WO2019112567A1 - Procédés et compositions associés à la sélection de variants de protéases - Google Patents

Procédés et compositions associés à la sélection de variants de protéases Download PDF

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WO2019112567A1
WO2019112567A1 PCT/US2017/064697 US2017064697W WO2019112567A1 WO 2019112567 A1 WO2019112567 A1 WO 2019112567A1 US 2017064697 W US2017064697 W US 2017064697W WO 2019112567 A1 WO2019112567 A1 WO 2019112567A1
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protease
moiety
probe
substrate
trypsin
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PCT/US2017/064697
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English (en)
Inventor
Brian M. Paegel
Duc T. TRAN
Valerie J. CAVETT
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The Scripps Research Institute
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Priority to PCT/US2017/064697 priority Critical patent/WO2019112567A1/fr
Publication of WO2019112567A1 publication Critical patent/WO2019112567A1/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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21004—Trypsin (3.4.21.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/976—Trypsin; Chymotrypsin

Definitions

  • Mass spectrometry is an extremelyly general and high-resolution platform for protein sequencing, identification, and mapping. Proteins are first enzymatically digested into smaller peptides that are suitable for chromatographic separation and ionization. A tandera mass analyzer then isolates individual peptide ons in a first analyzer, induces regular fragmentation along the peptide back bone, and scans the product ion series in the second analyzer. Mass differences between consecutive product Ion peaks in these series reveal the sequential identities of amino acids, and the type and location of any post- translational modifi cations (PTMs) .
  • PTMs post- translational modifi cations
  • Proteolytic digestion is the first step in preparing a sample for mass spectrometric analysis. Regions of protein sequence with sparse proteolytic cleavage sites are not detected and result in sequence coverage gaps. Trypsin is used almost exclusively because it is highly specific and efficient, and tryptic cleavage sites are common in many proteins. A few other proteases are used much less frequently because they are either too inefficient (AspN, GluC) or promiscuous (chymotry sin) to be generally useful The arsenal of viable proteases for sample preparation is shockingly small, and the absence of tryptic cleavage sites alone can render swathes of protein sequence completely invisible to the instrument. These sequence coverage gaps are a disabling obstacle because they can conceal key regulatory modifications, thwarting discovery and mapping efforts.
  • the present: invention provides protease activity assay matrices.
  • the matrices contain a solid support conjugated to a substrate moiety that can be Specifically cleaved by a protease,
  • the substrate moiety of the matrices is comprised of (1) a probe moiety capable of detecting cleavage of the substrate moiety by the protease and (2) a nucleotide moiety capable of priming replication of a polynucleotide sequence encoding the protease.
  • the substrate moiety can
  • the nucleotide moiet is a PCR oligonucleotide primer.
  • the solid support is a magnetic bead in some embodiments, the probe moiety contains a cleavage site of the protease and a detectable label.
  • the detectable label is a fluorescent label, e.g., a fluprogenic molecule.
  • the protease activity assay matrices can further include a polynucleotide sequence encoding the protease, e.g., a candidate mutant or variant of a target protease of interest in some of these embodiments the polynucleotide sequence can be jinked to the assay matrix via the nucleotide moiety, e.g , through hybridization .
  • the nucleotide moiety is a PCR oligonucleotide primer
  • the invention provides methods for identifying a variant protease that recognizes a desired substrate cleavage site. These methods entail (1 ) conjugating to a solid support a substrate moiety that contains (a) a probe moiety capable of detecting cleavage of the substrate moiety at the desired cleavage site and (b) a nucleotide moiety capable of priming replication of a polynucleotide sequence encoding a candidate variant protease, to generate a protease activity assay matrix, (2) emulsifying the protease acti vity assay matrix with a l ibrary of polynuc leotide sequences encoding a population of candidate proteases, to generate a library of matrix emulsified polynucleotide sequences, (3) performing emulsion PCR (emPCR) and emulsion in vitro transcription/translation
  • emPCR emulsion PCR
  • the substrate moiety can further include a linker moiety for connecting both the probe moiety and the nucleotide moiety to the solid support.
  • the nucleotide moiety Is a PCR oligonucleotide primer.
  • the employed solid support is a magnetic bead.
  • the emulsifying is performed with an emulsion formulation that comprises a continuous phase and an aqueous phase.
  • the emPCR is performed in the presence of a stabilizer in the aqueous phase and a stabilizer in the continuous phase
  • the ernIVTT is performed in the presence of a stabil izer in the continuous phase.
  • the employed probe moiety contains a cleavage site of the protease and a detectable label.
  • the detectable label is a fluorescent label, e.g.., a fluprogemc molecule.
  • the invention provides variant trypsin enzymes or enzymatic fragments thereof that have citridlme-dependent proteolytic activity, These enzymes typically contain an amino acid sequence that is at least 90% identical to the sequence of wild type trypsin. Further, relative to the wild type trypsin, the trypsin variants of the invention contain mutation D189S and one or more additional mutations selected from the group consisting of L7P, El 85K, and KI 88,4, or conservative substitution thereof. In some embodiments, the variant trypsin enzymes of the invention contain amino acid substitutions L7F, E185K, K188A and D189S, or conservatively substitutions thereof.
  • FIG. 1 shows activity assay bead synthesis, function, arid implementation in compartmentalized protease evolution.
  • the protease activity assay bead (top left) is prepared from 2.8-mhi- diameter, amino-functionalized magnetic resin, The resin
  • the bisamide probe displays two symmetric tripeptide arms. Cleavage only at the PI amide bond (dashed line) mediated by an active mutant protease (white pae-man) yields a fluorescent bead displaying the R1 10 fluorophore (white).
  • the beads are used in an in vitro compartmentalized protease evolution workflow (bottom), including preparation of a protease mutant gene library, emulsion PCR (emPCR) of the mutant gene l ibrary in the presence of the protease activity assay beads to yield a bead library ⁇ , each clonally displaying ⁇ l 0,000 copies of a progenitor mutant.
  • the bead library is washed and re-emulsified with in vitro transcription/translation reaction mixture (emiVTT).
  • Droplets housing beads that display an inactive protease gene result in translation of inactive protease (black pac-man) and no proteolytic activity directed toward the bead (black hex bead).
  • Droplets housing beads that display an active protease gene result in translation of the active protease, which then transforms bead-bound substrates to fluorescent bead-bound product (white hex bead).
  • the invention is predicated in part on the development by the present inventors a compartmentalized in vitro evolution platform to discover new proteases for mass spectrometry-based proteomics.
  • the platform is capable of generating and . screening over a million protease mutants, identifying mutants exhibiting desired proteolytic activity and rejecting those that exhibit off- target activity,
  • protease activity assay beads are prepared by functionalizing 2,8-pm magnetic amino beads with a bi functional linker that displays both a protease activity probe and a DNA oligonucleotide primer for PCR (Fig, i, top).
  • a protease activity- based probe such as the rhodamine 1 10- derived probe can be prepared with any type of side chain target for which mutant proteases that cleave C-terminal to that side chain are desired.
  • a mutant protease that can catalyze cleavage of that amide bond reveals the rhodamine 1 10 core fluorophore, generating a ⁇ I Ed- fold enhancement of fluorescence quantum yield on the bead.
  • Mutant library screening proceeds through a complex workflow of PCR mutagenesis, two emulsified biochemical reactions, and flow cytometry (Pig, 1 , bottom).
  • a mutant library is constructed by PCR, the mutant library is diluted and emulsified together with the protease activity assay beads in a PCR mix containing the opposite primer required to amplify the mutant protease gene. Genes and beads are diluted such that on average each droplet contains ⁇ 0.3 gene molecules and -1 bead, The emulsion PGR (emPCR) is then thermally cycled. In droplets that house both bead and gene, the bead becomes clonally populated with -I k copies of the progenitor mutant gene. Ail beads are harvested from emPCR and washed.
  • This bead library is emulsified with in vitro transcription/translation (IVTT) reagent.
  • IVTT in vitro transcription/translation
  • the bead-bound genes are transcribed to RNA, which is then translated to protease if the mutant protease is active (Fig. 1 ), it transforms the quenched bead-bound probe to the highly fluorescent bead-bound j 10 product. If the mutant protease is inactive (Fig. 1 ), the probe remains quenched and the head non- fluorescent.
  • the beads are harvested from the emiVTT, washed, and analyzed by
  • FACS fluorescence-activated cell sorting
  • the invention provides protease activity screening matrix that contain a solid support conjugated to a substrate moiety that can be specifically cleaved by a specific protease. Also provided in the invention are methods for utilizing the protease activit screening matrix described herein for identifying a variant protease that recognizes a desired substrate cleavage site, The invention additionally provides specific variant trypsin enzymes or enzymatic fragments that have eitrulline- dependent proteolytic activities.
  • emulsion PCR refers to a method for template amplification employed in multiple next-generation sequencing platforms. EmPCR is based on compartmenta!ization of DNA fragments in minute water dropieis/vesicies in a water-moil emulsion to a degree of dilution where there is only a single or a few template molecules per droplet. Ideally, each vesicle/droplet contains one sphere, one single-stranded template molecule, one of the primers bound to the sphere, and all other reagents necessary for the PCR reaction; the second primer remains in the solution to screen out molecules bound to same adapters. Thus, every droplet functions as an isolated PCR micro-reactor leading to generation of numerous copies of bound templates, facilitating signal amplification an detection during IVTT and FACS.
  • the organic compound citrulUne is an a-amino acid. It has the formula
  • HaNCCOiNHCCfbfjCH NlTijCOjH it is a key intermediate in the urea cycle, the pathway by which mammals excrete ammonia by converting it into urea.
  • Several proteins contain citrulline as a result of a postiranslationa] modification (PTM). These citrulline residues are generated by a family of enzymes called peptidylarginine deiminases (PADs), which convert arginine into citrulline in a process called citrullmatibn or deimination.
  • PTM postiranslationa] modification
  • Proteins that normally contain citrulline residues include myelin basic protein (MBP), fliaggrin, and several histone proteins, whereas other proteins, such as fibrin and viinentin are susceptible to citrul!ination during ceil death and tissue inflammation.
  • MBP myelin basic protein
  • fliaggrin fliaggrin
  • histone proteins whereas other proteins, such as fibrin and viinentin are susceptible to citrul!ination during ceil death and tissue inflammation.
  • a librar of protease variants or a combinatorial library of protease variants refers to a collection of protease mutants or variants having distinct and diverse amino acid mutations in its sequence with respect to the sequence of a starting template or wild type protease.
  • the mutations represented in the collection can be across the sequence of the starting protease or can be in a specified region or regions of the starting protease.
  • the mutations can be made randomly or can be targeted mutation designed empirically or rationally based on structural or functional information.
  • a template protease refers to a protease having a sequence of am ino acids that is used for mutagenesis thereof.
  • a template protease can be the sequence of a wild-type protease, or a eatalytically active portion thereof, or it can be the sequence of a variant protease, or eatalytically active portion thereof, for which additional mutations are made.
  • a specific variant protease identified in the selection methods herein can be used as a starting template for further mutagenesis to be used in subsequent rounds of selection,
  • random mutation refers to the introduction of one or more amino acid changes across the sequence of a polypeptide without regard or bias as to the mutation .
  • Random mutagenesis can be facilitated by a variety of techniques known to one of skill in the art including, for example, UV irradiation, chemical methods, and PCR methods e.g error-prone PCR).
  • a focused mutation refers to one or more amino acid changes in a specified region (or regions) or a specified position (or positions) of a polypeptide.
  • targeted mutation of the amino acids in the specificity binding pocket of a protease can be made.
  • Focused mutagenesis can be performed, for example, by site directed mutagenesis or multi-site directed mutagenesis using standard recombinant techniques known in the art.
  • desired specificity with reference to substrate specificity refers to cleavage specificity for a predetermined or preselected or otherwise targeted substrate.
  • protease refers to any peptide, polypeptide or peptide or polypeptide-containing substance that catalyzes the hydrolysis of a protein or peptide
  • the protease may be natural or non-naturally occurring and may be isolated from a natural source, may be recombinant or synthetic and is not required to be in any particular form.
  • Proteases include, for example, serine proteases, cysteine proteases, aspartic proteases, threonine and metallo-proteases depending on the catalytic activity of their active site and mechanism of cleaving peptide bonds of a target substrate.
  • proteases examples include trypsin, chymotrypsm, bromelain, cathepsin B, cathepsin D, cathepsin G, c!osiripain, collagenase, dispose, endoproteinase Arg-C, endoproteinase Asp-N,
  • endoproteinase Glu-C endoproteinase lys-C, factor Xa, kallikrein, papain, pepsin, plasm in, proteinase K, subtUisin, tbermolysin, thrombin, aeyiat ino-acid-releasing enzyme, aminopeptidase M, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, carboxypeptidase Y, cathepsin C, leucine aminopeptidase, and pyrog!utarnate
  • a "substrate” is a molecule that binds to the active site of a protease and is cleaved by the protease. After cleavage, part of the substrate may remain bound to the protease.
  • a zymogen refers to a protease that is activated by proteolytic cleavage, including maturation cleavage, such as activation cleavage, and/or complex formation with other proteih(s) and/or cofactor(s).
  • a zymogen is an inactive precursor of a proteolytic enzyme.
  • zymogens are converte to active enzymes by specific cleavage, including catalytic and autocatalytic cleavage, or by binding of an act ivating co-factor, which generates an active enzyme,
  • a zymogen thus, is an enzymatically inactive protein that is converted to a proteolytic enzyme fay the action of an activator.
  • Cleavage can be effected autocatalytically Zymogens, general ly, are. inactive and can be con verted to mature active polypeptides fay catalytic or autocatalytic cleavage of the proregion from the zymogen.
  • a protease domain is the catalytieally active portion of a protease.
  • Reference to a protease domain of a protease includes the single, two- and multi- chain forms of any of these proteins.
  • a protease domain of a protein contains all of the ; requisite: properties of that protein required for its proteolytic activity, such as for example, its catalytic center.
  • a catalytieally active portion or proteolytically active portion of a protease refers to the protease domain, or any fragment or portion thereof that retains protease activity.
  • the single chain forms of the proteases and catalytic domains or proteolytically active portions thereof exhibit protease activity.
  • a’’nucleic acid encoding a protease domain or catalytically active portion of a protease refers to a nucleic acid encoding only the recited single chain protease domain or active portion thereof, and not the other contiguous portions of the protease as a continuous sequence
  • active site of a protease refers to the substrate binding site where catalysis of the substrate occurs.
  • the structure and chemical properties of the active site allow the recognition and binding of the substrate and subsequent hydrolysis and cleavage of the seissile bond in the substrate.
  • the active site of a protease contains amino acids that contribute to the catalytic mechanism of peptide cleavage as well as amino acids that contribute to substrate sequence recognition, such as amino acids that contribute to extended substrate binding specificity.
  • the "substrate recognition sequence” or "cleavage site” refers to the sequence that is recognized by the active site of a protease and cleaved by a protease.
  • a cleavage sequence is made up of the P1 -P4 and PT-P4' amino acids in a substrate, where cleavage occurs after the PI position.
  • a cleavage site for a serine protease is six residues in length to match the extended substrate specificity of many proteases, but can be longer or shorter depending upon the protease.
  • the probe moiety' or probe peptide described herein typically contains a desired substrate recognition sequence or cleavage site.
  • target substrate refers to a substrate that is specifically cleaved at its substrate recognition site by a protease.
  • a target substrate includes the amino acids that make up the cleavage sequence.
  • a target substrate includes a peptide containing the cleavage sequence and any other amino acids
  • a target substrate includes a peptide or protein containing an addi tional moiety that does not affect cleavage of the substrate by a protease
  • a target substrate can include a four amino acid peptide or a full-length protein chemically linked to a fluorogenie moiety
  • altered specificity refers to a change in substrate specificity of a modified or selected protease compared to a starting wild-type or template protease
  • the change in specificity is a reflection of the change in preference of a modified protease for a target substrate compared to a wild type substrate of the template protease (herein referred to as a non-target substrate).
  • modified proteases or selected proteases provided herein exhibit increased substrate specificity for any one or more predetermined or desired cleavage sequences of a target protein compared to the substrate specificity of a template protease.
  • a modified protease or selected protease that has a substrate specificity ratio of 100 for a target substrate versus a non-target substrate exhibits a 10-fold increased specificity compared to a scaffol protease with a substrate specificity ratio of 10,
  • a modified protease that has a substrate specificity ratio of 10 exhibits a 10-fold increased specificity compared to a scaffol protease with a substrate specificity ratio of 10
  • a modified protease has a 1 5-fotd, 2-fold, 5-fold, lQ-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400- fold, 500-fold or more greater substrate specificity for any one of more of the predetermined target substrates.
  • primer refers to a nucleic acid molecule that can act as a point of initiation of template-directed DMA synthesis under appropriate conditions (e,g., in the presence of four different nucleoside triphosphates and a polymerization agent, such as DMA polymerase, RNA polymerase o reverse transcriptase) in an appropriate buffer and at a suitable temperature, it will be appreciated that a certain nucleic acid molecules can serve as a "probe” and as a “primer.”
  • a primer has a 3' hydroxyl group for extension, A primer can be use in a variety of methods, including, for example, polymerase chain reaction (PC ft), reverse-transcriptase (RT)-PCR, RNA PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3' and 5 RACE, in situ PCR, ligation- mediated PCR and other amplification protocols
  • primer pair refers to a set of primers that includes a 5’
  • upstream primer that hybridizes with the 5' end of a sequence to be amplified (e.g by PCR) and a 3’ (downstream) primer that hybridizes with the complement of the * end of the sequence to be amplified.
  • downstream primer that hybridizes with the complement of the * end of the sequence to be amplified.
  • the invention provides assay matrices (“assay media” or“substrate matrices”) and related methods that can be used to screen for modified or variant proteases that have an altered or desired substrate specificity relative to a known or wild type protease (reference or starting protease).
  • the assay matrix is contains a solid support onto which Is immobilized a substrate moiety.
  • the substrate moiety contains a probe moiety that harbors a designed peptide cleavage site that is desired to be specifically cleaved by a variant (or modified) protease of interest.
  • the probe moiety can additionally contain a detectable label that will generate a detectable signal upon cleavage of the probe by a variant protease of interest in addition to the probe moiety, the substrate moiety also contains a nucleotide moiety that is capable of priming replication of a
  • polynucleotide sequence encoding a candidate variant protease.
  • solid support or matrix materials can be used to prepare the solid assay matrix of the invention it can be any porous or non-porous material or matrix suitable for attaching macromolecules such as proteins, peptides, nucleic acids and the like.
  • the material can be formed in filters, membranes, flat surfaces, tubes, channels, we Us, sheets, beads, microspheres, columns, fibers (e.g.
  • the solid support can also be multiwell tubes (such as mierotiter plates) such as 12-well, 24-well 48-well, 96-well, 384-well, and 1537-well plates, in some embodiments, the solid support is a particle or bead.
  • Preferred beads are made of glass, latex, or a magnetic material (magnetic, paramagnetic, or stipe rmagnetic beads), in some embodiments, the sol id support can he a set of color coded microspheres such as those manufactured and sold by Luminex Corporation (Austin, ex,).
  • the solid support is particulate in this fashion, it is convenient to use individually addressable particulate structures that perm it identification and separation of the structures individually addressable structures are known in the art and include magnetic beads, radio-frequency tagged particles, fluorescently labeled microspheres and the like.
  • bound detection complex comprising the inhibitor bound to the protease
  • the presence of bound detection complex can be detected by virtue: of the presence of the label present on the inhibitor and the addressable moiety ⁇ present on the structure.
  • a fluorescently labeled bead can be detected using flow cytometry, as discussed in more detail below, in some preferred embodiments, the solid support employed in the invention is magnetic bead.
  • the substrate moiety can be bound covalently to the solid support by any technique or combination of techniques well known in the art.
  • the substrate moiety is conjugated to the solid support matrix via a linker moiety
  • the linker moiety can connect one or both of the probe moiety and the nucleotide moiety to the solid support.
  • the linker moiety is a chemical compound or small molecule group that reacts with both the substrate moiety and the solid support.
  • the linker can be a homobi functional or heterobi functional chemical group that connects the substrate moiety and the solid support.
  • the substrate moiety e.g., the probe
  • the linker moiety is first functionalized or reacte with the linker moiety before reacting with the solid support. In these embodiments the linker moiety can be considered part of the substrate moiety
  • any suitable compounds can be used as the linker moiety in the assay matrices of the invention, probes, e.g., peptides or organic compounds in some
  • linker moiety may be prepared from organic compounds such as alkyl chains, phenyl compounds, ethylene glycol, amides, esters and the like.
  • the linker moiety can typically comprise a chain length fro i to about 100 atoms, more preferably, from 5 to about 30 atoms.
  • suitable linker moieties include, but are not limited to ethanol amine, ethylene glycol, polyethylene with a chain length of 6 carbon atoms, polyethylene glycol with 3 to 6 repeating units, phe noxyethano I,
  • propanol a ide butylene glycol, butyleneglycolamide, propyl phenyl chains, and ethyl, propyl, hexyl, steryl, cetyl, and paimitoyl alkyl chains,
  • the probe moiety in the substrate assay matrix of the invention typically contains a cleavage site that is designed or desired for a variant protease of interest to cleave.
  • the cleavage site can be a specific peptide sequence (probe peptide) that is recognized by a variant protease that one wishes to identify : or select from a library of candidate variant protease.
  • the cleavage site can be a peptide sequence that contains post-translational modifications (PTMs).
  • the probe moiety can include a peptide sequence that contains a deim mated arginine (citrulllne).
  • the cleavage site can also contain other PTMs, e.g,, arginine methylation, lysine acetylation or metby!aiion.
  • the probe can additionally Include one or more detection labels (label moieties) whic can generate a detectable signal upon cleavage of the cleavage site.
  • the detectable label can either itself be detected or can produce a detectable signal upon reacting with another molecule under appropriate conditions. It can be any molecule, functional group or chemical moiety that displays or provides a signal that can be readily detected and/or measured. These: include, for example, radioactive isotopes fluorescent labels, chemiluminescent labels,
  • the label moieties can also be haptens that are be recognized by secondary reagents such as antibodies, peptides, direct chemical interactions, and other methods that are well known in the art.
  • the label moiety may also be an
  • the label moiety may also comprise two ehromophores bound in close proximity to utilize a phenomenon called fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the label in the probe moieties of the invention can be a fluorescent molecule, a chemiluminescent molecule (e.g., chemiluminescent substrates), a phosphorescent molecule, a radioisotope, an enzyme substrate, an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a chromogemc substrate, a contrast agent, an MR1 contrast agent, a positron emission tomography (PET) label (e.g., Techneimm-99m and fludeoxyglueose), a phosphorescent label, and the like.
  • chemiluminescent molecule e.g., chemiluminescent substrates
  • a phosphorescent molecule e.g., a radioisotope
  • an enzyme substrate e.g., an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a
  • the detectable label is : preferably a small moiety such as a detectable atom (e.g., a radioactive isotope), a small organic molecule, or a small reactive chemical moiety or functional group, as opposed to bigger molecules such as enzymes or other polypeptides.
  • the employed label moieties can be fluorophores, rhodamine moieties, and eoinnarin moieties (e.g., such as 7-arnino ⁇ 4 ⁇ carbamoyieoumarin, 7- amim 3-carbamoyimethy!-4-methylcoumarin, or 7 ⁇ am!no ⁇ 4 ⁇ methyle0umarin),
  • fluorophores that can be used in the invention: include, e.g., fluorescein, fluorescein analogs, BOD!PY -fluorescein, arginine, rhodamine ] 10, ihodamme-B, rhodamine -A, rhodamine derivatives, and the like.
  • fluorophores include, e.g., fluorescein, fluorescein analogs, BOD!PY -fluorescein, arginine, rhodamine ] 10, ihodamme-B, rhod
  • the detectable label is a long wavelength f!uorophore such as fluorescent dyes in the A!exa Fluor family (Thermo Fisher Scientific Inc.),
  • a fluorescein derivative such as 5-FAM-X-SE (6 - (Fluorescein - 5 - carboxamido)hexanoic acid, succimmidy! ester) can be used to label the probe moieties of the invention, This will l o result in the probe peptide being conjugated to a fluorophore, fluorescein - 5 - carboxamido)hexanoie acid (5-FAM-X).
  • the detectable label in the probe moieties can be a fluorogenie molecule as exemplified herein.
  • Fluorogenic molecules are fiuorophores in which fluorescence is activated by enzymatic activity, light, or
  • conjugation of the label moiety to the probe peptide results in quenching of the detectable signal (e.g., fluorescence) from the Sahel moiety
  • the detectable signal e.g., fluorescence
  • the cleavage site peptide sequence can be conjugated to rhodam e 1 10 (Ri 10).
  • these fluorogenic substrates contain an amino acid or peptide covalently linked to each of Rl 1 Q’s amino groups.
  • the quenched nonfluorescent probe moiety is converted to R 1 10 which displays a detectable fluorescence signal.
  • the detectable label in the substrate moiety is a fluorophore that generates a fluorescent signal upon, e.g., light excitation.
  • the detectable label or label moiety can be attached to the cleavage site (probe peptide) at any position in some embodiments, the detectable label is Jinked to a side chain of the probe peptide, In some embodiments, the detectable label is attached to the N-tennina! residue of the probe peptide, In some other embodiments, the detectable label is attached to the C -terminal residue of the probe peptide. As exemplified herein, some probe moieties of the invention have a detectable label that is coupled to the C- terminus of the probe peptide.
  • iluorophore-labeled peptide probes can be readily performed via protocols exemplified herein and/or method well known in the art. See, e.g., Weder et ah, J. Chromatogr. 698, 181 , 1995; Cavrois et ah, Nat. Biotechno! . 20,
  • labeled peptide probes can be prepared by either modifying isolated peptides or by incorporating the label during solid-phase synthesis.
  • f!uorophores can be conjugated to the N- or C- terminus of a resin-bound peptide before other protecting groups are removed and the labeled peptide is released from the resin.
  • Labeling of the peptide probes of the invention may also be achieved indirectly by using a biotinylated amino acid, in some other embod iments, the label moieties in the probe moieties of the invention are electroactive species for electrochemical detection or chemiluminescent moieties for chemiluminescent detection.
  • UV absorption is also an optional detection method, for which UV absorbers are optionally used, Phosphorescent, colorimetric, e.g., dyes, and radioactive labels can also be optionally attached to the probe peptides,
  • the substrate moiety of the assay matrices of the invention typically further contain a nucleotide moiety that can mediate or prime the replication or amplification of a polynucleotide sequence encoding a library of candidate variant proteases.
  • the assay matrices of the invention are useful for screening from a library of candidate proteases for a variant protease of interest that is capable to cleave the probe peptide of substrate moiety.
  • polynucleotides encoding a library of candidate proteases are reacted with an assay matrix to generate a heterogeneous population of assay matrices that can then express the candidate variant proteases via emulsion in vitro transcription/translation (IVTT).
  • IVTT emulsion in vitro transcription/translation
  • Variant protease of interest that can specifically cleave the probe peptide of the substrate moiety can then be identified.
  • the nucleotide moiety in the substrate moiety cart be any compound that can direct amplification of the library of enzyme- encoding polynucleotide sequences.
  • the nucleotide moiety is an oligonucleotide PCR primer sequence as exemplified herein,
  • the invention provides methods for screening a library of candidate variant proteases to identify a variant protease with desired catalytic specificity.
  • the methods involve first constructing an assay matrix as described herein that is intended for screening variants of a specific protease (e.g., trypsin) to identify a variant of interest with desired substrate specificity.
  • a specific protease e.g., trypsin
  • the substrate moiety on the assay matrix should contain a probe moiety or probe peptide having the desired cleavage site in addition, the nucleotide moiety of the substrate moiety should be able to prime amplification of polynucleotide sequences encoding a library of variants of the chosen or target protease in addition, the methods require polynucleotide sequences encoding a library of candidate variant proteases from which a desired variant protease of interest is to be identified.
  • Such polynucleotide sequences can he obtained by introducing mutations to the active site of the chosen protease, e.g., via PGR
  • variants are generated by randomizing two 4-amino at c solvent-exposed loops adjacent the enzyme’s active site.
  • Each of the 2 loop mutant libraries (each 4 20 :::: 160,000 members) can be used to generate solid support libraries displaying a citrulhne bearing probe moiety
  • the methods of the invention can be applied toward various proteases to identify variants with any cleavage site specificity.
  • the desired cleavage site can be any side chain in the substrate sequence, For example, it can be a site or side chain containing PTM
  • the desired cleavage site can also be any one or a subset of the ⁇ 20 biogenic amino acids.
  • the cleavage site can be at an amino acid residue with a charged side chain (acidic or basic amino acid residue), with an uncharged polar side chain, with a nonpolar side chain, with a beta-branched side chain or an aromatic side chain.
  • the substrate sequence contains (or is capable of sensing cleavage at) any of such desired amino acid side chains.
  • Proteases that can be employed in the methods of the invention can be any known class of protease capable of peptide bond hydrolysis.
  • Candidate proteases forScreening typically are wild type or modified or variant forms of a wild type candidate protease, or catalytically active portion thereof, including allelic variant and isoforms of any one protein. These include proteinases (endopeptsdases) and peptidases (exopeptidases).
  • the methods of the invention are employe for screening variants of a proteinase. Suitable proteinases include, e,g., the serine-, cysteine-, aspartic-, threonine- and metalio-type endopeptidases.
  • the library of variant proteases can be generated via various means.
  • Combinatorial libraries can be prepared in accordance with methods: routinely practiced in the art or the specific techniques exemplified herein. See generally, Combinatorial Libraries: Synthesis, Screening and Application Potential (Cortese Ed.) Walter de Gruyter, Inc., 1995:; Tietze and Lied, Curr. Opirs. Chem. Biol., 2(3):363-71 ( 1998); Lam, Anticancer Drug Des,, 12(3): 145-6? (1997); Blaney and Martin, Curr. C/pin. Chem. Biol., 1 ( I ):54 ⁇ 9 (1997); and Schultz and Schultz, Biotechnol. Prog., 12(6):729-43 ( 1996)). Methods and strategies: for- generating diverse libraries, including protease or enzyme libraries, including positional scanning synthetic combinatorial libraries (PSSCL), have been developed using molecular biology methods and/or simultaneous chemical synthesis methodologies. See, e.g.
  • the library of variant proteases can be generated via mutagenesis of a template or wild type enzyme.
  • mutagenesis methods include, for example, use of E. co!i XLl -red, UV irradiation, chemical modification such as by deamination, alkylation, or base analog mutagens, or PCR methods such as D A shuffling, cassette mutagenesis, site- directed random mutagenesis, or error prone PCR (see e.g, U.S, Application No.: 2006- 01 15874).
  • Such examples include, but are not limited to, chemical modification by hydroxy lam irie (Ruan, H,, et al. ( 1997) Gene 188:35-39), the use of dNTP analogs (Zaccolo, M., et al. (1996) I. Mol. Biol, 255:589-603), or the use of commercial ly available random mutagenesis kits such as, for example, GeneMorph PCR-based random mutagenesis kits (Stratagene) or Diversify random mutagenesis kits (Clonteeh).
  • Focused mutation can be achieved by making one or more mutations in a pre-determ ined region of a gene sequence, for example, in regions of the protease domain that mediate catalytic acti vity and/or substrate binding.
  • one or more amino acid residues in such regions of a protease can be mutated using any standard single or multiple site-directed mutagenesis kit such as for example QuikChange (Stratagene).
  • one or more amino acid residues of a protease can be mutated by saturation mutagenesis (see, e.g., Zheng et al. Nucl. Acids, Res,, 3 . 2; 115, 2004).
  • the chosen residues for mutagenesis are outside the active site the enzyme, as exemplified herein for trypsin.
  • the mutation may be made to residues in the active site of the enzyme.
  • substrate specificity and active site of a protease can be determined by molecular modeling based on three-dimensional structures of the complex of a protease and substrate.
  • the screening methods of the invention can typically be performed in a high throughput format. Any of the conventional techniques and equipment known in the art for screening a large number of compounds (e.g., automated library screening) can be employed in this screening assay of the present invention.
  • the methods are directed to screening for variant enzymes that have PTM- dependent protease activities, as exemplified herein for identifying citrulfine- dependent enzymes.
  • polynucleotides are contacted with the assay matrix.
  • the mixtures are then subject sequentially to emulsion PCR (emPCR) and emulsion in vitro transcription/translation femiVTT),
  • the variant protease sequences and the assay matrix are diluted such that on average each droplet contains around 0.3 polynucleotide per assay matrix (e.g., per magnetic bead).
  • the emulsion PCR emPCR
  • the assay matrix is harvested and washed before emulsified with in vitro transeription/translaflon (IVTT) reagents.
  • candidate variant proteases that are able to ⁇ cleave the probe peptide in the substrate moiety will generate a detectable signal (e,g., a fluorescent signal) that can be readily detected, e.g;, via FACS.
  • the identified assay matrix can then be subject to further thermal cycling to amplify the hound polynucleotide for activity assay and sequencing.
  • emulsion of the enzyme-encoding polynucleotide sequences and the assay matrix is performed with an emulsion formulation, e.g , a watgr-in-oi! emulsion.
  • the emulsion formulation typically contains a continuous phase arid a dispersed phase.
  • the continuous phase is an oil or silicone fluid.
  • the dispersed phase is aqueous.
  • the emulsion formulation can contain an aqueous phase containing the appropriate biochemical reaction mixture, e.g., reagents for the PCR and IVTT reactions.
  • the emulsion formulation contains a continuous phase-solubilized stabilizer (e.g., silicone hydrophobe surfactant or hydrocarbon hydrophobe surfactant) and an aqueous phase-soluble stabilizer.
  • a continuous phase-solubilized stabilizer e.g., silicone hydrophobe surfactant or hydrocarbon hydrophobe surfactant
  • an aqueous phase-soluble stabilizer e.g., aqueous phase-soluble stabilizer.
  • the continuous/oii phase stabilizer is present during both the emPCR and the ern!YTT reactions, while the dispersed/aqueous phase stabilizer is used onl in the emPCR reaction to enable generation of thermally stable emulsions.
  • the continuous/oil phase stabilizer employed in some of these embodiments can be KF-6Q38, and the employed dispersed/aqueous phase stabilizer can be KF-6012.
  • the methods disclosed herein have significant advantages that are applicable to any type of PTM for which a protease can be evolved to recognize for proteolysis.
  • digestion of purified protein with the PTM- ⁇ dependent protease and low-resolution mass analysis can provide some immediate insight, particularly if the PTM removes a tryptic cleavage site (e,g via arginine dei ination or methylafion, lysine acetylation or methylation, etc.).
  • PTM-dependent proteolysis Similar to the ubiquitous and uniform neutral loss of phosphate (80 or 98 Da) observed upon collision-acti vated dissociation (CAD) and MS2 analysis of certain phosphopeptides, PTM- dependent proteolysis generates peptides that should in theory yield a consistent y
  • Trypsin is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates where it hydrolyses proteins. Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cleaves peptide chains mainly: at the carboxyl side of the amino acids lysine or arginine, except when either Is followed by proline. It is used for numerous biotechnological processes, The process is commonly referred to as trypsin proteolysis or trypsin izatibn, and proteins that have been digested/treated with trypsin are said to have been
  • the aspartate residue (D189) located in the catalytic pocket (S l) of trypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine, and is, thus, responsible for the specificity of the enzyme.
  • the invention provides specific trypsin variants that are capable of cleave citruliine modified peptide sequences.
  • the trypsin variants of the invention contains amino acid mutations Di 89S and one or more additional mutations L7P, E185K an K ⁇ 88A, or conservative substitutions thereof.
  • the trypsin variant of the invention can ; include amino acid mutations DI 89S and L7P, or conservative substitutions thereof.
  • trypsin variants can additional contain mutation E185K or K188A, or conservative substitution " thereof, in some preferred embodiments, the trypsin variant of the invention can include amino acid mutations D189S, E185K, K188A and L7P, or conservative substitutions thereof In addition to these amino acid residue substitutions, the trypsin variants can have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% r 99% identical to the wild type trypsin sequence.
  • Some trypsin variants of the invention are trypsin fragments that contain one or more of the noted amino acid substitutions at the active site (DI 89S, E185K and K 188 A).
  • the variant trypsin enzymes of the invention exhibit strong citruliine- dependent protease activities
  • these variant trypsin molecules were identified by emulsifying a library of variant trypsin encoding sequences with a protease assay matrix bearing citruliine activity probe.
  • the library was then challenged in emiVTT, and analyzed by FACS as described above.
  • Highly fluorescent beads 53 beads were individually sorted, amplified and the PCR amp! icons assayed for cltrulline-dependeni proteolytic activity using the same iVTT reaction mixture and free-solution Rl 10 protease activity probe of the emiVTT challenge.
  • the variant trypsin proteases of the invention have advantageous util ities that are not previously available. As exemplified herein in MS/MS analysis experiments. PAD4, one of the above enzymes responsible for arginine deiraination, can also deiminate its own arginines (’'autode minaiion”) under conditions of high i !).
  • Digestion of the autodeiminated PAD4 (adPAD4) with the 806 protease followed by LC-MS/MS analysis of the digest reveal ed a series of peptides that unambiguously displayed characteristics of citrull inatian, Extracted ion chromatograms contained peaks corresponding to both the deiminate and unmodified peptide, with appropriate relative retention times (citrullmaied at later retention time relative to the unmodified peptide).
  • the citrulline- dependent proteolytic cleavage that the B06 protease catalyzes results in unique peptide fragments that bear a C-terminal citrulfine at modified sites.
  • the citrulfine-dependent proteases of the; invention greatly simplify the detection of a protein ⁇ s sites of citruilination. While there are abundant tools for isolating citrull inated proteins (e.g., anti-citrulline peptide antibodies and citruUine-specific chemical probes), these tools only facilitate pull-down of the target. Identification of the specific citrulhnated residue requires sequencing. Typically, citm! line-containing peptides can be identified as tryptic off-target cleavages at citrulfine, but the intensity ⁇ ' is very low because trypsin’s citrull ine-specific activity is almost immeasurable.
  • citruiline-speclfic enzymes described herein can directly cleave at citrullinated residues, resulting in fragments that terminate in citrulline.
  • the residual tryptic activity of the variant enzymes also permits it to cleave at arginine.
  • Probe-primer beads and a trypsinogen mutant gene library were used in emulsion PCR (emPCR) to generate beads clonal iy populated with ⁇ 10,000 copies of a gene library member (probe-gene beads) followed by emulsion in vitro transcription/translation (emIVTT) for highly parallel, low-volume protein expression an activity assay, FACS analysis isolated single beads encoding active proteases that dequehched bead-bound R 1 10 probes (Fig, I, Lower),
  • Control tryptic probe-primer beads displaying (acGPRLR I 10 that were digested with MS-grade or translated and enterokinase (EK)-activated trypsin and analyzed by flow cytometry exhibited good separation fro untreated and unfimctionalized beads.
  • EK enterokinase
  • Overexpressibn of wild-type trypsin was: unsuccessful under otherwise identical conditions. Installation of L7F alone onto trypsirr was sufficient to generate overexpress son product by SDS/PAGB, and the P7L trypsin* ® ' 1 revertant abolished overexpression product.
  • Tryptic and control digest XIC only contained the fbl ⁇ -length probe (9 and EK control digests of the BHQ-IPRAA-TMR probe yielded only full- length probe (987,75* ⁇ ! m/z) in the respective XICs, whereas the tryptic digest XIC contained new N- and C-terminal products of cleavage at Arg ( i ,03Q.58 + and 962, 33 f ro/z, respectively); the C -terminal fragment exists: as three regioisomerie TMR coupling products, which are chromatographically distinct but otherwise indistinguishable by MS, Trypsin* -51 * exhibited greatly enhanced citrul line-dependent activity in steady-state kinetic analyses.
  • the catalytic efficiency of BHQ-IPRAA-TMR cleavage by trypsin was 3.9 c 10 ⁇ M ⁇ Tryptic cleavage of BHQ-IPcttAA-FAM was undetectable.
  • a magnified view displays two example features unique to the PAD4 and adPAD4 trypsin tv!t digest.
  • the 1 ,387 m/2 ion (534 -T L REHN S F V E R- 544) (SEQ ID NO; 17) was prevalent in both PAD4 and adPAD4 trypsin +CiI digests, yet undetectable in the tryptic digest.
  • the peptide’s sequence yielde Us molecular formula and thereby the theoretical abundance of each isotopic peak based on the binomial distribution and natural abundances of each element.
  • the theoretical and observed isotopic envelopes agreed for both PAD4 and adPAD4 versions of the 1 ,387 m/z ion.
  • MS2 spectrum of this peptide contained 176.1030 m/z, the yi citrufline fragment, y I (cit), and its ammonia neutral loss, 159,0764 m/z,
  • a search for all MSI precursors generating the 176, 1030/159,0765 MS2 citruilination signatures yielded 66 unique MS I Of these 66, 28 produced two-peak XIC,, the earlier eluting peak
  • Example 4 Digestion identifies Citruilination in PAD4 -Treated Fibrinogen
  • Emulsification, PCRs contain FCR buffer ( 1 3 ⁇ 4), dNTPs (200 mM each), PEG-
  • disulfide enhancers 1 and 2 (14,9 pL each), and EK ( i /2G0 dilution , 4.1 pL).
  • the reaction 150 pL was combined w ith ice-cold oil (76% [wt/no ⁇ ] DMF-A-6es 20% [wt/vol j mineral oil: 4% [wt/vo!] KF-6038; 600 pL) and a stainless steel bead (6 mm diameter). Sample was loaded into a homogen izer (TsssueL-yser; QIAGEN), emulsified (10 s, 15 Hz; 10 $, 17 Hz), and placed on ice.
  • a homogen izer TsssueL-yser; QIAGEN
  • am plifications (5 ug) or plasm id tem plate (22 ng) was added to IVTT reactions (5 mE, above) and either (acIPcit) 2 R 1 10 (3 mM) or (cbzJPR)2R1 10 (3 mM). Reactions were incubated (37 °C, 1,000 min) with fluorescence monitoring (channel 1, CFX96; Bio-Rad).
  • lysozyme (Thermo Fisher Scientific), DNase I (New England BioLabs), RNase A (QIAGEN), urea (Bio-Rad), oxidized glutathione (GSSG), reduced glutathione (GSH; Thermo Fisher Scientific), sodium phosphate dibasic, sodium phosphate monobasic, 5 Prime PerfectPro Ni-NTA agarose (Thermo Fisher Scientific), imidazole, oUView Tris-Giycine precast gel (4-20%, NuSep; VWR), SDS, DL-1 ,4-DTT (Thermo Fisher Scientific), ammonium bicarbonate (AMBIC), sequencing grade trypsin (Promega Corp ), ActivX Desthiobiotin-FP Serine Hydrolase probe (Thermo Fisher Scientific), ⁇ Z-li6 ⁇ PrG“Arg)2R l I Q HCi salt (CPC Scientific), and iodoaeeta ide were used as provided.
  • Bead buffer (10 niM Tris, pH 8.3, 100 M NaCI, 0,5 tnM EDTA, 0.05% KF-6012), breaking buffer (I O mM Tris, pH 7.5, l OQ mM NaGl, 1 mM EDTA, 1% [wt/voi] SDS. 1 % [vol/vol] Triton X-100), binding and washing buffer with Tween- 20 (2x, BWBT, 10 mM Tris, pH 7,5.
  • Lysis buffer, lysate washing buffer, solubilization buffer, and renaturing buffer were sterilized (Q.22-pm syringe filter; HMD Millipore).
  • PGR buffer IO c , 100 mM Tris, pH 8.3, 500 mM KCI, 15 mM MgC!g; New England BioLabs), CutSmart Buffer (New England BioLabs), T4 DNA ligase buffer (New England BioLabs), and reducing Laemroli buffer (6*; Thermo Fisher Scientific) were used a provided. Oil for PCR and IVTT emulsification (76% [wt/voi] DMF- A-6cs;
  • 5-Azidopentanoic acid NHS ester was prepared by dissolving NHS (9.6 prnol) EDC (9 6 pmol), and 5-azidapentanoic acid (7.2 pmol) in DMF (26 4 pL) and incubating (30 min, 60 °C), The 5-az.idopentanoic acid NHS ester solution was added to the solution of 5' ⁇ am nohexyl -modified oligonucleotide and incubated (2 h, RT). A fresh aliquot of 5 ⁇ azidopentanoic acid NHS ester was prepared as described above, added to the acylation reaction, and the reaction incubated (1 h, RT).
  • reaction was quenched (3 M Tris, pH 7.6, 100 -xL) and incubated (5 min, 60 °C). 5 ! -Azido oligonucleotides were precipitated twice in ethanol. The pellet was dried (N2), resuspended (HPLC grade H2O, 200 pL).
  • Purified fluorogenic rhodamine probe core was diluted (2 mM in 0, 1 % TFA) and directly infused (50 pL/rnln) to an electrospray ionization (ESI) source of a mass spectrometer (LTQ-XL; Thermo Fisher Scientific).
  • ESI electrospray ionization
  • LTQ-XL mass spectrometer
  • Probe core resin S3 (30,5 mg) was transferred to a fritted syringe (2,5 fflL; Torviq) and the DMF drained.
  • Pmoc- trp!!ine-OH 1 mmol in i mL DMF
  • DIBA 2 mmol
  • COMU 1 mmol in 1 mL DMF
  • the activated acid was then added to resin, and the resin incubated with rotation (1 ,5 h, 50 °C, 8 rpm), The resin was washed with DMF until there was no visible trace of color in the DMF wash.
  • Probe construction proceeded via iterative cycles of solid-phase peptide synthesis.
  • Prnoc de- protection (20% [vol/vol] piperidine in DMF, 2 1 mL, 1 5 in each aliquot, RT, 8 rpm);
  • N-a-Fmoc-amino acid (0.2 mmol in 0,5 mL DMF) acti vation with COMU (0.2 mmol in 0.5 in i DMF) and DiEA (0.4 mmol) and incubation (30 s, RT); and
  • the TFA solution was expelled into ice-cold diethyl ether (22 ml,), incubated (-20 °C. 1 h), and centrifuged (5 min, 7,000 x g). The supernatant was decanted and the orange pellet dried in vacuo.
  • Probe core resin S3 (30.5 mg) was transferred to a fritted syringe (2.5 ml.; Torviq) and the DMF drained, Fmoe-Arg(pbf)-OH (1 mmol in I mL DMF) was combined with DIEA (2 mmol) and CQMU ( I mmol in 1 mL DMF), Incubated (30 s, RT), and the activated acid added to resin and the resin incubated with rotation ( 1.5 h, 50 °C, 8 rpm).
  • Rink Amide resin 160 pm, 0.44 mmol/g, 227 mg; Rapp-Poiymere was transferred to a fritted syringe (6 mL; Torviq), swelled in DMF ( 1 h, RT, 14 rpm), and washed (DMF, 2 x 2 ml.; DCM, 2 ⁇ * 2 mL; DMF, 2 c 2 mL).
  • Mtt was removed by washing the resin with DCM (.3 x 2 mL) and then deprotection mixture (1 % TFA, 5% TIPS, 94% dry DCM, 4 c 1.5 mL), and finally incubating the resin in deprotection mixture (3 x 2 mL, 5 min first two aliquots, 15 min third aliquot).
  • the deprotected resin was washed (4 2 mL DMF;4 x 2 mL DCM; 4 x 2 mL, DMF).
  • Rink Amide resin 160 gm, 0,44 mrool/g, 22.7 mg; Rapp-Polymere was transferred to a frited syringe (6 mL; Torviq), swelled in DMF (I h, RT, !4 rpm), and washed (2 x 2 mL DMF; 2 x 2 mL DCM; 2 x 2 ml, DMF) iterative cycles of peptide synthesis included (i) Fmoc deproteetion (20% [vol/vol] piperidine in DMF, 2 x 1 mL, 5 min each aliquot, RT, 8 rpm); (ii) N-a-Fmoc-amino acid (30 pmoj in 0,5 nil DMF) activation with COMU (30 mhio ⁇
  • Catalyst mix was prepared by combining C11SO4 (100 mM, 0.5 pL), TBTA ( 10 mM, 6 pL), and ascorbic aci (300 mM, 2.5 pL).
  • the beads were immobilized on magnet, the supernatant removed, azide mix and beads combined, catalyst mix added, and the reaction incubated (90 min, 50 °Q 14 rpm). Reactants were removed and the beads washed (2 c BWBT, 1 x 0,5 mL), resuspended in BWBT (1 mL), and incubated (90 min, 50 °C, 14 rpm).
  • oligonucleotide functionalized beads were washed (BWBT, 2 x 0,5 L; bead buffer 2 x 0.5 mL). Oligonucleotide- functionalized beads were exchanged to solvent (DMF, 2 * 0 5 mL, 1 * i ml), incubated (90 f ain, RT), and washed (DMF, 1 mL), Bead aliquots (0.5 mg) were further elaborated with (ae.IPcit) 2 Rl 10 probe S4,/(acGPR)2 H0 probe S5, or rhodamine probe core S3 (5 nmol probe in 20 pL DMF) Probes were combined with DIG (350 nmol in 5.5 pL DMF) and H O At (250 nmol in ⁇ m ⁇ .
  • GTATACCAAAGTTTGTAATTACGTGAACTGGATTCAGCAGACGATC- GCAGCGAACTAA underlined, italicized, double underlined, and underlined/italicized residues indicate propeptide, loop 1 , D189 and loop 2, respectively.
  • the porcine pancreatic trypsinogen ORP (GenBank accession no. NM_001 162891 ) was synthesized (GenScriptj with codon use optimized for bacterial expression.
  • the trypsinogen ORE was inserted between the Ncoi and Blpi sites of the pET45b vector (EMD Mil iipore) with the 5VATG-3' of the Ncoi corresponding to the site of translation initiation, Plasniid ( 1 rig) was combined with PCR buffer ( 1 V), dNTPs (200 mM each dNTP), Taq (0.05: U/pL), arid oligonucleotide primers 5 ?
  • the reactions were thermally cycled ([95 °C ⁇ 15 s; 62 °C, 20 s; 68 °G 60 s] c 20 cycles, C 1000; Bio-Rad) and the products confirmed Ori agarose gel. Products were purified (MinElute; QIAGEN) and quantitated by A26G.
  • Tryptic probe-primer beads (1 E6 beads) were combined with ftypsin (1 pg) and buffer (200 mM AMB1C, pH 8.3, 25 pL), incubated (37 °C, 3 1 h), digestion solution removed, and beads resuspended (0.5 rnL PBS), A separate tryptic probe-pri er head aliquot. (3 E6 beads) was transferred to an amplification reaction (150 pL) containing trypsinogen expression cassette tern- plate DNA (56 pg ⁇ 41 molecules/bead), PGR buffer (l x), dNTPs (200 mM each), PEG-8000 (4,8 mM), butanedioi (5% [vol/vol]).
  • Template standard solutions ( 1 E5-1 E-1 fg/pjL in log-scale dilutions) were prepared and added to separate amplification reactions (20 pL each).
  • Standards and samples were thermally cycied ([95 C, 20 s; 68 °C, 90 s] * 30 cycles, C 1000; B io-Rad) with fluorescence monitoring (channel 1, CFX96; Bio-Rad).
  • Samples were quantitated (CFX Manager, version 3, 1 ; Bio- Rad, baseline sub- traction). The number of trypsmogen templates per bead was calculated by dividing the qPCR result by the number of beads.
  • Tr psmogen-tem plated probe-primer beads (1.5E6 beads, probe- gene beads) were combined with IVTT components solution A (10 mE), solution B (7,5 m ⁇ .,), disulfide bond enhancer (1 mE each enhancer), and EK ( 1 /200 dilution, 0.5 pL).
  • Negative control beads (1 .5E6 beads) were combined with IVTT
  • Emulsion PCR Bead Library Preparation An amplification reaction ( 150 pL) was prepared containing PCR buffer ( l x), dNTPs (200 mM each), PEG-8000 (4.8 mM), butanediol :(5% [vol/voi]), KF-6012 (0.02% [vol/vol]), Taq (0.3 U/pL),
  • oligonucleotide primer 5 -TGCGTCCGG CGTAG AGG ATC-3' (SEQ ID NO; I I ),
  • PCR mix was combine with ice-cold emulsification oil mix (600 pL) and a stainless steel bead (6 mm diameter; Thomas Scientific).
  • Sample was loaded into a homogenizes (TissueLyser; QIAGEN) and emulsified (10 s, 15 Hz: 10 s, 17 Hz), The emulsion was placed on ice immediately and aliquots (50 pL) were transferred to PCR tubes for thermal cycling ([95 °C, 20 s; 68 °C, 90 s] x 30 cycles; 68 °C, 600 s; C 1000; Bio- Rad). Emulsion samples were pooled, combined with breaking buffer (750 pL), and mixed The beads were collected by centrifugation (5 min, 3,000 x g) and immobilized on magnet.
  • dNTPs 200 mM each ⁇ , Taq (0.05 Ij/uL), oligonucleotide primers 5 f -TGCGTCCGGCGT.A G A GG ATC-3 * (SEQ ID NOfi l ) and 5'-AGACCGAG- A T A G G G TTG A G TO TT G - 3 ' (SEQ ID NO: 12) (0.5 mM each), and 5V56-FAM/ ATG AAT ACC (SEQ ID NO: 13) /ZEN/TTT GIT CTG CTG GCA CTO CIO (SEQ ID NO: 14) /3iABkF:Q/ 3' S’ exonuclease assay probe (250 nM).
  • ampliflcatton reaction mixture (320 pL) was reserved for template standards (20 mE each, prepared in log dilutions as described).
  • template standards (20 mE each, prepared in log dilutions as described).
  • emPCR bead product (84 beads) was added, the sample vortexed, and aliquots (20 mE) quickly distributed to wells of a 96-well plate.
  • the plate was thermally cycled ([95 °C, 15 s; 68 °C, 80 s] x 40 cycles; CLOOO; Bio- Rad) with fluorescence monitoring (channel 1 , CFX96; Bio-Rad) and quantitated (CFX Manager, version 3, 1 ; Bio-Rad, Cq method), assigning Cq to each Well.
  • Wells with Cq within the standard curve were used to calculate the number of templates per bead.
  • Emulsion XVTT, Emulsion IViT, Beads from emPCR were washed (bead buffer, 3 x 100 L), resuspended in bead buffer ( 100 pL), and transferred to a 2-mL safe- lock tube (Thermo Fisher Scientific).
  • ⁇ U ⁇ T reaction components contain solution A (74,7 pL), solution B (56 m ⁇ ;.), disulfide enhancers 1 and 2 (14.9 m ⁇ each), and EK (1/200 dilution, 4.1 m ⁇ .. , ).
  • Fluorescence-Activated Cell Sorting Beads from ernlVTT were resuspended in PBS (1 mL, --5E6 beads/rnL).
  • Control ⁇ aeiPCit) 2 Rl 10 activity probe-primer beads were similarly prepared.
  • Samples were analyzed by FACS (FACSDiva 8.0.1 ; BD Bioscienees).
  • the control sample was analyzed (13,000 events) to establish a negative gate.
  • the positive gate was defined to yield a Hit rate of 0.004% Hit beads were sorted into individual wells of a 96- well microtiter plate.
  • Amplification reaction mixture for qPCR was prepared as described above and added to each well (20 pL); the plate was then thermally cycled and quantitated as above, PCR products were confirmed on agarose gel Weils exhibiting the correct PCR product size were purified (MinEh.rte;
  • the induced culture was incubated with shaking (30 °C, 4 h, 250 rpm). Aliquots (50 mL) were centrifuged, the supernatant discarded, and the cell pellet stored (-20 °C). One pellet of cells was thawed on ice (3 h), resuspended in lysis buffer (3 mL), and incubated (30 min, 4 °C). Egg hen: lysozyme (0, 1% [wt/vol]) was added and the cells incubated (35 min, 4 °C). DNase G (2 U) and RNase A (6 pg) were added and the cells Incubated (30 min. 4 °C).
  • the inclusion bodies were pelleted by centrifugation (30 min, 1 8,514 c g), the su pernatant discarded, and the pellets were washed with lysate washing buffer (9 mL, overnight, 4 3 ⁇ 4), The washed pellet was centrifuged: (30 min, 18,514 * g) and washed again with lysate washing buffer (9 mL, I h, 4 °C). After centrifugation (30 min, 4 °C, 1 8,514 * g), pellets were combined with solubilization buffer (9 mL) and incubated with rotation (6 h, 4 °C, 15 rpm).
  • Desthiobiotin-FP Serine Hydrolase probe 0, 5, 10, 20, 25, 50, or 1 00 mM; 1 m ⁇ ,) and incubating (2 h, 37 °G), Aliquots of (acIPCit) 2 Rl 10 (100 mM, 1 uL) were added to the equilibrated enzyme mixtures and fluorescent intensity monitored (Gemini XPS; Molecular Devices; l 3 ⁇ 4c - 500 nm, l3 ⁇ 4p ⁇ 525 nrn, automatic cutoff - 51 5 nm). Active site- concentration was estimated by linear extrapolation of observed slope (relative
  • sequencing-grade trypsin (6 mM) in combination with either (cbz- IPR)2R 1 10 probe or BHQ1-IPRAA- TMR probe S7 as substrates.
  • sequencing-grade trypsin 22 mM
  • buffer 100 mM AMBIC, pH 8,3, 10 L
  • substrate S4, 1.9 mM in DMSO, 2.5 m ⁇ .
  • the rnicroplate was analyzed (Gemini XPS; Molecular Devices, Xex - 494 nm think Xem ⁇ 525 ntn) monitoring fluorescence every 10 s for 400 s.
  • KM and k C at were determined using nonlinear regression with rate given in REUs/s and concentrations in mM, RFU was converted to concentration (mM) using the following empirical formula: [FAM] - (RFU FA M - 15.1 )/2,680 and [TMR] - (RFU TM R - ⁇ 39.8 ⁇ /1,600.
  • IPdtAA-FAM and BfiQl-iPRAA-TMR Sequencing-grade trypsin and trypsin c si stock solutions (6 mM, 50 pL) were prepared. Trypsin " ⁇ 11 was combined with EK (22 frao! and incubated (30 min, RT). Negative control enzyme solution (50 pL) . was prepared by combining EK (22 finol) and buffer (50 mM AMBIC, pH 8.3, 50 m ⁇ ,).
  • Digestion reactions were prepared in flat-botom black 384- well mieropiates (Coming, Inc.) by combining buffer (50 mM AMBIC, pH 8.3, 14 uL), protease stock (trypsin, EK-acti vated
  • the instrument was set to perform: one MS I scan (300-2,000 nVz) followed by one data-dependent MS2 and one data-dependent MS3 scan (most intense ion with minimum intensity of 2,500 counts, charge state of +2, isolation width window ⁇ 2 m/z, and normalized CID collision energy 35%).
  • instrument was set to perform MSI only (300-2,000 m/z).
  • Purified, recombinant human PAD4 (35 mM, 24 pL) was combined with calcium- containing buffer (100 mM Hepes, pH 7.6, 50 mM aCl, 10 mM CaCfl, 20 mM DTT, 156 pLJ and incubated with shaking (37 °C, 1 h, 400 rpm), A control sample was prepared by adding enzyme (24 pL) to HepeS buffer (1 56 m ⁇ ,), Autodeiminaied PAD4 (adPAD4) and control PAD4 (PAD4) were quenched by diluting aliquots of each (60 pL) in denaturing buffer (500 pL) in a molecular weight cutoff filter (10,000 IV1WCO; EMD Millipore) and centrifuged (20 min, RT, 14,000 x g).
  • calcium- containing buffer 100 mM Hepes, pH 7.6, 50 mM aCl, 10 mM CaCfl, 20
  • Reducing agent 200 mM DTT, 7 m ⁇ . was added to the filter, the sample incubated (2 h, 37 ° €, 400 rpm), and the filter centriftiged (20 min, RT, 14,000 x g), lodoacetamide (500 mM, 14 pL) was added to the filter and the sample incubated protected from light ( 1 h, RT). Denaturing buffer (200 pL) was added and the filter centrifuged (30 in, 14,000 x g).
  • Citrulline-dependent mutant protease 14 pM, 30 pL was combined with buffer (100 mM AMBIC, pH 8,3, 10 pL) and then combined with EK (30 fmol), Control enzyme solution was prepared by combining buffer (50 mM AMBIC, pH 8.3, 40 pL) and EK (30 fmol). Trypsin was prepared from stock (22 pM, 18 pL) by dilution In buffer ( 100 m ' AMBIC, pH 8.3, 22 pL) and E (1 /10 dilution, 4 pL) just before digestion, Buffer (50 mM AMBIC, pH 8.3, 200 pL) was applied to the filter and the filter centrifuged (20 min, 14,000 * g).
  • Buffer 50 mM AMBIC, pH 8.3, 100 pL was applied to the filter together with an aliquot (6 pL) of mutant protease, trypsin, or control enzyme solution
  • the filters were incubated with shaking (15 min, 37 °C, 750 rpm) and then incubated (37 °C, overnight).
  • Digestion volumes were adjusted to 100 pL with buffer(50 mM AMBIC, pH 8 3); an aliquot (50 pL) was removed for electrophoretic analysis; the filter centrifuged (20 min, 14,000 x g); and the filtrate collected.
  • the filtrate volume was adjusted to 80 pL (0.3% TEA in Di H20), reduced in volume to 30 pL (Speed-Vac;
  • adPAD4 tryptic digest (- 100 fmol) and adPAD4 mutant protease digest :( ⁇ 3 pmol) were analyzed by LC- MS/MS.
  • Peptides were concentrated and desalted on an RP precolumn (Acclaim PepMap 100 nanoViper, 0,075 c 20 mm; Thermo Fisher Scientific) and resolved using reversed-phase HPLC (Acclaim PepMap RL SC nano-Viper, 0,075 x 150 mm; Thermo Fisher Scientific), with gradient elution (300 nL/min, mobile phase A: 0.1 % [vol/vol] formic acid in HgO; mobile phase B: 0 1 % [vol/vol] formic acid and 80% [vol/vol] ACN in H2O; 5% B 3 min, 5-40% B 60 min, 40-80% B 2 min), Eiuate was directly infused into the ESI source of the tandem mass spectrometer (Q Exactive; Ther
  • the filter was centrifuged (20 min, 14,000 x g) lodoacetamide (500 M, 24 pL) was added to the filter, the sample incubated protected from light (1 h, RT), and the reactants re- moved by centrifugation (20 min, 14,000 x g).
  • Buffer SO mM AMBIC, pH 8.3, 230 pL
  • Mutant protease 14 pM, 30 mE
  • buffer 100 mM AMBIC, pH 8,3, ! O pLj and then activated by adding EK (30 fmoi).
  • Control enzyme solution was prepared by combining buffer (50 mM
  • AMBIC pH 8,3, 40 m ⁇ ., and EK, (30 fmo!). Trypsin was prepared from stock (22 mM, 18 pL) by dilution in buffer (100 mM AMBIC, pH 8.3, 22 pL) and EK (1/10 dilution, 4 pL) just before digestion. Buffer (50 mM AMBIC, pH 8.3, 100 pL) was applied to the filter together with an aliquot (6 pL) of Bod enzyme, trypsin, or control enzyme solution, and the filters incubated (37 °C, over- night). The filters were centrifuged (30 min, 14,000 x g) and the filtrate collected.
  • Denaturing buffer 50 pL was applied to the filter, the filter incubated (1 h, 37 °C; 5 min, 95 °C; 5 min, 37 °C), inverted, and centrifuged into a new' collection tube (5 min, 14,000 * g). Samples retained on the filter (50 m ⁇ .,) were combined with 6* Laemmii sample buffer (10 pL), incubated (5 min, 95 °G), and analyzed using SDS/PAGE (4-20% Mini-PROTEAN Tris- Glycine, 120 V, 60 min; Bio-Rad), Peptide solution volumes were adjusted to 90 pL with Di H2 . 0, evaporatively dried to 25 pl :
  • the samples were resolved using reverse-phase HPLC (0.075: * 150 mm Acclaim PepMap RLSC Nano Viper, EASY-nLC 1000; Thermo Fisher Scientific) with gradient elution (3Q0 nL/min, mobile phase A: 0.1% [vol/vol] formic acid in H2O; mobile phase B: 0.1% [vol/vol] formic acid, 80% [vol/vol] ACM in H2O; 5% B for 15 min; 5-40% B for 45 min, 40-80% B for 10 s, 80% B for 5 min), and eluent was directly infused to the ESI source of a tandem mass spectrometer (Orbitrap Fusion; Thermo Fisher Scientific).
  • Mass spectra were acquired in data-dependent MS2 mode using Top Speed precursor selection in a survey scan from 380 to 1 ,400 /z with Orbitrap detection.
  • Data-dependent MS2 was performed with HCO fragmentationfnormalized collision energy 30%) and detection in the Orbitrap.
  • Protein Identification was carried out using Mascot and X! Tandem (34, 35). Variable modifications NQR (deamidated) and M (oxidation) were permitted, carbarn idom ethylation of Cys was a fixed modification, one missed cleavage of a nonspecific enzyme was permitted, and mass tolerance of 5 ppm and 0,02 Da was set for precursor and fragment ions, respectively.
  • MS/MS raw files were searched against a customized database containing the amino acid sequences of the proteins of interest (UniProtKB accession nos. P02671, P0267I_2 S P02675, P02679, and Q9U M07).

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Abstract

La présente invention concerne des matrices de criblage d'activité de protéase qui contiennent un support solide conjugué à une fraction de substrat qui peut être clivée de manière spécifique par une protéase spécifique. L'invention concerne également des procédés d'utilisation des matrices de criblage d'activité de protéase fournies par la présente invention pour identifier un variant de protéase qui reconnaît un site de clivage de substrat souhaité. L'invention concerne en outre des variants d'enzymes de trypsine ou des fragments enzymatiques spécifiques qui ont des activités protéolytiques dépendantes de la citrulline.
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CN110426514A (zh) * 2019-08-28 2019-11-08 苏州新格诺康生物技术有限公司 肽酰基精氨酸脱亚胺酶(pad)的新型测活法
US11123735B2 (en) 2019-10-10 2021-09-21 1859, Inc. Methods and systems for microfluidic screening

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US20050014160A1 (en) * 2003-07-18 2005-01-20 Sriram Kumaraswamy Assays for protease enzyme activity
US9051612B2 (en) * 2006-09-28 2015-06-09 Illumina, Inc. Compositions and methods for nucleotide sequencing
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110426514A (zh) * 2019-08-28 2019-11-08 苏州新格诺康生物技术有限公司 肽酰基精氨酸脱亚胺酶(pad)的新型测活法
US11123735B2 (en) 2019-10-10 2021-09-21 1859, Inc. Methods and systems for microfluidic screening
US11247209B2 (en) 2019-10-10 2022-02-15 1859, Inc. Methods and systems for microfluidic screening
US11351544B2 (en) 2019-10-10 2022-06-07 1859, Inc. Methods and systems for microfluidic screening
US11351543B2 (en) 2019-10-10 2022-06-07 1859, Inc. Methods and systems for microfluidic screening
US11919000B2 (en) 2019-10-10 2024-03-05 1859, Inc. Methods and systems for microfluidic screening

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