WO2021025623A1 - Procédé de détection d'interactions protéines à protéines - Google Patents

Procédé de détection d'interactions protéines à protéines Download PDF

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WO2021025623A1
WO2021025623A1 PCT/SG2020/050458 SG2020050458W WO2021025623A1 WO 2021025623 A1 WO2021025623 A1 WO 2021025623A1 SG 2020050458 W SG2020050458 W SG 2020050458W WO 2021025623 A1 WO2021025623 A1 WO 2021025623A1
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protein
nucleic acid
interaction
template nucleic
variant
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PCT/SG2020/050458
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Farid John Ghadessy
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Agency For Science, Technology And Research
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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
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66

Definitions

  • the invention is in the field of protein biochemistry.
  • the invention relates to a method of detecting protein-protein interactions.
  • the invention also relates to a method of detecting protein ligase activity.
  • Protein-protein interactions can also be facilitated by protein ligases.
  • Protein ligases have recently emerged as important tools in the field of chemical biology.
  • peptide ligases which facilitate covalent linkage between the N- and C-termini of substrate proteins and peptides harbouring appropriate recognition sequences, have been used extensively in the development of new protein architectures and site-specific tagging. Protein ligases therefore allow a myriad of applications including the assembly of protein domains and the production of protein conjugates such as antibody-drug conjugates.
  • the conventional method of detecting protein ligase activity involves incubating substrate proteins in the presence of the enzyme and visualizing the product after separation by sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE sodium dodecyl sulphate -polyacrylamide gel electrophoresis
  • FRET fluorescence resonance energy transfer
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescence-activated cell sorting
  • a method of detecting an interaction between a first protein and a second protein comprising: a) providing a first fusion protein comprising the first protein connected to a nucleic acid amplifying enzyme; b) providing a second fusion protein comprising the second protein connected to a processivity enhancing protein; c) contacting the first protein of the first fusion protein to the second protein of the second fusion protein; d) performing a nucleic acid amplification reaction of one or more template nucleic acid under a condition in which the nucleic acid amplifying enzyme is not able to amplify the one or more template nucleic acid; e) detecting the interaction between the first protein and the second protein, wherein the interaction between the first protein and the second protein restores the activity of the nucleic acid amplifying enzyme and wherein the amplification of the one or more template nucleic acid under said condition is indicative of the interaction between the first protein and the second protein.
  • a method of determining activity of a protein ligase comprising: a) providing a first fusion protein comprising a first protein connected to a nucleic acid amplifying enzyme; b) providing a second fusion protein comprising a second protein connected to a processivity enhancing protein; c) ligating the first protein of the first fusion protein to the second protein of the second fusion protein with the protein ligase; d) performing a nucleic acid amplification reaction of one or more template nucleic acid under a condition in which the nucleic acid amplifying enzyme is not able to amplify the one or more template nucleic acid; e) determining the activity of the protein ligase, wherein the interaction between the first protein and the second protein restores the activity of the nucleic acid amplifying enzyme and wherein the amplification of the one or more template nucleic acid under said condition is indicative of protein ligase activity.
  • a bacterial cell comprising: a) a polynucleotide sequence encoding a first fusion protein comprising a first protein connected to a nucleic acid amplifying enzyme; b) a polynucleotide sequence encoding a second fusion protein comprising a second protein connected to a processivity enhancing protein.
  • a method of selecting one or more variants of an interacting protein pair, wherein the one or more variants have a higher affinity interaction relative to the wild type proteins comprising: a) providing a first fusion protein comprising a first protein variant of the interacting protein pair connected to a nucleic acid amplifying enzyme; b) providing a second fusion protein comprising a second protein variant of the interacting protein pair connected to a processivity enhancing protein; c) contacting the first protein variant of the first fusion protein to the second protein variant of the second fusion protein; d) performing a nucleic acid amplification reaction of one or more template nucleic acid under a condition in which the nucleic acid amplifying enzyme is not able to amplify the one or more template nucleic acid; e) detecting the interaction between the first protein variant and the second protein variant, wherein the interaction between the first protein variant and the second protein variant restores the activity of the nucleic acid amplifying enzyme and where
  • peptide refers to a polymeric form of amino acids. Proteins and polypeptides are understood to comprise more amino acids than peptides. Proteins and polypeptides typically comprise at least about 35 amino acids while peptides typically comprise from 2 to about 35 amino acids. Proteins may comprise 1 or more polypeptides and the individual polypeptide chains may be covalently or non-covalently linked. A portion of the protein or polypeptide may have or be capable of acquiring a three-dimensional arrangement by forming secondary, tertiary or quartemary structures. Peptides, polypeptides and proteins may be naturally-occurring or non-naturally occurring. Proteins may include moieties other than amino acids (e.g. may be glycoproteins) and may be otherwise processed or modified. In the context of this application, the terms “peptide”, “polypeptide” and “protein” may be used interchangeably.
  • interacting protein pair refers to 2 proteins that are capable of interacting covalently or non-covalently via protein -protein interactions.
  • Non- covalent interactions may include hydrogen bonds, ionic bonds, van der Waals interactions and hydrophobic bonds.
  • Examples of interacting protein pairs include SpyCatcher-SpyTag and the split NanoLuc lucif erase system.
  • a high affinity interaction refers to a protein-protein interaction that is has a dissociation constant (K d ) of less than 200 nM.
  • ligation refers to covalent linkage of two proteins mediated by an enzyme.
  • the ligation of two proteins involves the formation of a peptide or isopeptide bond between the two proteins.
  • An isopeptide bond is a type of peptide bond that forms between the carboxyl group of one amino acid and the amino group of another, where at least one of these joining groups is part of the side chain of one of these amino acids.
  • the enzyme mediating the ligation of proteins is known as “protein ligase” or “peptide ligase”.
  • ligation may also refer to native chemical ligation, which involves a chemo selective reaction between a first peptide having a C -terminal a-carboxythioester moiety and a second peptide having an N-terminal cysteine residue.
  • a thiol exchange reaction yields an initial thioester-linked intermediate, which spontaneously rearranges to give a native amide bond at the ligation site while regenerating the cysteine side chain thiol.
  • protein ligase or “peptide ligase” as used in the context of this application refer to enzymes which catalyse the formation of peptide and isopeptide bonds with site and substrate specificity. In the context of this application, the terms “protein ligase” and “peptide ligase” are used interchangeably. Protein ligases catalyse protein ligase reactions and are used in a myriad of applications including the assembly of protein domains, the production of therapeutic protein conjugates (e.g. antibody-drug conjugates) and the production of fusion proteins.
  • therapeutic protein conjugates e.g. antibody-drug conjugates
  • fusion protein refers to a protein comprising a polypeptide or a fragment thereof linked to another polypeptide.
  • a fusion protein can be made recombinantly by constructing a nucleic acid sequence encoding a polypeptide or fragment thereof in frame with a nucleic acid sequence encoding a different protein, and then expressing the fusion protein.
  • a fusion protein can be generated by connecting a polypeptide or fragment thereof with another protein via covalent bonds or non-covalent interactions.
  • fusion proteins can be generated by chemical methods such as cross-linking/native chemical ligation or can be generated by ligation by a protein ligase.
  • the term “variant” in the context of a protein refers to a protein comprising a mutation of one or more amino acids as compared to a reference protein.
  • the term “mutation” refers to a modification to the amino acid sequence resulting in a change in the amino acid sequence of the protein compared to a reference amino acid sequence.
  • the mutation may involve one or more amino acid residues and may be selected from the group consisting of substitution, insertion, deletion, truncation and combinations thereof.
  • a library of protein variants may be generated from mutation of the protein. The library may be randomised or semi-randomised. A randomised library is generated by random mutation of the amino acid residues of a protein.
  • a semi-randomised library is generated by random mutation of specific amino acid residues of a protein.
  • Protein variants may be functional or non-functional.
  • a functional protein variant is one that retains the activity of the wild type protein or has increased activity compared to the wild type protein.
  • a non-functional protein variant is one that has decreased activity or complete loss of activity compared to the wild type protein.
  • the activity of a functional protein variant may refer to the affinity with which the functional protein variant interacts with another protein.
  • the term “inhibit” in the context of inhibiting the activity of an enzyme refers to a partial decrease or a complete loss in the activity of the enzyme when compared to the enzyme’s original activity.
  • the term “restore” in the context of restoring the activity of an enzyme refers to the reversal of a decrease in the activity of the enzyme.
  • the term “restore” may refer to a partial recovery or a full recovery of the enzyme activity after a loss of activity.
  • the conditions that inhibit or restore the activity of an enzyme may include but are not limited to temperature, pH, salt concentration and reaction durations.
  • PCR polymerase chain reaction
  • PCR cycles are well established in the art.
  • Thermostable polymerases such as Taq DNA polymerase and its variants are commonly used.
  • the versatility of PCR has led to a large number of variants of PCR such as real-time/quantitative PCR, reverse transcription PCR, nested PCR, assembly PCR and multiplex PCR.
  • PCR can be used in various research applications including genotyping, cloning, sequencing, mutagenesis and microarrays.
  • Amplification may be clonal or non-clonal.
  • Clonal amplification refers to an amplification from a single template DNA molecule only.
  • Non-clonal amplification refers to an amplification from a mixture of template DNA molecules comprising more than one template DNA molecule.
  • amplicon refers to a piece of DNA or RNA that is the source and/or product of amplification or replication events.
  • the term “amplicon” may be used to describe an amplification product. Amplicons can be formed from PCR reactions.
  • copy number in the context of an amplicon refers to the number of copies of the amplicon. The copy number of an amplicon can be used to quantify the amplification reaction.
  • the term “not able to amplify” or “unable to amplify” in the context of a template nucleic acid refers to a nucleic acid amplifying enzyme not being able to amplify the full length of the template nucleic acid or amplicon.
  • a nucleic acid amplifying enzyme such as a polymerase
  • reaction parameters that limit the function of the enzyme, such as cycling conditions in a polymerase chain reaction (PCR). Cycling conditions may refer to PCR annealing times, or PCR extension times, or both.
  • PCR cycling conditions for optimal polymerase function such as annealing time and extension time
  • annealing time and extension time will depend on the length of the template nucleic acid. For example, for a shorter amplicon, the polymerase would require a shorter annealing time and shorter extension time to carry out amplification of the full length of the template nucleic acid. For longer amplicons, the polymerase would require a longer annealing time and longer extension time to carry out amplification of the full length of the template nucleic acid.
  • An example of a reaction parameter that limits the function of the nucleic acid amplifying enzyme is accelerated cycling conditions.
  • accelerated cycling conditions refers to PCR cycling times that are shorter than the minimum cycling times required for the polymerase to amplify the full length of the template nucleic acid.
  • accelerated cycling conditions may refer to a PCR annealing time that is shorter than the minimum annealing time required for the nucleic acid amplifying enzyme to amplify the full length of the one or more template nucleic acid, or a PCR extension time that is shorter than the minimum extension time required for the nucleic acid amplifying enzyme to amplify the full length of the one or more template nucleic acid, or both. It will be understood by a person skilled in the art that a polymerase is unable to generate full-length amplicons under the accelerated cycling conditions.
  • the term “ Stoffel” or “ Stoffel fragment” as used herein refers to a protein that makes up amino acid residues 293 to 832 of full length Taq polymerase and is also produced as a recombinant protein in Escherichia coli.
  • Stoffel DNA polymerase is around 2-fold more thermostable than Taq DNA polymerase and works over a broader range of magnesium ion concentrations.
  • CSR Complementaryised Self Replication
  • CSR refers to a technique originally developed to select for thermostable nucleic acid polymerase variants with improved functionality.
  • CSR is a technique based on the self replication of polymerase genes by the encoded polymerases within discrete compartments.
  • CSR entails clonal encapsulation of bacteria expressing a library of polymerase variants into the aqueous compartments of a heat-stable emulsion. Subsequent thermal cycling permits amplification of a polymerase gene only by the particular enzyme it encodes, thereby quantitatively linking activity of the constituent library members to the copy number of their respective genes. Genotype-phenotype linkage is therefore maintained.
  • CH2R Compartmentalised 2-Hybrid Replication
  • CH2R may be used to couple the interaction between an interacting protein pair to polymerase read-out.
  • CH2R involves expressing candidate proteins as respective fusions to a polymerase and a processivity clamp. Protein-protein interaction between the interacting protein pair brings the processivity clamp into close proximity with the polymerase, allowing DNA amplification in conditions that are otherwise prohibitive to the function of the polymerase.
  • CH2R may also be used in the co evolution, or the selection of variants of, interacting protein pairs.
  • Fig. 1 shows an overview of the peptide ligase detection paradigm.
  • the Stoffel fragment is unable to PCR- amplify template DNA in presence of high salt concentrations (top panel).
  • Ligation of the two fusion proteins results in tethering of Sso7d to Stoffel, and rescue of activity in the presence of high salt (middle panel).
  • a fusion protein comprising Sso7d-Stoffel hybrid will PCR amplify template DNA under high salt concentrations (lower panel).
  • Fig. 2 shows that the coupling of Sso7d and Stoffel fragment mediated by SpyCatcher-SpyTag interaction facilitates PCR in high salt buffer conditions.
  • indicated proteins were (co)-expressed in E.coli and cells directly used in PCR reactions with increasing KC1 concentrations (0, 100, 200, 300 mM).
  • S7d-S Sso7d - Stoffel fusion
  • S7d-SC Sso7d- SpyCatcher fusion
  • ST-S SpyTag-Stoffel fusion
  • S Stoffel.
  • B shows SDS-PAGE analysis of uninduced/induced E.coli cell lysates (co)-expressing indicated proteins.
  • FIG. 3 shows Compartmentalised Self Replication (CSR) used in the selection of peptide ligase activity.
  • CSR Compartmentalised Self Replication
  • Fig. 4 shows that novel SpyTag variants selected for using the method of the invention are functional.
  • Indicated proteins were (co) expressed in E. coli and cell lysates analysed by SDS-PAGE.
  • Lanes 4-7 indicate that novel SpyTag variants indicated (residues shown replace XXXX in sequence GAHXXXXDAYKP) can form a covalent bond with SpyCatcher (indicated by top arrow).
  • Ligation of endogenous SpyTag to SpyCatcher is shown in lane 2. Note that SpyCatcher and SpyTag components are respectively fused to Sso7d and Stoffel fragment.
  • Fig. 5 shows that processivity-clamp fusion enhances polymerase activity in high salt buffer conditions.
  • A shows PCR amplicon yields at indicated KC1 concentrations in reaction buffer using E. coli cells expressing either Stoffel fragment (S) or a Topoisomerase V HhH processivity domain - Stoffel fusion protein (H-S).
  • B shows PCR amplicon yields at indicated KC1 concentrations in reaction buffer using E. coli cells expressing Sso7d - Stoffel fusion protein with induction at 37°C for 3 hours (lane 1), 37°C overnight (lane 2) and room temperature overnight (lane 3).
  • Fig. 6 shows that the coupling of Sso7d and Stoffel fragment mediated by reconstitution of split Nanoluc luciferase facilitates PCR in high salt buffer conditions.
  • A shows the structure of NanoLuc highlighting the large (light grey) and small (darker grey) fragments of split Nanoluc. Peptide sequences of the endogenous (NS6) and engineered small fragments (NS1-NS5) along with affinity constants indicated to the right.
  • (B) shows PCR amplification in absence (top panel) and presence (lower panel) of 100 mM KC1 by indicated co-expressed proteins.
  • S7d-SC Sso7d-SpyCatcher fusion
  • ST-S SpyTag-Stoffel fusion
  • S Stoffel
  • S7d-NB Sso7d-NanoLuc large fragment fusion
  • NS(l-6)-S NanoLuc small fragment- S toff el fusion.
  • Fig. 7 shows a Compartmentalised 2-Hybrid Replication (C2HR) selection paradigm.
  • C2HR Compartmentalised 2-Hybrid Replication
  • Fig. 8 shows a C2HR model selection. E. coli cells co-expressing either Sso7d-NB + NS1-S or Sso7d-NB + S were mixed at different ratios prior to emulsification and CSR in high KC1 buffer (left panel) or direct PCR in high salt buffer (open control).
  • Fig. 9 shows C2HR selection of functional SpyTag and related variants.
  • (B) shows consensus sequence logos derived from 500 most abundant sequences selected from libraries 1 and 2.
  • Fig. 10 shows that SpyTag variants selected by C2HR retain function.
  • S7d-SC Sso7d- SpyCatcher
  • ST wild-type SpyTag
  • the core “IVMVD” motif has been omitted for clarity (replaced with vertical bar).
  • Highlighted bands represent 1: S7d-SC-ST-S fusion protein; 2: Stoffel fragment; 3: S7d-SC. All selectants yield correct size fusion protein corresponding to wild-type SpyTag control (band 1).
  • B the same expressor cells highlighted in (A) were used directly in PCR reactions ⁇ KC1 (100 mM). As with wild-type SpyTag, all SpyTag variants enabled PCR in high-salt buffer.
  • Fig. 11 shows the directed co-evolution of SpyCatcher and SpyTag.
  • A shows the two underlined phenyalanine residues in SpyCatcher and the underlined isoleucine in SpyTag were randomized prior to selection. The corresponding positions of these residues in the binary complex are shown on the right.
  • B shows consensus sequence logos for naive and library selectants after one or two rounds of C2HR. Frequency of endogenous (FF/I) and other enriched motifs indicated.
  • Fig. 12 shows a pull-down assay of Sso7D-SpyCatcher protein (S7D-SC) by endogenous (ST) and selected (STL2) biotinylated SpyTag peptides.
  • S7D-SC Sso7D-SpyCatcher protein
  • ST endogenous
  • STL2 selected biotinylated SpyTag peptides.
  • Covalently bound Sso7D- SpyCatcher protein indicated by arrow in SDS-PAGE gel. Streptavidin beads with no peptides bound used as control. Lower protein band corresponds to streptavidin monomer co-eluted from beads.
  • Fig. 13 shows that coupling of Sso7d and Stoffel fragment mediated by reconstitution of split Nanoluc luciferase facilitates PCR with accelerated cycling conditions. This figure shows that high affinity interactions can be detected under conditions of accelerated cycling counditions.
  • A shows PCR amplification of a 1545 bp amplicon using shortened annealing and extension times (15 and 10 seconds respectively) and
  • B shows PCR amplification using longer annealing and extension times (30 and 120 seconds respectively) by indicated co-expressed proteins.
  • S7d-SC Sso7d-SpyCatcher fusion.
  • the present invention refers to a method of detecting an interaction between a first protein and a second protein, the method comprising: a) providing a first fusion protein comprising the first protein connected to a nucleic acid amplifying enzyme; b) providing a second fusion protein comprising the second protein connected to a processivity enhancing protein; c) contacting the first protein of the first fusion protein to the second protein of the second fusion protein; d) performing a nucleic acid amplification reaction of one or more template nucleic acid under a condition in which the nucleic acid amplifying enzyme is not able to amplify the one or more template nucleic acid; e) detecting the interaction between the first protein and the second protein, wherein the interaction between the first protein and the second protein restores the activity of the nucleic acid amplifying enzyme and wherein the amplification of the one or more template nucleic acid under said condition is indicative of the interaction between the first protein and the second protein.
  • the first protein of the first fusion protein and the second protein of the second fusion protein may interact in the presence or absence of a protein ligase.
  • the method as described herein further comprises providing a protein ligase; wherein the contacting of the first protein of the first fusion protein to the second protein of the second fusion protein in step c) comprises the ligation of the first protein to the second protein by the protein ligase; and wherein the amplification of the one or more template nucleic acid in step e) is indicative of protein ligase activity.
  • the first protein of the first fusion protein or the second protein of the second fusion protein is a protein ligase; wherein the contacting of the first protein of the first fusion protein to the second protein of the second fusion protein in step c) comprises the ligation of the first protein to the second protein by the protein ligase; and wherein the amplification of the one or more template nucleic acid in step e) is indicative of protein ligase activity.
  • the present invention refers to a method of determining activity of a protein ligase, the method comprising: a) providing a first fusion protein comprising a first protein connected to the a nucleic acid amplifying enzyme; b) providing a second fusion protein comprising a second protein connected to a processivity enhancing protein; c) ligating the first protein of the first fusion protein to the second protein of the second fusion protein with the protein ligase; d) performing a nucleic acid amplification reaction of one or more template nucleic acid under a condition in which the nucleic acid amplifying enzyme is not able to amplify the one or more template nucleic acid; e) determining the activity of the protein ligase, wherein the interaction between the first protein and the second protein restores the activity of the nucleic acid amplifying enzyme and wherein the amplification of the one or more template nucleic acid under said condition is indicative of protein ligase activity.
  • the method as described herein may be used to determine the activity of a protein ligase.
  • protein ligases include SpyCatcher, Sortase, butelase-1 (CtAEPl) and OaAEPlb.
  • the first protein of the first fusion protein and the second protein of the second fusion protein are ligated.
  • the ligation of the first protein and the second protein restores the activity of the nucleic acid amplifying enzyme and facilitates amplification of the one or more template nucleic acid as described herein. Therefore, amplification of the one or more template nucleic acid is indicative of protein ligase activity.
  • the activity of the protein ligase is coupled to the amplification of the template nucleic acid.
  • the protein ligase activity may be quantified in relative terms based on the copy number of the amplicons.
  • the first protein and the second protein may be naturally occurring or mutated.
  • the protein ligase may be naturally occurring or mutated.
  • the mutation may be selected from the group consisting of substitution, insertion, deletion, truncation and combinations thereof.
  • the mutation may be introduced by targeted mutation, random mutation, or combinations thereof.
  • Libraries of protein variants may be generated by mutation of the proteins. Protein variants may be functional or non-functional.
  • the first fusion protein may comprise the first protein connected to the N-terminus or the C-terminus of the nucleic acid amplifying enzyme.
  • the second fusion protein may comprise the second protein connected to the N- terminus or the C-terminus of the processivity enhancing protein.
  • the interaction between the first protein and second protein is a covalent interaction or a non-covalent interaction.
  • Non-covalent interactions may include hydrogen bonds, ionic bonds, van der Waals interactions and hydrophobic bonds.
  • a covalent interaction may result from a ligation reaction.
  • the nucleic acid amplifying enzyme may be a DNA polymerase or an RNA polymerase.
  • the nucleic acid amplifying enzyme is a Stoffel fragment of a Taq DNA polymerase.
  • the nucleic acid amplifying enzyme is the functionally equivalent domain of Stofflel fragment from a family A or family B polymerase, such as polB from Thermococcus kodakarensis or polA-DNA polymerase I from Thermus thermophilus.
  • the processivity of a nucleic acid amplifying enzyme refers to the number of nucleotides that the nucleic acid amplifying enzyme can incorporate into the nucleic acid during a single template-binding event, before dissociating from the nucleic acid template.
  • the processivity of a nucleic acid amplifying enzyme can be increased by the binding of a processivity enhancing protein.
  • the processivity enhancing protein is Sso7d or topoisomerase V HhH. In another embodiment, the processivity enhancing protein is Sso7d.
  • the first protein and the second protein are respectively SpyCatcher and SpyTag, or SpyTag and SpyCatcher.
  • the first protein and the second protein are respectively the large peptide fragment (NB) and small peptide fragment (NS) of split NanoLuc luciferase, or the small peptide fragment (NS) and the large peptide fragment (NB) of split NanoLuc luciferase.
  • the method of detecting an interaction between a first protein and a second protein or the method of determining activity of a protein ligase as described herein is carried out in vitro.
  • step a) to step c) of the method as described herein is carried out in a cell.
  • the cell may be a mammalian cell, a yeast cell or a bacterial cell.
  • the cell is a bacterial cell.
  • the bacterial cell may be an Escherichia coli cell.
  • the first fusion protein and the second fusion protein are expressed in a cell. It will generally be understood that the polynucleotide sequences encoding the fusion proteins are transformed into the cells and that the expression of the fusion proteins are induced in the cell.
  • the first fusion protein and the second fusion protein are expressed in a cell comprising a protein ligase.
  • the protein ligase may be native to the cell or the polynucleotide sequence encoding the protein ligase may be transformed into the cell and expressed in the cell.
  • the one or more template nucleic acid as described herein is template DNA.
  • the template nucleic acid may be a predetermined template nucleic acid.
  • the one or more template nucleic acid as described herein comprises the polynucleotide sequences encoding the first protein, or the polynucleotide sequence encoding the second protein, or both.
  • the one or more template nucleic acid as described herein comprises the polynucleotide sequence encoding the protein ligase.
  • step a) to step c) of the method as described herein is carried out in a cell, it is possible to maintain genotype-phenotype linkage when each cell contains only a single variant of the first protein and when the template nucleic acid is the gene encoding the variant of the first protein.
  • the variant of the first protein is a functional variant that is able to interact with the second protein, clonal amplification of the template nucleic acid occurs during CSR. Since the template nucleic acid includes the gene encoding the variant of the first protein, a functional variant of the first protein generates amplification of its own gene.
  • the affinity of interaction of the variant of the first protein with the second protein is therefore quantitatively linked to the copy number of the variant of the first protein after CSR. It will be understood by a person skilled in the art that the method as described herein may also be used in a situation where each cell contains a single variant of the second protein and when the template nucleic acid is the gene encoding the variant of the second protein.
  • the method as described herein may also be used in a situation where each cell contains a single variant of a protein ligase and when the template nucleic acid includes the gene encoding the variant of the protein ligase.
  • the method can be used to screen a library of variants of a protein ligase by coupling the activity of the protein ligase variant to the amplification of the one or more template nucleic acid. Clonal amplification of the template nucleic acid (e.g. during CSR) is indicative of ligation of a first protein of an interacting protein pair to the second protein of the interacting protein pair.
  • step a) to step c) of the method may be carried out in a cell and the template nucleic acid in each cell is the gene encoding the said protein ligase variant.
  • ligation of the first protein to the second protein results in amplification of the gene encoding said protein ligase variant during CSR, thereby quantitatively linking activity of the protein ligase variants to the copy number of their respective genes.
  • the activity of the protein ligase variants can therefore be compared with the activity of wild type or other variants of protein ligases. Therefore, the method as described herein may be used for the screening of protein ligases.
  • the cell as described herein is located in a water- in-oil droplet.
  • the water-in-oil droplet comprising the cell further comprises a buffer, salt, deoxynucleotides (dNTPs) and primers within the same droplet.
  • dNTPs deoxynucleotides
  • the water-in-oil droplet has a diameter of between 1 pm and 20 pm. In another embodiment, the water-in-oil droplet has a diameter of between 1 pm and 10 pm.
  • the nucleic acid amplification reaction is polymerase chain reaction (PCR).
  • condition of step d) of the method as described herein is a salt concentration of at least 50 mM, or accelerated cycling conditions, or both. .
  • the condition is a salt concentration between 50 mM to 300 mM.
  • the condition may be a salt concentration of about 50 mM, about 100 mM, about 150 mM, about 200 mM or about 300 mM.
  • the salt concentration is about 100 mM.
  • the salt is potassium chloride.
  • Cycling conditions may refer to PCR annealing times, or PCR extension times, or both.
  • the accelerated cycling conditions is a PCR annealing time that is shorter than the minimum annealing time required for the nucleic acid amplifying enzyme to amplify the full length of the one or more template nucleic acid, or a PCR extension time that is shorter than the minimum extension time required for the nucleic acid amplifying enzyme to amplify the full length of the one or more template nucleic acid, or both. It will be understood that the nucleic acid amplifying enzyme is unable to amplify the full length of the one or more template nucleic acid under the accelerated cycling conditions.
  • the detection of the interaction between the first protein and the second protein in step e) of the method as described herein may be based on the detection and quantification of amplicons to determine the strength of the interaction relative to the interaction of a reference pair of interacting proteins.
  • the reference may be an interacting protein pair, such as a wild type interacting protein pair, which serves as a positive control.
  • the reference may be a non interacting protein pair which serves as a negative control.
  • the determination of protein ligase activity in step e) of the method as described herein may be based on the detection and quantification of PCR amplicons to determine the activity of a protein ligase relative to a reference activity of a protein ligase.
  • the reference may be a functional protein ligase, such as a wild type protein ligase, which serves as a positive control.
  • the reference may be any protein ligase with known activity.
  • the reference may be a non-functional protein ligase which serves as a negative control.
  • the present invention refers to a bacterial cell comprising: a) a polynucleotide sequence encoding a first fusion protein comprising a first protein connected to a nucleic acid amplifying enzyme; b) a polynucleotide sequence encoding a second fusion protein comprising a second protein connected to a processivity enhancing protein.
  • the bacterial cell as described herein further comprises a polynucleotide sequence encoding a protein ligase.
  • the present invention refers to a method of selecting one or more variants of an interacting protein pair, wherein the one or more variants have a higher affinity interaction relative to the wild type protein pair, the method comprising: a) providing a first fusion protein comprising a first protein variant of the interacting protein pair connected to a nucleic acid amplifying enzyme; b) providing a second fusion protein comprising a second protein variant of the interacting protein pair connected to a processivity enhancing protein; c) contacting the first protein variant of the first fusion protein to the second protein variant of the second fusion protein; d) performing a nucleic acid amplification reaction of one or more template nucleic acid under a condition in which the nucleic acid amplifying enzyme is not able to amplify the one or more template nucleic acid; e) detecting the interaction between the first protein variant and the second protein variant, wherein the interaction between the first protein variant and the second protein variant restores the activity of the nucleic acid ampl
  • the method as described herein can be used to screen a library of variants of a first protein of an interacting protein pair by coupling the interacting affinity of the first protein variant to nucleic acid amplification readout during clonal amplification.
  • Clonal amplification may be facilitated by Compartmentalised Self Replication (CSR) which entails clonal encapsulation of bacterial cells into the aqueous compartments of a heat-stable emulsion.
  • Clonal amplification may also be facilitated by depositing one bacterial colony in one reaction well or tube. Screening of clonal amplification may be conducted using robotic or manual methods.
  • CSR Compartmentalised Self Replication
  • Amplification of the template nucleic acid is indicative of an interaction between the variant of the first protein with the second protein of the interacting protein pair.
  • step a) to step c) of the method is carried out in a cell and the template nucleic acid is the gene encoding the said variant of the first protein
  • a high affinity interaction between the first protein variant and the second protein results in amplification of the gene encoding said first protein variant, thereby quantitatively linking the interacting affinity of the first protein variants to the copy number of their respective genes.
  • variants of the first protein with high interacting affinity can be selected.
  • the method as described herein can be used to select variants of both members of an interacting protein pair, or co-evolving an interacting protein pair, by coupling the interacting affinity of the first protein variant and second protein variant to nucleic acid amplification readout.
  • Clonal amplification of the template nucleic acid is indicative of an interaction between the variant of the first protein with the variant of the second protein of the interacting protein pair.
  • Clonal amplification may be facilitated by Compartmentalised Self Replication (CSR) which entails clonal encapsulation of bacterial cells into the aqueous compartments of a heat-stable emulsion.
  • Clonal amplification may also be facilitated by depositing one bacterial colony in one reaction well or tube.
  • step a) to step c) of the method is carried out in a cell and the template nucleic acid comprises the gene encoding said variant of the first protein and the gene encoding said variant of the second protein
  • a high affinity interaction between the first protein variant and the second protein results in amplification of the gene encoding said first protein variant and the gene encoding said second protein variant, thereby quantitatively linking the interacting affinity of the protein variants to the copy number of their respective genes.
  • variants of the first and second proteins with high interacting affinity can be selected.
  • the interacting protein pair is SpyCatcher-SpyTag.
  • the interacting protein pair is the large peptide fragment (NB) and small peptide fragment (NS) of split NanoLuc luciferase.
  • Oligonucleotides and genes were from Integrated DNA Technologies; restriction enzymes, T4 polynucleotide kinase and T4 DNA ligase were from NEB; Pfu DNA polymerase (Agilent Technologies) and Taq DNA polymerase (Bioline) were used for DNA amplification. Nucleic acid purification kits were from Qiagen and chemicals from Sigma. Electrocompetent TGI and BL21 cells were obtained from Lucigen.
  • Taq pET22b(+) was generated via amplification of the Taq polymerase gene with primers TAQNdel-F and TAQXholR, followed by infusion into pET22b(+) via Ndel and Xhol sites.
  • Inverse PCR was carried out on Taq pET22b(+) with primers pET-ATG-R and Stoff-F, followed by intramolecular ligation to produce Stoffel pET22b(+) - which encodes only the Stoffel fragment.
  • HhH-Stoffel pET22b(+) which encodes for Topoisomerase V HhH processivity domain - Stoffel fusion, was produced via amplification of the processivity domain gene using primers HhH-Stoff-F and HhH-GGG-Stoff-R, followed by infusion into Stoffel pET22b(+) via Ndel site. Inverse PCR and intramolecular ligation were carried out on Taq pET22b(+) with primers StoffAPWP-F and KALEtoLPETGGG-R to generate Exo-Stoffel pET22b(+).
  • Sso7d-Stoffel pET22b(+) encoding for Sso7d - Stoffel fusion, was constructed via infusion cloning Sso7d gene with primers SS07DINF-F and SS07DINF-R into an inverse PCR product from amplification of Exo-Stoffel pET22b(+) generated using primers pET-ATG- R and EXOLPETV2-F.
  • Complementary primer pair SPYTINF-TOP and SPYTINF-B were annealed to form an oligo duplex which was cloned into Sso7d-SpyCatcher Stoffel pETDuet-1 via Ndel site to yield Sso7d-SpyCatcher SpyTag- S toff el pETDuet- 1.
  • Fib 1 and Fib 2 were created by amplifying Sso7d-SpyCatcher Stoffel pETDuet-1 with primers FPETGG-Sall-F and SPYTR6.2, and FPETGG-Sall-F and SPYTR5.2 respectively.
  • Fib 3 was created by overlap extension PCR of two PCR products - the first with primers FPETGG-Sall-F and SpyC-NNKl-R and the second with primers SpyC-NNK2-F and SpyT-NNK-R on the same vector. All resultant library PCR products were then cloned into Sso7d-SpyCatcher Stoffel pETDuet-1 via Sail and Spel.
  • Constructs expressing Stoffel, HhH-Stoffel fusion protein and Sso7d-Stoffel were transformed into E. coli BL21 (DE3) competent cells. Cells expressing HhH-Stoffel were induced for 3 hours at 37°C with 1 mM IPTG. Cells expressing Sso7d-Stoffel were induced with 1 mM IPTG with different temperature and duration as described in text. 1 mL of culture was then harvested by centrifugation, washed with PBS twice and resuspended in 50 pL of PBS.
  • CSR was carried out using different primers pairs (BIOOLS79- duetMCS2-F and Spytag-Spel-R2 for test selection, BIO-OLS79-LPETGG-Sall-F and Spytag-Spel-R2 for selection of Lib 1 and 3, BIO-OLS79-LPETGG-Sall-F and NESTSpyTag- Spel-R3 for selection of Lib 2) at 95°C for 5 mins, followed by 10 cycles of 95°C for 5 s, 55°C for 30 s and 72°C for 1 min. The aqueous phase was extracted twice with 900 pL ether and treated with 10 pL exonuclease and 2 pL Dpnl overnight at 37°C.
  • the aqueous phase was then incubated with 25 pL streptavidin M280 beads (Invitrogen) for 1 hour with rotation at room temperature before 3 washes with 200 pL of PBSBT [PBS + 0.1% (w/v) BSA, 0.1%(v/v) Tween 20] and 3 washes with 200 pL of PBS.
  • the beads were then resuspended with PCR reactions containing different primer pairs (NESTOLS79-duetMCS2-F and Spytag-Spel-R2 for test selection, NESTOLS79-LPETGG-F and Spytag-Spel-R2 for selection of Lib 1 and 3, NESTOLS79-LPETGG-F and NESTSpyTag-SpeI-R4 for selection of Lib 2) and subjected to a rescue PCR (95°C for 5 mins followed by 20 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 1 min).
  • a rescue PCR 95°C for 5 mins followed by 20 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 1 min.
  • the Sso7D-SpyC construct was cloned with a N-terminal 6xHis-tag and transformed into Escherichia coli BL21(DE3) (Invitrogen) competent cells. These were grown in LB medium at 37°C and induced at OD600 nm ⁇ 0.6 at 25°C with 1 mM IPTG and incubated overnight. Cells were then harvested by centrifugation, sonicated and heated at 65 °C for 15 min before clarification by centrifugation. The clarified cell lysate was applied to a His-TrapFF column (GE Healthcare) and purified using a gradient elution.
  • the fractions containing the protein were pooled and dialyzed into buffer A solution (20 mM Tris, pH 8, 1 mM DTT) using HiPrep 26/10 desalting column, and loaded onto anion-exchange Resource Q 1 mL column (GE Healthcare) pre-equilibrated in buffer A. The column was then washed in 60 column volumes of buffer A and bound protein was eluted with a linear gradient in buffer comprising 1 M NaCl, 20 mM Tris pH 8, and 1 mM DTT over 30 column volumes. Protein purity as assessed by SDS-PAGE was ⁇ 95%, and the protein was concentrated using Amicon-Ultra (3 kDa MWCO) concentrator (Millipore).
  • the SpyTag-Stoffel construct was cloned with a C-terminal 6xHis-tag and transformed into Escherichia coli BL21(DE3) (Invitrogen) competent cells. These were grown in LB medium at 37°C and induced at OD600 nm ⁇ 0.6 at 30°C with 0.5 mM IPTG and incubated overnight. Cells were then harvested by centrifugation, sonicated, then heated at 65°C for 15 min and clarified by centrifugation. The clarified cell lysate was applied to a His- TrapFF column (GE Healthcare) and purified using a gradient elution.
  • the fractions containing the protein were pooled and buffer exchanged into buffer with 50 mM Tris pH 8, 150 mM NaCl, 1 mM DTT and run on a size exclusion Hi Load 16/600 Superdex S200 column. Fractions were pooled and protein purity as assessed by SDS-PAGE was ⁇ 95%. The protein was concentrated using Amicon-Ultra (10 kDa MWCO) concentrator (Millipore).
  • Activity assay was carried out by co-incubating purified proteins (Sso7D-SpyC and SpyTag-Stoffel, 5 mM each) at room temperature for 30 minutes. 1 pL of the reaction mixture was subjected to polymerase activity assay.
  • Biotin-labelled peptides 100 pM were incubated with streptavidin beads (50 pL) for 2 hours at room temperatures prior to washing with 3 washes of PBS + 0.1% (v/v) Tween 20. Beads were next incubated at 4°C overnight with 500 pM of Sso7d-SpyCatcher protein, followed by 3 washes with PBS + 0.1% (v/v) Tween 20 and then 3 washes with PBS. Bound protein was eluted by boiling in SDS buffer prior to analysis by SDS-PAGE.
  • Example 1 Peptide ligase detection protocol
  • FIG. 1 is a schematic of the peptide ligase detection protocol.
  • the substrate pair of interest were respectively fused to the C-terminus of SSo7D and the N-terminus of the Stoffel fragment (residues 293 to 832 of Taq polymerase).
  • Ligation of the substrate pair effected by a peptide ligase or intrinsic activity resulted in a fusion protein capable of amplifying DNA in the presence of high (50-200mM) salt concentrations. In the absence of ligation, no amplification products were detected.
  • the ligation reaction can be carried out using purified components, or within the confines of a bacterial cell within which the enzyme/substrate proteins have been expressed.
  • Example 2 Coupled polymerase read-out of protein-peptide interactions using model interactants.
  • the Spycatcher-Spytag protein-peptide pair associate with relatively high affinity to form a complex with exceptional stability due to interlinking isopeptide bond formation.
  • Sso7d-Spycatcher and SpyTag-Stoffel fusion proteins were co-expressed in E.coli and polymerase activity assayed by adding cells directly to other standard PCR components and carrying out thermal cycling in buffer with increasing salt concentrations.
  • Covalent association between Spycatcher and bound SpyTag peptide resulted in an Sso7d-Spycatcher-SpyTag- Stoffel fusion protein competent for PCR in high salt buffer (FIG.2A).
  • Example 3 Clonal amplification by Compartmentalised Self Replication (CSR)
  • CSR Compartmentalised Self Replication
  • the collection of cells was then emulsified to yield on average one bacterial cell per aqueous compartment that also comprised buffer, high salt, dNTPS and oligonucleotide primers flanking the gene encoding the peptide ligase.
  • Thermal cycling first lysed cells (and destroyed non-thermostable E.coli proteins) and allowed for clonal amplification of peptide ligase genes that encoded functional enzymes. These amplicons were harvested post-CSR and further analysed (secondary assay/sequencing) or re-cloned into appropriate expression plasmid for further rounds of selection.
  • Example 5 Model selections for interacting proteins and peptides using the Compartmentalised Self Replication (CSR) platform.
  • CSR Compartmentalised Self Replication
  • the dynamic read-out of the reporter polymerase was next evaluated in the CSR platform.
  • a test selection was carried out using E. coli cells co-expressing either Sso7d-NB + Stoffel or Sso7d-NB + NSl-Stoffel (FIG. 7). Cells were mixed at different ratios prior to emulsification and thermocycling in high salt buffer using a primer pair common to both expression constructs flanking the NS1 cassette. In the absence of emulsification, the Sso7d- NB -NS 1 -Stoffel complex amplified from both expression plasmid templates as expected (FIG. 8).
  • C2HR enabled clonal amplification/enrichment of the NS1 cassette in plasmids expressing NSl-Stoffel (upper arrowed band) over those expressing Stoffel only (lower arrowed band). This was readily apparent at the 1:100 ratio of cells, with selection for the NS1 gene cassette occurring only when C2HR is used.
  • the panel of cells co-expressing Sso7d-NB and NS-Stoffel variants (FIG. 6) were next combined equally and one round of C2HR carried out.
  • Example 6 Selection of functional SpyTag variants from a semi-randomised library using CSR.
  • the selection strategy shown in FIG. 3 was implemented to select for randomized Spytag variants capable of binding to and forming a covalent bond with Spycatcher.
  • the endogenous Spytag amino acid sequence is: GAHIVMVDAYKP (SEQ ID NO: 20).
  • the underlined hydrophobic residues are important for high affinity interaction with Spycatcher.
  • Lane 1 is positive control (Sso7d-Stoffel fusion protein). Lane 2 is positive control (Sso7d-Spycatcher and WTSpyTag-Stoffel fusion proteins expressed). Lane 3 is negative control (no SpyTag fused to Stoffel fragment). Lanes 4-7 are same as lane 2 however the variant selected Spytag sequences are fused to Stoffel. This showed that the SpyTag variants selected for are functional, further validating the selection method.
  • Example 7 Selection for functional SpyTag peptide variants using Compartmentalised 2-Hybrid Replication (C2HR).
  • a library of SpyTag-Stoffel variants was created wherein the hydrophobic “IVMV” (SEQ ID NO: 90) motif in SpyTag essential for high affinity interaction with SpyCatcher (FIG. 11A) was randomised.
  • This library (Lib 1) was co-expressed in E.coli along with Sso7d- SpyCatcher prior to encapsulation in emulsion compartments containing oligonucleotide primers flanking the randomized region of SpyTag along with other requisite PCR components (dNTPs, high-salt buffer).
  • Ten rounds of thermal cycling were carried out to facilitate clonal amplification of genes encoding functional SpyTag core motifs, following which amplicons were harvested and sequenced en masse.
  • Example 8 Co-evolution of an interacting protein-peptide pair using C2HR
  • the C2HR platform may be further adapted to select for other classes of proteins whose activity directly or indirectly facilitates co-localisation of polymerase and processivity factor components. These include peptide ligases belonging to the hydrolase and transglutaminase families and intein domains that regulate protein splicing. Nucleic acid modifying enzymes, particularly DNA recombinases could also be engineered by C2HR. In this case, enzyme activity fuses the otherwise split processivity and polymerase gene cassettes, leading to expression of the requisite fusion protein. Whilst this approach has been previously described using other reporter genes, dynamic read-out afforded by polymerase function may expedite selections.
  • Example 9 High affinity interactions can be detected under conditions of accelerated cycling conditions
  • Results shown in FIG. 13 indicate amplification of large -1.5 Kb amplicon (arrowed) under conditions of short annealing and extension time (15 and 10 seconds respectively) only by cells co-expressing high affinity interactants: ST and S and NS 1/4/5 and NB. Under normal annealing and extension times (30 and 120 seconds respectively) amplification occurs irrespective of interactions.

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Abstract

La présente invention concerne un procédé de détection d'une interaction entre une première et une seconde protéine, le procédé comprenant : a) la fourniture d'une première protéine de fusion comprenant la première protéine connectée à une enzyme d'amplification d'acide nucléique ; b) la fourniture d'une seconde protéine de fusion comprenant la seconde protéine connectée à une protéine d'amélioration de processivité ; c) la mise en contact de la première protéine de la première protéine de fusion avec la seconde protéine de la seconde protéine de fusion ; d) la réalisation d'une réaction d'amplification d'acide nucléique d'un ou de plusieurs acides nucléiques de matrice dans une condition dans laquelle l'enzyme d'amplification d'acide nucléique n'est pas capable d'amplifier le ou les acides nucléiques de matrice ; e) la détection de l'interaction entre la première et la seconde protéine, l'interaction entre les première et seconde protéines restaurant l'activité de l'enzyme d'amplification d'acide nucléique et l'amplification du ou des acides nucléiques de matrice sous ladite condition indiquant l'interaction entre la première protéine et la seconde protéine.
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Title
MEHLA J. ET AL.: "A Comparison of Two-Hybrid Approaches for Detecting Protein-Protein Interactions", METHODS ENZYMOL, vol. 586, 5 January 2017 (2017-01-05), pages 333 - 358, [retrieved on 20201102], DOI: 10.1016/BS.MIE. 2016.10.02 0 *
PU J. ET AL.: "RNA Polymerase Tags To Monitor Multidimensional Protein- Protein Interactions Reveal Pharmacological Engagement of Bcl-2 Proteins", J AM CHEM SOC, vol. 139, no. 34, 2 August 2017 (2017-08-02), pages 11964 - 11972, XP055791727, [retrieved on 20201102], DOI: 10.1021/JACS.7B06152 *

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