • Individual peptide molecules which remain in the supernatant of the fourth system are identified via sequencing of the nucleic acid contained within the amplifiable genetic moiety in the fourth system, wherein any sequences that remain in the supernatant of the fourth system comprise an internal cleavage site for the protein cleavage reagent.
  • the transferase comprises E. coli AaT
  • the fixed N-terminal residue of the peptide library comprises Arg or Lys.
  • the transferase comprises V. vulnificus BpT
  • the N-terminal residue of the peptide library comprises Asp or Glu.
  • the amplifiable genetic moiety comprises a phage virion, a living cell, mRNA, a plasmid, or a paused ribosome.
  • the alkyne in the alkyne-containing biotin derivative comprises a dibenzocyclooctyne moiety.
  • the biotin-binding molecule comprises avidin, streptavidin, an anti-biotin antibody, or derivatives thereof.
  • the protein-cleavage reagent comprises a proteolytic enzyme. In other embodiments, the protein-cleavage reagent comprises a chelated metal or other small molecule.
  • Also included in the invention is a method of determining one or more cleavage sites for a protein-cleaving reagent.
  • the method comprises the following steps.
  • a random peptide library is constructed comprising a fixed N-terminal residue followed by at least one randomized residue.
  • the peptides in the library are physically linked to an amplifiable genetic moiety.
  • the identity of each peptide in the library is specified by the sequence of a corresponding nucleic acid contained within the amplifiable genetic moiety.
  • the library is contacted with a transferase and a derivative of an azido- containing amino acid or salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the fixed N-terminal residue of the peptide library, thereby yielding a first system.
  • a protein-containing fraction is isolated from the first system.
  • the first system, or isolated protein-containing fraction thereof is subjected to a protein-cleaving reagent capable of cleaving the internal cleavage site, thereby yielding a second system.
  • a protein-containing fraction is isolated from the second system.
  • the second system or isolated protein-containing fraction thereof, is contacted with an alkyne-derivatized bead or surface, under conditions whereby the azido and alkyne groups react to form a triazole group, thereby yielding a third system.
  • Individual peptide molecules which remain in the supernatant of the third system are identified via sequencing of the nucleic acid contained within the amplifiable genetic moiety in the third system, wherein any sequences that remain in the supernatant of the third system comprise an internal cleavage site for the protein cleavage reagent.
  • the transferase comprises E. coli AaT
  • the fixed N-terminal residue of the peptide library comprises Arg or Lys.
  • the transferase comprises V. vulnificus BpT
  • the N-terminal residue of the peptide library comprises Asp or Glu.
  • the amplifiable genetic moiety comprises a phage virion, a living cell, mRNA, a plasmid, or a paused ribosome.
  • the alkyne comprises a dibenzocyclooctyne moiety.
  • the protein-cleavage reagent comprises a proteolytic enzyme. In other embodiments, the protein-cleavage reagent comprises a chelated metal or other small molecule.
  • the invention includes a kit comprising a transferase, a derivative of an azido-containing amino acid or a salt thereof, a dibenzocyclooctyne-containing biotin derivative, and an instructional material for use thereof.
  • the instructional material comprises instructions for a method of determining whether a protein of interest is present in a sample, and the method comprises contacting the sample with the transferase and the derivative of an azido-containing amino acid or a salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the N-terminus of the protein of interest, thereby yielding a first system.
  • the method optionally comprises comprises isolating a protein-containing fraction from the first system.
  • the method further comprises contacting the first system or protein-containing fraction thereof with the dibenzocyclooctyne-containing biotin derivative, under conditions whereby the azido and dibenzocyclooctyne groups react to form a triazole group, thereby yielding a second system.
  • the method optionally comprises isolating a protein-containing fraction from the second system.
  • the method further comprises contacting the second system or protein- containing fraction thereof with a solid surface comprising an avidin-containing molecule immobilized thereon, under conditions whereby biotin and the avidin-containing molecule bind to each other.
  • the method further comprises identifying any combination
  • the invention further includes a kit comprising a transferase, a derivative of an azido-containing amino acid or a salt thereof, a dibenzocyclooctyne-containing biotin derivative, and an instructional material for use thereof.
  • the instructional material comprises instructions for a method of determining whether a protein comprising an internal cleavage site is present in a sample, and the method comprises contacting the sample with the transferase and the derivative of an azido-containing amino acid or salt thereof, under conditions whereby the transferase transfers the azido-containing amino acid to the N-terminus of the protein, thereby yielding a first system, wherein the first system is substantially free of a synthetase.
  • the method optionally comprises isolating a protein-containing fraction from the first system.
  • the method further comprises contacting the first system or protein-containing fraction thereof with a
  • the method optionally comprises isolating a protein-containing fraction from the second system.
  • the method further comprises submitting the second system or protein- containing fraction thereof to reagents capable of cleaving the internal cleavage site, thereby yielding a third system.
  • the method optionally comprises isolating a protein- containing fraction from the third system.
  • the method further comprises contacting the third system or protein-containing fraction thereof with a solid surface comprising an avidin-containing molecule immobilized thereon, under conditions whereby biotin and the avidin-containing molecule bind to each other.
  • the method optionally comprises determining whether a protein remains in the supernatant, wherein, if a protein remains in the supernatant, a protein comprising the internal cleavage site is present in the sample.
  • adenosyl amino acid donor compounds may be carried out on any natural or non-natural amino acid with appropriate acid-labile protecting groups (e.g., N-Boc).
  • Acid-labile protecting groups include, but are not limited to, tert- butyloxycarbonyl, tert-butyl ester and 2-phenylisopropyl ester.
  • Scheme 1 exemplifies a non-limiting method of preparing adenosyl donors useful within the invention.
  • 5 '-DMT-protected adenosine may be reacted with a cyanomethyl ester (la-lz) or a succinate ester (2a) to yield acylated adenosine derivative (3a-3z).
  • acylated adenosine derivative (3a-3z).
  • Deprotection of (3a-3z) with TFA yields adduct (4a-4z).
  • This adduct which acts as the reaction donor in the N-terminal modification reaction, may be reacted with a peptide substrate (containing a N-terminus free amino group) in the presence of the aminoacyl transferase to yield the product (5a-5z).
  • a label compound may be bound directly or via a spacer to any functional group, such as an amino, thiol, carboxyl, hydroxyl, aldehyde, allyl, or halogenated alkyl group of a natural or non-natural amino acid.
  • any functional group such as an amino, thiol, carboxyl, hydroxyl, aldehyde, allyl, or halogenated alkyl group of a natural or non-natural amino acid.
  • suitable functional group such as an amino, thiol, carboxyl, hydroxyl, aldehyde, allyl, or halogenated alkyl group of a natural or non-natural amino acid.
  • reagents for labeling amino groups include a succinimide ester, isothiocyanate, sulfonyl chloride, NBD-halide, and dichlorotriazine.
  • substances that may be used as reagents for labeling thiol groups include an alkyl halide,
  • substances that may be used as reagents for labeling carboxyl groups include a diazomethane compound, aliphatic bromides, and carbodiimide.
  • succinimide ester is introduced to a label compound directly or via a spacer; on the other hand, an amino group is introduced to an aromatic ring of an amino acid, and then the amino acid and the label compound can be bound to each other by means of amide bonds.
  • An example of an amino acid comprising an aromatic ring to which an amino group has been introduced is aminophenylalanine.
  • a functional group used in such a case may be suitably selected and introduced, and a binding method can also be suitably selected.
  • forming the amide bond at about pH 5 enables selective reaction with an amino group on the side chain of aminophenylalanine, even if another amino group is present in the amino acid molecule.
  • another amino group may be protected with Boc or the like, and the protecting group can be removed after the reaction of amino group on the side chain.
  • the labeled amino acid of the present invention has properties of its label substance. Accordingly, desired functions can be imparted to the labeled amino acid through the selection of a label compound having desired functions.
  • a variety of label moieties can be used to incorporate into proteins and polypeptides, including affinity handles (e.g., biotin), immunoprobes, isotopic labels, heavy-atom derivatives, PEG moieties, fluorescein derivatives, and other non-natural constituents.
  • any detectable label that can be incorporated into a substrate e.g., biotin labeled peptide esters
  • a free N-terminus e.g., alpha-amino group of a polypeptide generated through proteolysis
  • fluorescent substances have particularly high usefulness as labels for a protein. Further, a luminescent substance in the visible light range can be detected by a detector extensively and commonly used. Furthermore, a variety of highly sensitive detectors have already been developed and extensively used. These fluorescent substances are very useful as label compounds for labeling cells or the like since they are not affected by interferential actions caused by fluorescence emission in cells.
  • fluorescent substances that can be used in the present invention include all known fluorescent substances including rhodamine, fluorescein (FITC), Texas Red, acridine orange, SYBR Green, Cy3, Cy5, a BODIPY compound, and a derivative thereof.
  • the detectable label may be directly detectable or indirectly detectable, e.g., through combined action with one or more additional members of a signal producing system.
  • directly detectable labels include radioactive, paramagnetic, fluorescent, light scattering, absorptive and colorimetric labels.
  • isothiocyanate, rhodamine, phycoerythrin phycocyanin, allophycocyanin, gamma- phthalaldehyde, fluorescamine and the like are all exemplary fluorescent labels.
  • Chemiluminescent labels i.e., labels that are capable of converting a secondary substrate to a chromogenic product
  • indirectly detectable labels For example, horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenate, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucoamylase, acetylcholinesterase, luciferin, luciferase, aequorin and the like are all exemplary protein-based chemiluminescent labels.
  • Luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester and the like are exemplary non- protein-based chemiluminescent labels.
  • Another non-limiting and commonly used example of an indirectly detectable label is an affinity ligand, i.e., a label with strong affinity for a secondary binding partner (e.g., an antibody or aptamer), which may itself be directly or indirectly detectable
  • a detectable label may be visualized or detected in a variety of ways, with the particular manner of detection being selected based on the particular detectable label, where representative detection means include, e.g., scintillation counting, autoradiography, measurement of paramagnetism, fluorescence measurement, light absorption measurement, measurement of light scattering and the like.
  • a pre-conjugated label may contain one or more reactive moieties (e.g., carboxyl or reactive ester, amine, hydroxyl, aldehyde, sulfhydryl, maleimidyl, alkynyl, azido, etc. moieties).
  • reactive moieties e.g., carboxyl or reactive ester, amine, hydroxyl, aldehyde, sulfhydryl, maleimidyl, alkynyl, azido, etc. moieties.
  • these reactive moieties may, in certain embodiments, facilitate the conjugation process.
  • Specific examples include peptidic labels bearing alpha-terminal amine and/or epsilon-amine lysine groups. It will be appreciated that any of these reactive moieties may be artificially added to a known label if not already present.
  • a suitable amino acid e.g., a lysine
  • the conjugation process may be controlled by selectively blocking certain reactive moieties prior to conjugation.
  • any method that allows the detection of labeled polypeptides may be used to identify, isolate, or analyze the labeled polypeptides.
  • the skilled artisan will recognize that alpha-amino groups of polypeptides labeled with a peptide ester containing a biotin label can be isolated or detected using avidin-related proteins such as avidin itself, streptavidin, and neutravidin.
  • avidin-related proteins such as avidin itself, streptavidin, and neutravidin.
  • neutravidin beads may be used to isolate biotin labeled polypeptides from complex mixtures or streptavidin linked to horseradish peroxidase may be used to identify biotin labeled polypeptides after protein separation by a procedure such as electrophoresis and avidin blotting.
  • mass spectrometry is an analytical technique used to measure the mass-to- charge ratio of gaseous ions. It can be used to determine the composition of a biological sample by generating a mass spectrum representing the masses of sample components such as peptides and proteins. It can additionally be used to determine the structure of components in mixtures by observing the fragmentation of each peptide or protein present in the sample.
  • ESI electrospray ionization
  • MALDI matrix-assisted laser desorption/ionization
  • intact proteins are ionized by either of the two techniques described elsewhere herein, and then introduced directly to a mass analyzer.
  • proteins are enzymatically digested into smaller peptides using an agent such as trypsin or pepsin. The collection of peptide products is then introduced to the mass analyzer.
  • the labeled proteins and polypeptides of the present invention can be part of a very complex mixture of other proteins, polypeptides, and molecules that coexist in a biological medium such as a cell extract.
  • a resin comprising a moiety that binds to the affinity label may be used to isolate labeled proteins and polypeptides.
  • neutravidin beads may be used to isolate proteins and polypeptides resulting from proteolysis that have been labeled with peptide esters containing a biotin moiety.
  • the data generated from mass spectrometry analyses can be compared to sequence databases using computer programs available to the skilled artisan to determine the identity of labeled proteins.
  • labeled or modified peptides can be readily identified in MS/MS data by the presence of characteristic N-terminal modifications, such as characteristic di-peptide modifications.
  • a protein can be expressed with an N-terminal Cys and reacted with a synthetic thioester, yielding a semi-synthetic protein with an N-terminal chemical modification.
  • modifications may include non-natural amino acids with useful fluorescent or chemical properties and segmental isotopic labeling for infrared (IR), nuclear magnetic resonance (NMR or MRI), and positron emission tomography (PET) imaging.
  • IR infrared
  • NMR or MRI nuclear magnetic resonance
  • PET positron emission tomography
  • This approach may also be used to semi-synthesize proteins with homogeneous post-translational modifications such as glycosylation.
  • Strategy 2 requires an N-terminal Cys from the expressed protein portion. This can be avoided in Strategy 1 by using non-natural amino acids such as selenocysteine (Sec), which can be converted to Ala by treatment with TCEP, making the ligation "traceless.”
  • Sec selenocysteine
  • the transferase enzymes AaT and BpT may deliver an amino acid like C*sp (a protected Sec derivative) to the protein N-terminus, whereby it can be used in ligation and then deselenized using TCEP ( Figure 2A).
  • C*sp a protected Sec derivative
  • TCEP TCEP
  • This approach allows the implementation of traceless ligation on expressed proteins.
  • analogs of Sec which are deselenized to generate other natural amino acids at the ligation site may be developed.
  • homocysteine (Hcs) can be added to the N-terminus, used as a ligation handle, and then converted to Met by alkylation.
  • the methods of the invention are useful for identifying a proteolyzed or modified protein through a direct pull-down process (as illustrated in Figure 23).
  • a protein or peptide of interest is submitted to proteolysis (such as selective digestion by a sequence-specific protease), yielding a protein or peptide fragment, wherein the N-terminus amino acid of the protein or peptide fragment has a "X" side chain.
  • the N-terminus amino acid in the protein or peptide fragment may be recognized by a transferase.
  • AaT recognizes and effectuates amino acyl transfers to peptides or proteins with Arg or Lys as the N-terminus amino acid
  • BpT recognizes and effectuates amino acyl transfers to peptides or proteins with Asp or Glu as the N-terminus amino acid.
  • the invention further contemplates the use of any other natural or site-directed mutant transferase, which effectuates amino acyl transfers to peptides or proteins with any type of N-terminus amino acid.
  • the amino acyl donor is Azf-A
  • the resulting protein or peptide comprises 4-azido-Phe as the N-terminus residue.
  • the resulting azido-containing protein or peptide may then be reacted with a
  • the reaction between the azido-containing protein or peptide and the dibenzocyclooctyne (DBCO)-containing biotin derivative comprises a copper-free Click reaction.
  • the triazole-containing protein or peptide may then be pulled down using a biotin-containing reagent that is immobilized on a solid surface.
  • the pulled-down protein or peptide may then be digested (using for example trypsine) and sequenced.
  • the methods of the invention may be used to identify a proteolyzed protein in a system via elimination of other unproteolyzed proteins from the system ( Figure 24).
  • one protein has an internal Zi site
  • another protein has an internal Z 2 site
  • proteolysis may be used to cleave internal Z 2 site with selectivity over internal Zi site.
  • Both proteins have a N-terminus amino acid that is recognized by a transferase (the N-terminus amino acid of the two proteins may be the same, or distinct from each other).
  • AaT transfers to R or K
  • BpT transfers to D or E.
  • the transferase may be used to attach an azido-containing amino acid, such as but not limited to Azf, to the N-terminus of the protein, using Azf-A as a donor.
  • the Azf-modified protein is then reacted with a dibenzocyclooctyne- containing biotin derivative, as DBCO biotin, whereby the azido and dibenzocyclooctyne groups undergo a Click reaction to form a triazole group.
  • the reaction is copper-free.
  • Proteolysis is then performed under conditions wherein the Zi internal site is not cleaved but the Z 2 internal site is cleaved. Under such conditions, the protein comprising the internal Z 2 site has its biotin label cleaved, but the protein comprising the internal Z site retains its biotin label.
  • Chloroacetonitrile, ⁇ , ⁇ -diisopropylethylamme (DIPEA), 5 '-0-(4,4'- Dimethoxytrityl) adenosine ((DMT)-A), tetrabutyiammonium acetate (TBAAc), trifluoroacetic acid (TFA), and triisopropyl silane (TIPSH) were purchased from Sigma- Aldrich (St. Louis, MO).
  • DMT- A previously available from Sigma- Aldrich, may be obtained from ChemGenes Corporation (Wilmington, MA). It was additionally synthesized following the protocol outlined by Ogilvie et al (Ogilvie et af 1967, J Org ( hem 32:2365-2366).
  • E. coli BL21(DE3) cells were purchased from Strata-gene (La Jolla, CA). The pEG6 piasmid, containing His] 0-tagged E. coli AaT was obtained. Sequencing- grade trypsin was purchased from Promega (Madison, WI). All other reagents were purchased from Fisher Scientific (Pittsburgh, PA).
  • Matrix-assisted laser desorption ionization (MALDI) mass spectra were collected using a Bniker Ultraflex I I I MALDI -TOF-TOF mass spectrometer (Bilierica, MA). UV absorbanee spectra were obtained with a Hewlett-Packard 8452A diode array spectrophotometer (currently Agilent Technologies; Santa Clara, CA).
  • MALDI Matrix-assisted laser desorption ionization
  • Donor molecule purification was conducted on a BioCad Sprint FPLC (GMI Inc.; Ramsey, MN; originally from Perseptive Biosystems) with a Waters Sunfire Prep C18-prep OBD column, 5 iim, 17 x 150 mm (Milford, MA).
  • N-Boc-L-leucine Boc-Leu-OH
  • N-Boc-L-/?-azidophenylalanine Boc- Azf-OH
  • N-Boc-L-naphthylalanine Boc-Nap-OH
  • N-Boc-, N-Me-L-phenylalanine Boc-Mef-OH
  • N-Acetyl-L-phenylalanine Acf-OH
  • N-Fmoc- methoxycoumarinylalanine Fmoc-Mcm-OH
  • the carboxy-terminus of the amino acid (or aannaalloogg)) wwaass aaccttiivvaatteedd aass aa ccyyaannoommeetthhyyll eesstteerr uussiinngg cchhlloorrooaacceettoonniittrriillee ttoo yyiieelldd llbb--llzz.
  • Boc-L-Cys-OH (221 mg, 1.0 mmol) was dissolved in H 2 0/THF (50%v/v, 20 mL). To the solution was added 10 N sodium hydroxide aq. (1.2 mL, 12 mmol) and 2-propane thiol (929 uL, 10 mmol) at 0 °C. Then, a solution of iodine in 95% EtOH was added dropwise until the color of the reaction system changed from colorless to brown. The mixture was stirred for 1 d with warming to room temperature. After removing THF under reduced pressure, reaction mixture was neutralized by 1 N HC1 aq. until pH 2-3. The solution was extracted with EtOAc. The extract was washed with saturated aq.
  • Boc-L-Csm-OH (100 mg, 0.5 mmol) was dissolved in THF (1 mL). To the solution were added chloroacetonitrile and diisopropylamine (350 mg, 5.0 mmol). The mixture was stirred for over-night under N 2 atmosphere.
  • Boc-L-Csp-OH (100 mg, 339 ⁇ ) was dissolved in THF (1 mL). To the solution were added chloroacetonitrile (1.08 mL, 16.2 mmol) and diisopropylamine (118 uL, 647 ⁇ ). The mixture was stirred for over-night under N 2 atmosphere.
  • Boc-L-Hcm-OH 43 mg, 153 ⁇ was dissolved in THF (1 mL). To the solution were added chloroacetonitrile (484 ⁇ , 7.65 mmol) and diisopropylamine (53 ⁇ , 306 ⁇ ). The mixture was stirred overnight under N 2 atmosphere. After 18 h, additional chloroacetonitrile (484 L, 7.65 mmol) was added.
  • Tetrahydrofuran (5 mL) was added to Boc-Azf-OCH 2 CN lc (101 mg, 0.290 mmol), (DMT)-A (43 mg, 75 ⁇ ), and TBAAc (2 mg, catalyst) and stirred for 24 h. Solvent was removed under vacuum and preparative TLC (100% ethyl acetate), afforded 44 mg of white solid in 69% yield. R f 0.3 -0.5 in ethyl acetate; 1H and 13 C NMR for 3c shown in Figure 8; LRMS (ESI) m/z calcd for (M + Na) + 880.3, found 880.4.
  • Tetrahydrofuran (5 mL) was added to Boc-Nap-OCH 2 CN Id (58 mg, 0.16 mmol), (DMT)-A (26 mg, 46 ⁇ ), and TBAAc (6.4 mg, catalyst) and stirred for 24 h. The solvent was removed under reduced pressure and preparative TLC (5% methanol in ethyl acetate), afforded 36 mg of a white solid in 91% yield.
  • Tetrahydrofuran (3.5 mL) and DIPEA (44 mg, 60 ⁇ , 0.35 mmol) were added to Boc-Mef-OCH 2 CN le (1 10 mg, 0.347 mmol), (DMT)-A (50 mg, 89 ⁇ ), and TBAAc (3 mg, catalyst) and stirred for 12 h.
  • the solvent was removed under reduced pressure and Si0 2 flash chromatography (50 -100% ethyl acetate in hexanes), afforded 10 mg of a white solid in 14% yield.
  • Tetrahydrofuran (3.5 ml) and DIPEA (44 mg, 60 ⁇ , 0.35 mmol) were added to Boc-Mcm-OCH 2 CN lh (56 mg, 0.15 mmol), (DMT)-A (50 mg, 89 ⁇ ), and TBAAc (3 mg, catalyst) and stirred for 12 h.
  • the solvent was removed under reduced pressure and Si0 2 flash chromatography (50 -100% ethyl acetate in hexanes), afforded 35 mg of a white solid in 43% yield.
  • Trifluoroacetic acid (1 mL), tetrahydrofuran (1 mL) and TIPSH (99 mg, 0.13 mL, 0.63 mmol) were added to 3b (123 mg, 0.157 mmol) following the general deprotection procedure.
  • HPLC/MALDI analysis m/z calcd CieftsNeOs (M + H) + 381.2; Gradient 2 as described elsewhere herein; retention time 14.9 min, found 381.1; retention time 16.6 min, found 381.1.
  • Trifluoroacetic acid (1 mL), tetrahydrofuran (1 mL) and TIPSH (33 mg, 42 ⁇ , 0.21 mmol) were added to 3c (44 mg, 52 ⁇ ) following the general
  • HPLC/MALDI analysis m/z calcd C 2 iH 2 5N 6 0 6 (M + H) + 457.2, (M + Na) + C 2 iH 2 4N 6 Na0 6 479.2; Gradient 2 as described above; retention time 18.2 min, found 457.3, 479.3; retention time 19.5 min, found 457.3, 479.3.
  • TSUf-A tetrahydrofuran-3-yl 2-azido-3-phenylpropanoate
  • Trifiuoroacetic acid (1 mL) and TIPSH (21 mg, 30 ⁇ , 0.13 mmol) were added to 3g (25 mg, 33 ⁇ ) following the general deprotection procedure.
  • Trifiuoroacetic acid (1 mL), tetrahydrofuran (1 mL) and oxalic acid (3.9 mg) were added to 3e (31 mg, 37 ⁇ ) following the general deprotection procedure, except was worked up in water only and water-soluble portion was HPLC purified. No TIPSH was used in this reaction.
  • HPLC/MALDI analysis m z calcd C23H25N6O8 (M + H) + 513.2, C23H 2 4N 6 0 8 Na (M + Na) + 535.2; Gradient 2 as described elsewhere herein; retention time 19.9 min, found 513.1 , 535.1 ; retention time 20.9 min, found 513.1 , 535.1.
  • Each ligation reaction 125 ⁇ , total volume, contained the following reagents: aminoacyl adenosine donor (1 niM), recombinant Hisl O-tagged E. coli aminoacyl transferase (2.26 ⁇ ), and LysAlaAcm (100 ⁇ , ⁇ ) in the AaT Ligation Buffer (50 mM HEPES pH 8,0, 150 mM C1, 10 mM MgCl 2 ).
  • the reaction mixtures were incubated at 37 °C for four hours and quenched with 1% acetic acid.
  • the proteins were extracted from the reactions via acetone precipitation.
  • the reactions were precipitated using 4x reaction volume of acetone and cooled at -20 °C for 1 h.
  • the reactions were centrifuged at 13,200 rpm at 4 °C for 20 mm to separate the reaction from precipitated protein.
  • the supernatant was transferred to fresh 1.5 mL centrifuge tubes and allowed for acetone evaporation overnight at room temperature. After acetone evaporation, the supernatant was dried in a Speedvac (Savant, Thermo Scientific, Fisher Inc.) for 30 mi to remove residual acetone.
  • the resulting reaction volume was dissolved up to 1.2 mL using Milli-Q water and analyzed by HPLC (gradient below) to determine ligation yield by integration of separated peak intensities monitored at 325 nm. Collected HPLC fractions were characterized through MALDI MS analysis.
  • Reactions were performed with 6 trials on at least two protein preparations for ail successful reactions, 3 trials for failed reactions. Reactions using Phe-pdCpA as donor were carried out in an identical fashion (Ellman, et ah, 1991, Methods Enzymol. 202:301-336).
  • Ligation test reactions with V, vulnificus Bpt were conducted in a manner identical to the AaT LysAlaAcm ligation assay with the following exceptions, 2.26 uM BpT was used rather than 2.26 ⁇ AaT, 100 ⁇ AspAlaAcm was used rather than 100 ⁇ LysAlaAcm, and BpT Ligation Buffer (50 mM HEPES, pH 8, 150 mM C ' I. lOmM MgCl 2 ) was used rather than AaT Ligation Buffer.
  • adenosyl compounds adenosine, AMP, ATP, or pdCpA
  • Adenosyl compound concentrations tested were 1 mM, 2.5 mM, and 5 mM.
  • HPLC analysis was used to determine inhibition of ligation by integration of HPLC reagent and product peaks.
  • the LysAlaAcm ligation assay was used to analyze the kinetics of AaT with substrate 4a.
  • the ligation assay was modified for ease of analysis as follows: The total reaction volume was scaled up to 590 ( uL and ail reagents were maintained at the same
  • concentrations as mentioned above. Five concentrations of 4a were monitored for a total reaction time of 30 min, with concentrations ranging from 0.05 to I mM.
  • Fcontroi is the fluorescence reading of the reaction with water added instead of 4d. Calibration details are given in Supporting Information. Measurements were taken on the Gary Eclipse fluorometer in the "Kinetics" mode at 37 °C, acquiring data every 15 s. The excitation and emission slit widths were 5 nm and the averaging time was I s. a-Casein N-Termin al Mo d ification
  • a-Casein (4.8 mg) was modified with Azf-A (4c) in a reaction volume of 1 nil . in modified AaT buffer (50 mM HEPES pH 8.0, 150 mM KCl, 10 mM MgC! 2 ) and AaT (0.05 mg).
  • reaction mixture was incubated at 37 °C for 12 h, AaT was removed using Ni 2" resin (Ni-NTA Superflow, Qiagen), and then buffer exchanged four times into phosphate buffered saline (PBS, 12 mM NaH 2 P0 4 , 50 mM NaC!, 4.7 mM KCl, pH 8.0) by Spectra/Por 1 dialysis tubing (Spectrum Laboratories; Raiicho Dominguez, CA). Azf-modified a-casein was used directly after buffer exchange into PBS.
  • a-Casein-Azf (33 ⁇ ) or a-casein (33 ⁇ ) was digested with sequencing grade modified Trypsin (0.6 u.g) in 27 ⁇ of 25 mM ammonium bicarbonate pH 7.5 (freshly prepared). Digestions were carried out at 37 °C for 14 h. Trypsin digest aliquots ( 1 p L) were combined with a-cyano-4-hydroxycitinamic acid ( 1 ⁇ of a saturated solution in 1 : 1 H 2 0/CH 3 CN with 1% TFA) and analyzed by MALDI MS. ⁇ -Casein Labeling in Cleared Cell Lysate.
  • AaT was expressed in E. coli BL21-Goid (DE3) cells as previously described.
  • a cleared cell lysate was obtained by centrifugation following cell lysis using sonication. Protein were modified in a final reaction volume of 110 p L with Azf-A (4c, 3 fflM) using AaT (25 from cleared lysate in AaT buffer (50 niM HEPES pH 8.0, 150 mM C1, 10 mM MgCi 2 ).
  • Aa-Casein (0.012 mg or 0.12 mg) was added to the reaction and incubated at 37 °C for 2 h, after 1 h, an additional 0.3 ⁇ of 4c was added. For control reactions, equivalent volumes were replaced with water.
  • the amino acid analog donor (4a-4z) was suspected to be hydrolytically unstable and was tested in mock LysAlaAcm reactions without enzyme present and then analyzed by HPLC.
  • the mock LysAlaAcm reactions were performed as described in the LysAlaAcm Ligation Assay section; however, the AaT solution was replaced with water.
  • Phe- A solutions were analyzed at 30 min, 1 h, and 4 h.
  • the sample was diluted to 1,200 and injected onto the CI 8 HPLC column using Gradient 1. As set forth in Figure 16, Phe-A was completely hydro lyzed after 4 h. Additionally, 1 mM A and 1 mM Phe were injected on the HPLC to serve as standards to verify retention times.
  • E. coli AaT was expressed from the pEG6 plasmid in E. coli BL21-Gold (DE3) cells using a procedure adapted from Graciet et al (Graciet, et al, 2006, Proc Natl Acad Sci U S A 103:3078-3083). E. coli were grown in a primary culture of 5 mL LB at 37 °C to OD 6 oo of 0.5 and then were rediluted into a secondary culture of 500 mL LB and grown to OD 6 oo of 0.6. AaT expression was induced using 0.1 mM isopropyl ⁇ -D- thiogalactoside and cells were grown at 25 °C for -16 h.
  • Cells were pelleted at 6,000 RPM using a GS3 rotor and Sorvall RC-5 centrifuge. Cell pellets were resuspended in the Ni-NTA binding buffer (50 mM Tris, 10 mM imidazole, 300 mM KCl, and 5 mM ⁇ - mercaptoethanol, pH 8.0) and included protease inhibitor cocktail, 1 mM PMSF, and 10 units/mL DNAsel -Grade II. Following resuspension, the cells were lysed using sonication. Soluble proteins were collected via centrifugation at 13, 200 RPM for 15 min. Collected soluble protein was gently shaken for 1 h at ambient temperature with Ni- NTA resin.
  • Ni-NTA binding buffer 50 mM Tris, 10 mM imidazole, 300 mM KCl, and 5 mM ⁇ - mercaptoethanol, pH 8.0
  • protease inhibitor cocktail 1 mM PMSF
  • the resin was prepared by rinsing with Ni-NTA binding buffer and then washed with four volumes of Ni-NTA wash buffer (50 mM Tris, 50 mM imidazole, 300 mM KCl, and 5 mM ⁇ -mercaptoethanol, pH 8.0).
  • Ni-NTA wash buffer 50 mM Tris, 50 mM imidazole, 300 mM KCl, and 5 mM ⁇ -mercaptoethanol, pH 8.0.
  • the proteins were eluted with elution buffer (50 mM Tris, 250 mM imidazole, 300 mM KCl, and 5 mM ⁇ -mercaptoethanol, pH 8.0). Pure elution fractions of E.
  • coli AaT were dialyzed overnight in AaT buffer (50 mM Tris, 30% glycerol, 120 mM (NH 4 ) 2 S0 4 , 5 mM ⁇ -mercaptoethanol, pH 8.0). The dialyzed enzymes were stored at -80°C. Protein concentrations were determined using the Bradford assay and a bovine serum albumin standard curve according to the manufacturer's instructions (Bradford, 1976, Anal Biochem 72:248-254).
  • V. vulnificus BpT was expressed from the pEG145 plasmid in E. coli
  • E. coli were grown at 37 °C to OD 600 of 0.5-0.6, followed by incubation at 42 °C for 1 h and at 0°C for 30 min. Expression was induced with 0.25 mM isopropyl ⁇ -D-thiogalactoside, and was carried out at 25°C for 5 h.
  • Ni-NTA binding buffer 0.3 M KCl/10 mM imidazole/5 mM ⁇ - mercaptoethanol/50 mM Tris, pH 8.0
  • Soluble proteins were collected via centrifugation at 13,200 RPM for 15 min, and incubated for 1 h at 4 °C with Ni-NTA resin preequilibrated in the Ni-NTA binding buffer. The resin was washed with Ni-NTA binding buffer, followed by a second wash with the binding buffer plus 50 mM imidazole. The proteins were eluted with the Ni- NTA binding buffer plus 0.25 M imidazole.
  • BpT Purified BpT was dialyzed against BpT buffer (10% glycerol/0.3 M KCl/15 mM -mercaptoethanol/50 mM Tris, pH 8.0), and stored at - 80 °C. Protein concentrations were determined by using the Bradford assay. AaT Ligation Reaction Analysis by HPLC.
  • Fc on troi is the fluorescence reading of the reaction with water added instead of donor 4d. This calibration was used to determine the time-dependent concentration of the NapLysAlaAcm product as described elsewhere herein.
  • Equation 1 The reaction scheme for the catalysis of N-terminal aminoacylation by AaT is shown in Equation 1.
  • the sequential bisubstrate AaT reaction can be treated as pseudo-first order when carried out with saturating concentrations of one substrate (acceptor peptide) (Abramochkin et al, 1996, J. Biol. Chem. 271 :22901-22907). Therefore, the reaction was characterized in terms of the Michaelis-Menten kinetic scheme in Equation 2.
  • Phe was ligated to a-casein using 4a as described elsewhere herein with the following modifications.
  • the substrate (1 mM) was 10 times more concentrated, and the total volume was 4 times greater. This reaction was run overnight; at the 4 h timepoint, an additional 50 dose of 25 mM 4a donor was added to the reaction.
  • the Hisio-tagged AaT was separated from the reaction via nickel bead purification: 100 Ni-NTA resin was added at a ratio of 25 per 12.5 ⁇ AaT, the beads were shaken with the reaction for 2 hours, then separated via centrifugation at 13,200 RPM for 2 min.
  • the supernatant was removed, diluted to 1 mL with Milli-Q water, and dialyzed against IX PBS (Hyclone, Fisher) overnight at 4 °C to remove any residual 4a.
  • the protein was electroblotted onto PVDF membrane and Edman Degradation analysis was conducted.
  • Tris-(3-hydroxypropyltriazolylmethyl)amine was also synthesized as previously described (Hong et al, 2009, Angew. Chem. Int. Ed. 48:9879- 9883). Briefly, a solution of 3-bromo-propanol (2 g, 14.4 mmol) in dichloromethane (10 mL) was added to Ac 2 0 (2.94 g, 28.8 mmol) and NEt 3 (2.91 g, 28.8 mmol). The reaction was stirred continuously and monitored by TLC. An aqueous solution of NaHC0 3 was added and the phases were separated. The organic layer was washed once more with NaHC0 3 and twice with brine. Solvent was removed under reduced pressure and 3- bromopropyl acetate was afforded as a colorless oil.
  • THPTA acetyl-protected THPTA (397 mg, 0.711 mmol) in 2.0 M ammonia in MeOH (10 mL) was continuously stirred overnight at 40 °C. Solvent was removed under reduced pressure. The solid was washed and filtered four times with acetonitrile and dried under reduced pressure to afford THPTA as a white solid.
  • the AcMetAspValPhe peptide was synthesized using a manual, Fmoc- based solid-phase procedure on 2-chlorotrityl chloride resin (100-200 mesh; 0.6 mmol substitution/g). For each coupling, 5 equiv of amino acid, 5 equiv of HBTU, and 10 equiv of DIPEA in dimethylformamide (DMF) were stirred for 30 min at room temperature. After rinsing the resin three times with DMF, 20% piperidine in DMF was used to deprotect the Fmoc-group from the coupled amino acid. The deprotection solution was collected for UV analysis to quantify the coupling efficiency.
  • DMF dimethylformamide
  • the N-terminus of the peptide was acetylated by two successive 10 min incubations with a capping solution of acetic anhydride, N-methylmorpholine, and DMF (5:3:42 v/v).
  • a capping solution of acetic anhydride, N-methylmorpholine, and DMF 5:3:42 v/v.
  • the capped N-terminal peptides were incubated for 1 h with acetic acid (AcOH), trifluoroethanol (TFE), and CH 2 CI 2 (1 : 1 :8 v/v).
  • the cleaved peptide solution was dried by rotary evaporation and rinsed with CH 3 CN three times to remove AcOH.
  • the crude peptide (1 equiv, 2 mM) was dissolved in tetrahydrofuran (THF), and thiophenol (3 equiv, 6 mM) was added to the solution. After stirring for 5 min, PyBOP (3 equiv, 6 mM) and DIPEA (3 equiv, 6 mM) were added to the reaction mixture. The solution was allowed to stir for 1 h at room temperature, at which point the solvent was removed by rotary evaporation.
  • reaction mixture was sparged with argon for 15 min and stirred under an argon atmosphere at room temperature.
  • aliquots 50 ⁇ were taken periodically at regular intervals and diluted to 800 ⁇ ⁇ with 0.1 % TFA in water. Each sample was analyzed by analytical HPLC.
  • adenosyl donor compounds shown in Scheme 1, can be carried out on any amino acid with appropriate acid-labile protecting groups (e.g., N- Boc).
  • acid-labile protecting groups e.g., N- Boc
  • This synthesis is not limited to a-amino acids, and the present donor synthesis has also enabled to test the transfer of analogs such as ⁇ -azidophenylalanine (N 3 f, 4g).
  • the Phe donor 4a was synthesized from both the cyanomethyl ester (la) and N-hydroxysuccinimidyl (OSu) ester (2a).
  • AH other adenosyl donors were synthesized from the cyanomethyl ester (lb-z).
  • TFA trifluoroacetic acid
  • CH 2 C1 2 washing or Et 2 0 precipitation
  • LysAlaAcm starting material ehited at 11.8 min as depicted in Figure 2A; hydrophobic products eluted at 12 to 15 min (PheLysAlaAcm 5a elutes at 13.0 min in Figure 2B, NapLysAlaAcm 5d elutes at 14.1 min in Figure 2D).
  • Sisido has shown that AaT mutants can use substrates with larger side chains; some of these mutations are currently explored (Taki et al., 2008, ChemBioChem 9:719-722). While transfer yields after 4 h varied, most reactions could be driven to completion by a bolus of donor molecule as depicted in Figure 2C.
  • adenosyl donors over in situ tRNA aminoacylation with a tRNA synthetase is that it is not limited by the substrate specificity of the synthetase.
  • non-amino acid substrates can be attached to adenosyl donors to assess their transferability by AaT.
  • N-Methyl phenylalanine (Mef, 4e) was transferred, albeit with low yield.
  • N-acetyl phenylalanine (Acf, 4f) and a- azidophenylalanine (4g) have also been tested and found to be poor substrates.
  • the present experiment was also achieved to monitor product formation in real time based on partial qu enching of coumarin fluorescence upon ad dition of Nap to the LysAiaAcm peptide to form 5d.
  • NapLysAlaAcm is 28% of the fluorescence of LysAiaAcm, and the overall fluorescence of mixtures of the two peptides can be used to determine the proportions of each peptide if the total concentration is known.
  • the change in fluorescence intensity was used to monitor NapLysAlaAcm formation in real time, and HPLC injection of the reaction end points were used to confirm the final product distribution. It was found that exogenous adenosine did not significantly inhibit the reaction, even at concentrations up to 1 mM as shown in Figure 3A.
  • the enzyme efficiency (k c K 1.35 x 10 3 M _1 s _1 ) was relatively low (Fersht, 1998, Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding, 3rd ed.). While K M was higher for Phe-A than what Shrader reported for aminoaeyl ⁇ .RNA Leu ⁇ 4 (0.3 ⁇ ), the kcat was comparable (0.13 x 10 _1 s _1 ) (Abramochkin et al,, 1996, J. Biol. Chem. 271 :22901-22907). Since it is trivial to run the present reactions at donor concentrations well above KM, the lower apparent affinity for the present substrates should not affect the utility of the reaction. The binding affinity, which gave a KM of 124 ⁇ for 4a, arises primarily from Phe binding interactions since the K ⁇ for adenosine inhibition was greater than 5 niM.
  • results presented herein demonstrate that E. coli aminoacyl t NA transferase (AaT) modifies the N-terminus of a protein under conditions that maintain folding and activity of the protein.
  • AaT E. coli aminoacyl t NA transferase
  • the results demonstrate that aminoacyl tRNA transferase is useful for conjugation to a protein.
  • Enzymological analysis demonstrated that AaT can use the Xaa-A substrates with turnover numbers comparable to full-length aminoacy! tRNAs.
  • the major obstacle to efficient transfer is donor hydrolysis, but this can be overcome by a second addition of donor molecule since the &i for inhibition by adenosine is greater than 5 niM.
  • the present AaT-only reaction sequence allows exploring the compatibility of Phe analogs lacking a primary a-amine with transfer by AaT.
  • the wild type transferase seems to have a strong preference for the a-amine, as only N-methyl- phenylalanine was transferred, in contrast, the side chain pocket is quite permissive: transfer of tricyclic amino acid side chains was observed and Sisido has reported mutants capable of transferring tricyclic amino acids (Taki et al, 2008, ChemBioChem 9:719- 722).
  • the present invention can transfer larger side chains including but not limited to
  • the present method can be used to transfer reactive handles to the N-terrninus under "protein friendly" (pH ⁇ 7, high salt, 37 °C) conditions and that the labeled protein product can be easily purified afterward.
  • protein friendly pH ⁇ 7, high salt, 37 °C
  • further mutation of AaT allows for the transfer of larger side chains that can permit direct transfer of fluorescent probes or affinity tags.
  • crystal structures can be used to redesign AaT to act on other N-terminal sequences through a combination of rational design and selection.
  • AaT can modify a-casein in crude lysates coupled with the cell-permeability of the present moderately polar donors.

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Abstract

L'invention concerne un procédé sélectif de modification de l'extrémité N-terminale d'une protéine au moyen d'une aminoacyl-ARNt transférase.
PCT/US2013/045594 2012-06-14 2013-06-13 Nouveaux procédés d'identification de protéines par étiquetage n-terminal et extraction sélective WO2013188635A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104529898A (zh) * 2015-01-15 2015-04-22 成都丽凯手性技术有限公司 氮杂二苯并环辛炔类化合物及其制备方法
CN116183899A (zh) * 2023-04-28 2023-05-30 天津市协和医药科技集团有限公司 一种吖啶酯标记抗体保存液的制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2644474A1 (fr) * 2006-03-03 2007-09-13 California Institute Of Technology Incorporation specifique de site d'acides amines dans des molecules
CA2645159A1 (fr) * 2006-03-10 2008-06-26 Michael S. Urdea Fractionnement multiplex de proteines
CN103153927B (zh) * 2010-04-27 2017-02-15 西纳福克斯股份有限公司 稠合的环辛炔化合物及其在无金属点击反应中的应用

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104529898A (zh) * 2015-01-15 2015-04-22 成都丽凯手性技术有限公司 氮杂二苯并环辛炔类化合物及其制备方法
CN104529898B (zh) * 2015-01-15 2016-07-27 成都丽凯手性技术有限公司 氮杂二苯并环辛炔类化合物及其制备方法
CN116183899A (zh) * 2023-04-28 2023-05-30 天津市协和医药科技集团有限公司 一种吖啶酯标记抗体保存液的制备方法
CN116183899B (zh) * 2023-04-28 2023-06-30 天津市协和医药科技集团有限公司 一种吖啶酯标记抗体保存液的制备方法

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