WO2021028577A1 - Système de sélection bactérienne pour des protéases spécifiques à une cible - Google Patents

Système de sélection bactérienne pour des protéases spécifiques à une cible Download PDF

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WO2021028577A1
WO2021028577A1 PCT/EP2020/072893 EP2020072893W WO2021028577A1 WO 2021028577 A1 WO2021028577 A1 WO 2021028577A1 EP 2020072893 W EP2020072893 W EP 2020072893W WO 2021028577 A1 WO2021028577 A1 WO 2021028577A1
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caspase
enzyme
protease
specifically
bacterial
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PCT/EP2020/072893
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Rainer Schneider
Alois Jungbauer
Petra ENGELE
Christina KRÖSS
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Universität Innsbruck
Universität Für Bodenkultur Wien
Acib Gmbh
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    • 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)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention refers to a method for the selection of target-specific proteases. Specifically, the method of the present invention uses a circularly permuted, essential bacterial enzyme, comprising at one of its termini one or more foreign amino acids inactivating its enzymatic activity and a protease recognition site or target site for specific proteolytic cleavage. Respective exact proteolytic removal of the inhibitory additional foreign amino acids reactivates the enzyme and allows for bacterial growth in suitable selective media.
  • the present invention also refers to circular permuted bacterial enzymes and bacterial host cells, specifically for use in the selection method or to identify inhibitors of proteases.
  • protease substrate specificity and selectivity has a huge potential concerning analytical, biotechnological or therapeutic applications (Pogson MW, et al., Curr Opin Biotechnol. 2009 Aug;20(4):390-7. Epub 2009 Aug 24. Review).
  • Pogson MW, et al., Curr Opin Biotechnol. 2009 Aug;20(4):390-7. Epub 2009 Aug 24. Review Despite the technological advances in molecular biology that offered the fast generation of large libraries of protease variants, the necessary high throughput screening methods are tedious and require robotic assistance and sophisticated counter selections.
  • US6846628B1 discloses a method of selecting proteases using a fusion construct of a metabolically important enzyme fused to a bulky protein via a peptide recognition sequence to its natural termini for positive cellular selection in E. coli.
  • the metabolically important enzyme is inactivated because of steric hindrance by the bulky protein (p. 18, lines 6-18).
  • a large fusion protein of the catalytic subunit of aspartate transcarbamoylase was created, comprising b-galactosidase attached to its amino terminus and gene 10 (capsid protein) from bacteriophage T7 attached to its carboxy terminus via the HIV protease recognition decapeptide (Example VII).
  • W02000/66615A1 discloses an assay system for detecting in vitro the presence of a protease in a sample, e.g. a blood sample.
  • the assay system utilizes a chimeric protease detector protein composed of three domains: (1) a repressor domain, (2) a protease cleavage domain specific for the protease to be assayed, and a reporter domain.
  • the reporter domain is not detectable when linked to the repressor domain, but becomes detectable upon release from the repressor domain by protease-mediated cleavage.
  • Steroid hormone receptors, large proteins, are used as repressor domains.
  • W02008/045148A2 also discloses methods of detecting in vitro specific proteases in a sample, using protease trap polypeptides which upon cleavage by the protease forms a stable complex with the protease, inhibiting and trapping it.
  • An efficient bacterial in vivo selection system is provided herein that selects for crucial properties such as, but not limited to, substrate specificity or P1’ tolerance of a protease.
  • the in vivo selection of proteases has several advantages over in vitro screening systems: while in vitro screening systems can only achieve high throughputs by robotic assistance and several rounds of selection, the present bacterial in vivo selection system has the advantage that within one round of selection millions of mutants can be analyzed, because only those bacteria that can cope with the selection criteria can form colonies. Furthermore, in the case of selecting for specific proteases, undesired nonspecific protease variants will not pass the selection because of severe toxic effects on the host due to proteolytic destruction of crucial host proteins and enzymes.
  • the inventive selection system specifically relies on the use of a sterically inhibited circularly permuted recombinant essential bacterial enzyme that can only be reactivated by an exact and specific cut by a protease.
  • a circularly permuted bacterial enzyme which harbors its termini within its core structure can be completely inhibited by fusing only a few, or even just one, additional amino acids at its N- or C-terminus. Reactivation of the enzyme can only occur if the additional amino acid residues are removed via the respective protease by precisely cutting at the cleavage site and thereby restoring the authentic N- or C-terminus of the enzyme.
  • a bacterial strain defective in an essential enzyme and supplemented with a vector expressing a circular permuted enzyme inhibited by an N- or C-terminal fusion tag can only grow significantly on a minimal medium, if simultaneously transformed with a protease that can remove the inhibiting fusion tag.
  • This method specifically allows for the selection of proteases programmed for a desired property, such as recognizing a recognition site different from the wild type recognition site, improved specificity for a certain recognition site and improved stability from a library of modified proteases.
  • the present invention refers to a method for the selection of target-specific proteases.
  • the method of the present invention uses a circularly permuted, essential bacterial enzyme, comprising at one of its termini one or more foreign amino acids inactivating its enzymatic activity and a protease recognition site or target site for specific proteolytic cleavage. Respective exact proteolytic removal of the inhibitory additional foreign amino acids reactivates the enzyme and allows for bacterial growth in suitable selective media.
  • proteases can be selected which have cis- or trans-cleaving properties. Specifically, proteases showing trans-cleavage are selected herewith.
  • the in vivo bacterial selection system as described herein using an essential circularly permuted bacterial enzyme to select for desired protease properties is an efficient novel method to generate proteases with tailored properties for various applications, e.g. but not limited to analytics, biotechnology and therapeutics.
  • the selection assay described herein specifically has quantitative and qualitative advantages over existing screening systems. It specifically provides
  • the selection method can also be adapted to select for optimized proteases which are resistant towards certain proteinaceous inhibitors, specifically by co-expressing such an inhibitor during the selection.
  • a method for the selection of a target- specific protease comprising the steps of i. providing bacterial host cells comprising at least one circularly permuted bacterial enzyme essential for growth of said host cells under selection conditions, comprising at its N- or C-terminus one or more additional amino acids inactivating its enzymatic activity and a protease recognition site for target-specific proteolytic cleavage, wherein, upon target-specific proteolytic cleavage and removal of the additional amino acids, its enzymatic activity is restored, and wherein said host cells do not comprise said bacterial enzyme in enzymatically active form, ii. introducing at least one protease into the host cells, iii.
  • the circularly permuted bacterial enzyme comprises one or more foreign amino acids at its N- or C-terminus, preferably its N-terminus.
  • the circularly permuted (cp) bacterial enzyme comprises at its N-terminus a sequence of foreign amino acids comprising the following structure from C- to N-terminus i. a recognition site for proteolytic cleavage, comprising at least 4 amino acids of the sequence P1-P2-P3-P4 and a cleavage site P1’-P1 , wherein P1 is the first foreign amino acid and P1’ is the first wild-type N-terminal amino acid of the enzyme, and ii. optionally a linker, and iii. optionally a tag sequence.
  • the cp enzyme therefore comprises at its N-terminus a recognition site and cleavage site of the sequence: P4-P3-P2-P1-P1’ (read from N- to C-terminus).
  • the circularly permuted (cp) bacterial enzyme comprises at its C-terminus a sequence of amino acids comprising the following structure from N- to C-terminus i. a recognition site for proteolytic cleavage, comprising a cleavage site P1- P1’, wherein at least P1’ is a foreign amino acid, and ii. optionally a linker, and iii. optionally a tag sequence.
  • P1 represents the native C-terminus of the circular permuted essential bacterial enzyme and at least one foreign amino acid is added, namely P1’, to inactivate the essential bacterial enzyme.
  • the entire recognition site e.g. P4-P3-P2-P1 , is part of the sequence of the cp enzyme.
  • the recognition site may be part of the endogenous sequence of the cp enzyme, or it may be engineered to replace a number of amino acids of the enzyme’s terminus.
  • P4-P3 may be part of the endogenous sequence and P2-P1 may be heterologous amino acids replacing two endogenous amino acids.
  • P4-P3-P2 are endogenous
  • P3-P2-P1, or P1 are heterologous amino acids replacing the endogenous amino acid residues of the C-terminus.
  • the heterologous amino acids replace the enzyme’s natural terminal amino acids and are compatible with the activity of the enzyme.
  • the cp enzyme comprises at its C- terminus one or several amino acids which can be removed one after the other by one or more carboxypeptidases.
  • the cp enzyme therefore comprises at its C-terminus a recognition site and cleavage site, specifically for endopeptidases, of the sequence: P4-P3-P2-P1- P1’ (read from N- to C-terminus), or one or several amino acids which can be removed by exopeptidases.
  • the at least one circularly permuted bacterial enzyme and the protease are introduced into the host cell simultaneously or sequentially using expression vectors.
  • the at least one circularly permuted bacterial enzyme is stably integrated into the genome of the host cell.
  • an expression cassette comprising the circularly permuted bacterial enzyme and the protease fused to the N-terminus of the circularly permuted bacterial enzyme is introduced into the host cell.
  • the host cells comprise a variety of circularly permuted bacterial enzymes, each differing in at least one amino acid residue.
  • the host cells comprise a variety of circularly permuted bacterial enzymes, each comprising a different P1’ amino acid residue.
  • the host cells are contacted with more than one protease.
  • the host cells are contacted with a library of proteases, each protease differing in its encoding sequence in at least one nucleotide.
  • the proteases are encoded by a repertoire of vectors each expressing one or more proteases.
  • the bacterial host cells are selected from the group consisting of £. coli, Salmonella, Streptomyces, Bacillus, Mycobacterium, Listeria, Lactococcus, Lactobacillus, Staphylococcus and Streptococcus.
  • the bacterial host cells do not comprise the functional bacterial enzyme due to a mutation or deletion within the endogenous gene encoding said bacterial enzyme.
  • the bacterial enzyme is circularly permuted aspartate transcarbamoylase (cpATCase), specifically comprising at its N-terminus one or more foreign amino acids inactivating its enzymatic activity and a recognition site for proteolytic cleavage.
  • cpATCase circularly permuted aspartate transcarbamoylase
  • the bacterial host cells are £ coli cells comprising a modification such as a mutation or deletion in the endogenous pyrB gene (SEQ ID NO:1), and wherein the cp bacterial enzyme is cp pyrB, preferably comprising SEQ ID NO:2 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:2, comprising at its N-terminus one or more foreign amino acids inactivating its enzymatic activity and a recognition site for proteolytic cleavage.
  • SEQ ID NO:1 endogenous pyrB gene
  • the circularly permuted bacterial enzyme is cp pyrB comprising at its N-terminus a caspase-14 recognition site, a GSG linker and a His-tag, preferably comprising SEQ ID NO:4 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:4.
  • the bacterial enzyme is cp pyrB comprising at its N-terminus a caspase-6 recognition site, a GSG linker and a His-tag, preferably comprising SEQ ID NO:6 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:6.
  • the circularly permuted bacterial enzyme comprises at its N-terminus a caspase-3 recognition site, a GSG linker and a His-tag.
  • the circularly permuted bacterial enzyme comprises at its N-terminus a caspase-2 recognition site, a GSG linker and a His-tag.
  • the protease is codon-optimized for expression in a bacterial host, preferably it is codon-optimized for expression in £. coli.
  • the protease provided herein is a caspase selected from the group consisting of caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14 and any chimera comprising a small subunit and a large subunit of any caspase, specifically a caspase 6-14 chimera, comprising a small caspase-6 subunit and a large caspase-14 subunit.
  • the caspase is a circularly permuted caspase (cp caspase) selected from the group consisting of cp caspase-1 , cp caspase- 2, cp caspase-3, cp caspase-4, cp caspase-5, cp caspase-6, cp caspase-7, cp caspase- 8, cp caspase-9, cp caspase-10, cp caspase-11 , cp caspase-12, cp caspase-13, cp caspase-14 and a cp caspase chimera comprising a small subunit and a large subunit of any caspase, specifically a cp caspase 6-14 chimera, comprising a small caspase-6 subunit and a large caspase-14 subunit, or variants thereof.
  • cp caspase circularly permuted caspase
  • the protease described herein is selected from the group consisting of carboxypeptidases and aminopeptidases.
  • the protease is Npro, SARS-CoV-2 Mpro, HIV-protease, and TEV-protease, or variants thereof.
  • the circularly permuted bacterial enzyme is cpATCase and the selection condition is a selection medium devoid of pyrimidines.
  • a circularly permuted bacterial enzyme essential for growth under selection conditions, comprising at its N- or C-terminus one or more foreign amino acids inactivating its enzymatic activity and a recognition site for proteolytic cleavage, wherein upon proteolytic cleavage of the one or more foreign amino acids its enzymatic activity is restored.
  • said selection conditions are selected from the group consisting of selection temperature, selection oxygenation and selection media, preferably media devoid of one or more essential amino acids or essential nutrients or pyrimidines.
  • the cp enzyme comprises at its N-terminus a sequence of foreign amino acids comprising the following structure from C- to N-terminus i. a recognition site for proteolytic cleavage, comprising at least 4 amino acids of the sequence P1-P2-P3-P4 and a cleavage site of the sequence P1-P1’, wherein
  • P1 or P1’ is the first foreign amino acid, and ii. optionally a linker, and iii. optionally a tag sequence.
  • P1’ is the amino acid naturally occurring at the new N-terminus of the cp enzyme.
  • P1 is the first additional, also referred to as foreign, amino acid, i.e. P1 is the first amino acid not naturally occurring at the N-terminus of the cp enzyme.
  • the cp enzyme comprises at its N-terminus a sequence of foreign amino acids comprising the following structure from C- to N-terminus i. one or several amino acids, which can be removed one after the other by one or more aminopeptidases, and ii. optionally a linker, and iii. optionally a tag sequence.
  • the cp enzyme comprises at its C-terminus a sequence of foreign amino acids comprising the following structure from C- to N-terminus i. optionally a tag sequence, ii. optionally a linker, and iii. a cleavage site of the sequence P1-P1’ and a recognition site for proteolytic cleavage, comprising at least 4 amino acids of the sequence P1-P2-P3-P4, which amino acids P1 , P2, P3, P4 are either part of the enzyme’s sequence or compatible with the enzyme’s activity; or one or several amino acids which can be removed one after the other by one or more carboxypeptidases.
  • the cp enzyme as described herein has a recognition site for proteolytic cleavage comprising at least 4 amino acids of the sequence P4-P3-P2-P1 and a cleavage site of the sequence P1’-P1 , wherein
  • P1 ’ can be any amino acid, preferably it is G, A, L, M, Q, E, S, P, V, I, C, R, N, D,
  • P1 can be any amino acid, preferably it is D, E;
  • P2 can be any amino acid, preferably it is A, H, S or V, K, I, T;
  • P3 can be any amino acid, preferably it is V, I or E, F, Y, G; and
  • P4 can be any amino acid, preferably it is D, E or W, H, Q.
  • the cp enzyme therefore comprises at its N-terminus a recognition site and cleavage site of the sequence P4-P3-P2-P1-P1’.
  • the bacterial enzyme is cp aspartate transcarbamoylase (cpATCase).
  • the enzyme is a cpATCase derived from E. coli comprising SEQ ID NO:2 or a variant thereof having at least 75%, 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:2.
  • cp enzyme described herein in a screen to identify a target-specific protease, an inhibitor of a target-specific protease, or optimized expression tags.
  • a bacterial host cell comprising the circularly permuted bacterial enzyme essential for growth under selection conditions comprising at its N- or C-terminus one or more foreign amino acids inactivating its enzymatic activity and representing a recognition site for proteolytic cleavage described herein, wherein said cell does not comprise a respective endogenous bacterial enzyme in enzymatically active form.
  • the host cell comprises a vector expressing said circularly permuted bacterial enzyme or wherein said circularly permuted bacterial enzyme is stably integrated into the genome of said cell.
  • said cell does not comprise said bacterial enzyme in enzymatically active form due to a mutation or partial or full deletion of the endogenous gene.
  • said circularly permuted bacterial enzyme is cpATCase and wherein said cell comprises a mutation or deletion of the endogenous gene encoding ATCase.
  • said bacterial cell is an £. coli cell comprising a mutation or deletion of its endogenous pyrB gene (SEQ ID NO:1) and wherein said circularly permuted bacterial enzyme is a circularly permuted aspartate transcarbamoylase catalytic subunit enzyme (cp-pyrB), specifically comprising SEQ ID NO:2, comprising at its N- or C-terminus one or more foreign amino acids inactivating its enzymatic activity and a recognition site for proteolytic cleavage.
  • SEQ ID NO:1 an endogenous pyrB gene
  • cp-pyrB circularly permuted aspartate transcarbamoylase catalytic subunit enzyme
  • Figure 1 Selected nucleotide and amino acid sequences.
  • Figure 2 Multiplication of ATCase/pyrB deficient £. coli cells transformed with cp caspase-6 and different substrate constructs during 21 hour growth in minimal medium with special supplements (6H: His-tag, DEVD (SEQ ID NO:31), VEID (SEQ ID NO:6) and WEHD (SEQ ID NO:4): recognition sites of caspase-3, -6 and -14, respectively.
  • Figure 3 Number of colonies harboring active cp caspase-6-S (SEQ ID NO:29) per transformation containing different amounts of cp caspase-6-S DNA. As background 25 ng of DNA containing inactive chimera were used.
  • Figure 4 Scheme of selection system for caspases co-expressed with tagged cpATCase in £ coli BL21(DE3) ApyrBI knock-out cells. Active caspase cleaves the tag from the ATCase and the knock-out cells can produce pyrimidines to survive. Inactive caspase does not cleave off the tag and the ATCase is blocked.
  • Figure 5 Comparison of the growth behavior of original inactive chimera, isolated active chimera variant isolated from the library and an active site mutant thereof.
  • Figure 6 Growth of cells containing cp caspase-2 co-expressed with cpATCase substrates containing the caspase-3 recognition site DEVD, the caspase-6 recognition site VEID or the caspase-14 recognition site WEHD. Multiplication of starting OD600 in M9 medium after cultivation at 30 °C for 22 h.
  • the positive control contained empty pACYCDuet-1 vector and cpATCase
  • the negative control contained empty pACYCDuet-1 vector and VDVAD-ATCase.
  • Figure 7 In vitro cleavage of cp caspase-2 with E2 fusion protein containing a VDVAD or DEVD cleavage site respectively. Cleavage of 1 mg/ml VDVAD and DEVD-E2 by 0.01 mg/ml cp caspase-2.
  • Figure 8 Comparison of in vivo activity of variants S9, mS9 Pro, and cp caspase-2 co expressed with VDVAD-AM-P-cpATCase (SEQ ID NO:18).
  • Figure 9 PT tolerance of mS9 Pro and cp caspase-2 mutants with single mutations in % compared to cp caspase-2.
  • Figure 10 The N-terminal tag fused to the 25 kDa protein E2 via the cleavage site DEVD is removed by incubation with caspase-7 variants cp caspase-7 and cp caspase-7A.
  • Figure 11 The activity of cp caspase-7A towards EISD is retained (a) and cp caspase- 7’s activity towards HYID is lost in cp caspase-7A (b).
  • Figure 12 Example Michaelis-Menten kinetic measured by FRET assay.
  • amino acids refer to twenty naturally occurring amino acids encoded by sixty-one triplet codons. These 20 amino acids can be split into those that have neutral charges, positive charges, and negative charges:
  • Alanine (Ala, A) nonpolar, neutral;
  • Asparagine (Asn, N) polar, neutral
  • Cysteine (Cys, C) nonpolar, neutral
  • Glutamine (Gin, Q) polar, neutral
  • Glycine (Gly, G) nonpolar, neutral
  • Leucine (Leu, L) nonpolar, neutral
  • Methionine (Met, M) nonpolar, neutral
  • Phenylalanine (Phe, F) nonpolar, neutral;
  • Proline (Pro, P) nonpolar, neutral
  • Serine (Ser, S) polar, neutral
  • Threonine (Thr, T) polar, neutral
  • Tryptophan (Trp, W) nonpolar, neutral;
  • Tyrosine (Tyr, Y) polar, neutral
  • Valine (Val, V) nonpolar, neutral
  • Histidine (His, H) polar, positive (10%) neutral (90%).
  • the “positively” charged amino acids are:
  • Arginine (Arg, R) polar, positive
  • Lysine (Lys, K) polar, positive.
  • the “negatively” charged amino acids are:
  • a method for the selection of a target- specific protease comprising the steps of providing bacterial host cells comprising at least one circularly permuted bacterial enzyme essential for growth of said host cells under selection conditions, comprising at its N- or C-terminus one or more foreign amino acids inactivating its enzymatic activity and a protease recognition site for target-specific proteolytic cleavage, wherein, upon target-specific proteolytic cleavage and removal of the foreign amino acids, its enzymatic activity is restored, and wherein said host cells do not comprise said bacterial enzyme in enzymatically active form, introducing at least one protease into the host cells, maintaining said host cells under conditions to allow target- specific cleavage of the circularly permuted bacterial enzyme by the protease, selecting host cells growing under the selection conditions, and isolating the target
  • the protease recognition site is (a) a sequence naturally constituting one of the termini of the cp bacterial enzyme, specifically the C-terminus of the enzyme, or (b) the protease recognition site is composed of foreign amino acids, introduced at one of the termini of the cp enzyme by recombinant means.
  • at least one foreign amino acid specifically two or more foreign amino acids are introduced at the C- terminus of the cp enzyme.
  • the first foreign amino acid following the natural C-terminus is the P1’ residue and the amino acid residue of the natural C-terminus is the P1 residue.
  • one or more additional N- or C- terminal foreign amino acids may be introduced.
  • the first foreign amino acid before the natural N-terminus is the P1 residue and the amino acid residue of the natural N-terminus is the P1’ residue
  • the first foreign amino acid introduced after the natural C-terminus is the P1’ residue and the amino acid of the natural C-terminus is the P1 residue (read from N- to C-terminus).
  • the cp enzyme may comprise an N-terminal methionine.
  • the N-terminal methionine is deleted.
  • Proteases also known as proteinases, are peptidases that are able to hydrolyze the peptide bond between amino acid residues in a polypeptide chain. Proteases are found in all organisms and are involved in all areas of metabolism. Proteases can be classified by three criteria: the reaction catalyzed, the chemical nature of the catalytic site, and their evolutionary relationships. Endopeptidases cleave the target protein internally. Exopeptidases remove single amino acids from either the amino- or carboxy- terminal ends of a protein. Exopeptidases are divided into carboxypeptidases or aminopeptidases depending on whether they digest proteins from the carboxy- or amino- terminus, respectively. Proteases are also divided based on their catalytic site architecture.
  • Serine proteases have a serine in their active site that covalently attaches to one of the protein fragments as an enzymatic intermediate.
  • This class includes the chymotrypsin family (chymotrypsin, trypsin, and elastase) and the subtilisin family.
  • Cysteine proteases have a similar mechanism but use cysteine rather than serine. They include the plant proteases (papain, from papaya, and bromelain, from pineapple) as well as mammalian proteases such as calpains and caspases.
  • Aspartic proteases have two essential aspartic acid residues that are close together in the active site although far apart in the protein sequence. This family includes the digestive enzymes pepsin and chymosin. Metalloproteases use metal ion cofactors to facilitate protein digestion and include thermolysin. Threonine proteases have an active-site threonine.
  • target specific or “substrate specific” in connection with the proteases as referred to herein are proteases which are specific to a selected target proteolytic cleavage site within a polypeptide.
  • Target specificity in this context refers to proteases which specifically need a defined cleavage recognition site for binding and subsequent proteolytic cleavage of a polypeptide or protein, specifically of the cp bacterial enzyme.
  • caspasses are the key enzymes in the initiation and execution of apoptosis and inflammation; hence their activity has to be tightly controlled. Although the sequences of caspases do differ (e.g. human caspase-1 and -2 have only 27 % amino acid identity and 52 % similarity), their active sites and tertiary structure are highly conserved. All caspases are synthesized as relatively inactive single-chain zymogens (procaspases), which comprise a prodomain (2-25 kDa), as well as a large and a small subunit of 17- 21 kDa and 10-13 kDa respectively.
  • procaspases relatively inactive single-chain zymogens
  • the executioner caspases (caspases-3, -6, -7) and caspase-14 have a short, while all other caspases have a long prodomain.
  • wild-type caspases first need to dimerize through hydrophobic interactions, then their intersubunit linker is cut and the prodomain removed by proteolytic cleavages after aspartate residues.
  • a main difference between the activation of executioner and initiator caspases is that the latter are already active after dimerization and the autocatalytic separation of their subunits is only necessary for stabilization.
  • Active wild-type caspases are homodimers of heterodimers. Each heterodimer consists of a large and small subunit derived from a single protein chain.
  • the enzyme is formed by a central twelve-stranded b-sheet, to which each of the four subunits contributes. From this core four loops protrude which contain the active site and form the binding pockets. In all caspases the catalytic center is in the large subunit.
  • the substrate recognition site is formed by amino acids from both subunits, though the small subunit contributes the main residues which are responsible for differing substrate specificity between caspases. The cleavage of the inter-subunit linker causes a rearrangement of the active site loops, allowing the binding pockets to form and to make the active cysteine solvent accessible.
  • Bacterial host cells or cell lines as referred to herein can be any bacterial cells functionally depleted of an essential enzyme, whereby these cells can only grow if conditions are created to compensate for their deficiency or if they are provided with the functional essential enzyme. Said depletion of the essential enzyme may result in growth defects due to lack of certain nutrients such as, but not limited to amino acids, essential nutrients, salts etc. Growth deficiency may also be due to changes in temperature, oxygenation or any other conditions at which the respective enzyme is essential for bacterial growth. Specifically, selection media lacking a certain component such as pyrimidines, specifically thymine, cytosine or uracil, can be used for such purpose. As an example, a pyrimidine auxotroph £ coli mutant can only survive in media supplemented with pyrimidines or when the cells are complemented with a functional ATCase.
  • pyrimidine auxotroph £ coli mutant can only survive in media supplemented with pyrimidines or when the cells are
  • Suitable bacterial host cells are, but are not limited to, caulobacteria, phototrophic bacteria, cold adapted bacteria, pseudomonads such as Pseudomonas sp.] halophilic bacteria such as Halomonas elongate ; streptomycetes such as Streptomyces sp] nocardia; mycobacteria; coryneform bacteria, such as Corynebacterium sp, Brevibacterium sp] bacilli such as Bacillus subtilis, Bacillus brevis, Bacillus licheiformis, Bacillus amyloliquefacians] lactic acid bacteria such as Lactococcus sp., Lactobacillus sp.] proteobacteria such as £. coli being the most prominent one.
  • the bacterial cells described herein comprise at least one circularly permuted bacterial enzyme essential for growth under selection conditions.
  • £ coli cells are comprising a mutation or deletion in the endogenous pyrB gene (SEQ ID NO:1), and a cp pyrB, specifically comprising SEQ ID NO:2 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:2, comprising at its N-terminus one or more foreign amino acids inactivating its enzymatic activity and a recognition site for proteolytic cleavage.
  • the circularly permuted bacterial enzyme is cp pyrB comprising at its N-terminus a caspase-14 recognition site, a GSG linker and a His-tag, specifically comprising SEQ ID NO:4 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID N0:4.
  • the circularly permuted bacterial enzyme is cp pyrB comprising at its N- terminus a recognition site comprising the amino acid sequence VEID, a GSG linker and a His-tag, preferably comprising SEQ ID NO:6 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:6.
  • the circularly permuted bacterial enzyme is cp pyrB comprising at its N-terminus a recognition site comprising the amino acid sequence VDVAD or DEVD, and optionally comprises a GSG linker and/or a His tag.
  • Circular permutation refers to a changed order of amino acids in the peptide sequence of proteins.
  • Circular permuted proteins have a changed order of amino acids in their protein sequence, such that the sequence of the first portion of one protein (adjacent to the N-terminus) is related to that of the second portion of the protein (near its C-terminus), and vice versa. The result is a protein structure with different connectivity, but overall similar three-dimensional (3D) shape.
  • Circular permutation (CP) has first been discovered in natural proteins in 1979.
  • Circularly permuted (cp) proteins arise by covalent linkage of native N- and C-terminus and the introduction of new termini by cleavage elsewhere in the protein. In nature this either happens by duplication/deletion or fission/fusion events at the gene level.
  • the new variants have an altered order of amino acids but maintain the same tertiary structure.
  • Circular permuted enzymes as referred to herein encompass any essential bacterial enzymes comprising a reorganized polypeptide chain due to CP, specifically due to covalent ligation of the wild type N- and C-termini and intramolecular cut of the protein backbone at a different position to create new N- and C-termini, which leads to swapped domains.
  • the native protein termini are connected via a covalent linker or a deletion and new ends are introduced through the cleavage of an existing peptide bond.
  • a circularly permuted ATCase was designed whose scaffold was changed by circular permutation of the catalytic subunit, i.e. covalent ligation of the wild type N- and C-termini and intramolecular cut of the protein backbone at a different position to create new N- and C-termini.
  • the cut in the protein backbone was made before residue 227 (number of amino acids in the wild type catalytic chain, SEQ ID NO:11) in a b-sheet of the C-terminal domain, ranging from amino acid residue 150 to residue 248 of SEQ ID NO:11.
  • a domain of at least about 70 to 80 amino acids in length was taken from the C-terminal end of the enzyme and fused to the natural N-terminus, thereby becoming the new N-terminus.
  • the essential bacterial enzyme is circularly permuted by taking a sequence of amino acids of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 residues in length, from one of the termini and in silico or in vitro joining it to the opposite terminus.
  • the resulting circularly permuted essential bacterial enzyme is enzymatically active as described herein and provides the significant advantage that its enzymatic activity can be inhibited by addition of a small number of amino acids at the new C- or N-terminus.
  • Inactivation of regular ATCase requires addition of large inhibitory domains, e.g. to provide steric hindrance.
  • Prior art strategies thus encompassed addition of whole proteins or large protein domains in order to inactive the bacterial enzyme, thereby generating bulky and large fusion proteins that are difficult to produce and handle.
  • large inhibiting fusions cannot be used for the selection of amino- or carboxypeptidases which usually remove only one or a few amino acids from the respective termini.
  • the inactivated cp enzymes provided herein are significantly smaller, since inactivation is achieved by addition of very few amino acids, e.g. as few as only one foreign amino acid, and are thus easier to generate and express in culture and can be used to select for optimized exopeptidases.
  • the circular permuted enzymes provided herein are rendered biologically inactive enzymes by addition of one or more foreign N- or C-terminal amino acid residues.
  • Said cp enzymes further comprise a protease recognition site adjacent to said one or more foreign amino acids.
  • the proteases are exopeptidases the additional foreign N- or C-terminal amino acids are the proteolytic target themselves.
  • secondary and tertiary structures are very important for recognition, too. Therefore, the skilled person may consider that in vivo proteins are preferably cleaved at solvent accessible loops, but a significant amount is also cleaved within a-helices.
  • said cp enzyme can be reactivated and enzyme activity can cure the host cell’s deficiency.
  • the term “foreign” refers to a number of amino acids which are not natively associated with a respective polypeptide, in particular with the essential bacterial enzyme described herein.
  • the term foreign refers to additional amino acids which are not part of the wild type protein, and which are added to the N- or C-terminus of the circular permuted essential bacterial enzyme described herein.
  • the number of foreign amino acids to be added at the N- or C-terminus of the enzyme as described herein depend on the N-terminal and C-terminal structure of the respective enzyme. Specifically, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more foreign amino acid residues can be linked to the N- or C-terminus.
  • the respective number of foreign amino acids can be determined by the skilled person.
  • said amino acid residues are histidine residues, more specifically 1 , 2, 3, 4, 5, 6 histidine residues.
  • a short linker as described in more detail below may be introduced between the foreign amino acids and the protease recognition site for providing more flexibility and accessibility.
  • protease recognition site or “cleavage recognition site” refers to an amino acid sequence of at least 3, preferably at least 4 or 5, amino acid residues of a substrate, which is specifically recognized by the circularly permuted protease described herein.
  • the at least three substrate amino acids which are targeted and bound by the cp caspase provided herein and which form the recognition site are termed P3 - P1 or P3 P2 P1 , P4 - P1 or P4 P3 P2 P1 for a recognition site comprising 4 substrate amino acids, P5 - P1 or P5 P4 P3 P2 P1 for a recognition site comprising 5 substrate amino acids, P6 - P1 or P6 P5 P4 P3 P2 P1 for a recognition site comprising 6 substrate amino acids, P7 - P1 or P7 P6 P5 P4 P3 P2 P1 for a recognition site comprising 7 substrate amino acids, and so on.
  • the circular permuted protease provided herein interacts with its substrate in a target-specific manner by specifically recognizing and binding the recognition site comprising at least 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues comprised in the sequence of the substrate.
  • the recognition site amino acid residues occupy specific pockets on the caspase, numbered with the matching S designation S1, S2, S3, S4, S5 etc.; ST, S2’ etc, each of which may be constructed of several amino acid residues.
  • the objective of this interaction mode which almost always binds the cleavage region in an extended peptide conformation, is to align the substrate accurately into register with the catalytic machinery.
  • cleavage site refers to the amino residues P1/PT wherein cleavage occurs at the residue of the amino terminal scissile bond P1 and the one to the carboxy-terminal side PT. Specifically, the cleavage site is adjacent to the recognition site. Proteolytic cleavage of the substrate happens after the P1 residue. Specifically, the amino acids following the P1 residue are referred to as PT-P4' residues, also termed the prime side. For many proteases, the prime side of the substrate is important for substrate recognition or processing, specifically the P1 ' residue. The PT-P4' residues can under certain circumstances influence binding by steric hindrance.
  • Cp caspase-2 the PT residue is close to the active site and in particular branched (e. g. leucine or valine) and polar amino acids (e. g. threonine or aspartate) in this position can compete for space with the catalytic cysteine and negatively influence the cleavage.
  • Cp caspase- 2 prefers a glycine residue at the PT site.
  • variants of target-specific proteases, specifically caspases can be selected for increased PT tolerance.
  • Wild-type caspases have a high preference for aspartate in the P1 position.
  • the P2 and P3 positions are less selective and a variety of residues is accommodated, although many caspases have the highest activity with a glutamate residue at the P3 position.
  • the P4 position is crucial for distinction between caspase classes: Inflammatory caspases and caspase-14 prefer hydrophobic residues, initiator caspases and caspase-6 aliphatic residues, and executioner caspases as well as wild-type caspase-2 favor aspartate.
  • the prime side positions of substrates have not been investigated as intensively, although studies have shown that the PT site has an influence on cleavage, as certain residues can reduce the activity up to 1000-fold.
  • All wild-type caspases prefer substrates with small residues (glycine, serine, alanine), but large hydrophobic amino acids (phenylalanine, tyrosine) are also surprisingly well tolerated. Most likely the PT site is not necessary for efficient binding, but certain residues can hinder it. The prime sites further away (P2-P4') from the cleavage site have little influence. However, whether a substrate is cleaved by a caspase or not, does not only depend on the mere presence or absence of a recognition site, as many proteins are processed at non-canonical sites. Secondary and tertiary structures of the substrate are very important for recognition. In vivo proteins are preferably cleaved at solvent accessible loops, but a significant amount is also cleaved within a-helices.
  • catalytically active refers to the ability of the caspase described herein to catalyze the hydrolysis of the substrate’s peptide bond.
  • Caspases are endopeptidases capable of forcing formation of a tetrahedral intermediate by promotion of a cysteine residue to a nucleophile in order to cleave its substrate.
  • the catalytic efficiency of a protease is defined as the rate of hydrolysis and can be determined using the Michaelis Menten equation (kcat/Kivi).
  • the Michaelis constant, KM is equal to the substrate concentration at which the enzyme converts substrates into products at half its maximal rate and hence is related to the affinity of the substrate for the enzyme.
  • the catalytic constant (kcat) is the rate of product formation when the enzyme is saturated with substrate and therefore reflects the enzyme's maximum rate. The rate of product formation is dependent on both how well the enzyme binds substrate and how fast the enzyme converts substrate into product once substrate is bound. An equation with a low KM value indicates a large binding affinity, as the reaction will approach Vmax, the maximal rate of the reaction, more rapidly. An equation with a high KM indicates that the enzyme does not bind as efficiently with the substrate, and Vmax will only be reached if the substrate concentration is high enough to saturate the enzyme.
  • the catalyst rate constant (kcat) measures the number of substrate molecules turned over by enzyme per second. The reciprocal of kcat is then the time required by an enzyme to turn over a substrate molecule. The higher the kcat is, the more substrates get turned over in one second. When kcat is divided by KM, a measure of enzyme efficiency is obtained. The enzyme efficiency can be increased as kcat has high turnover and a small number of KM.
  • catalytic efficiency constants is used as a measure of the preference of an enzyme for different substrates, i.e. substrate specificity.
  • substrate specificity the higher the specificity constant, the more the enzyme "prefers" that substrate.
  • catalytic activity of the protease described herein can be measured by examining cleavage of the caspase substrate.
  • cleavage activity of the protease described herein can be examined by methods well known in the art.
  • the cp bacterial enzyme described herein comprises the following structure from C- to N-terminus: a recognition site for proteolytic cleavage, comprising at least 4 amino acids of the sequence P4-P3-P2-P1 , wherein the amino acid residue C-terminal to P1 is PT and wherein P1 ’ is the first foreign amino acid or the wild-type N-terminal amino acid of the enzyme.
  • proteolytic cleavage happens between the PT residue and the recognition site P1-P4.
  • the authentic N-terminus of the essential bacterial enzyme is restored and the amino acid at the PT position is its N-terminal amino acid residue.
  • a linker can further be placed N- terminal to the proteolytic cleavage site, said linker specifically being a GSG linker.
  • a tag sequence can also be placed directly at the N-terminus of the proteolytic cleavage site or adjacent to the linker.
  • the cp bacterial enzyme described herein comprises at least 4 amino acids of the sequence P4-P3-P2-P1 and the cleavage site is P1-P1’, wherein P1’ can be any amino acid, preferably it is G, A, L, M, Q, E, S, P, V, I, C, R, N, D, T; P1 can be any amino acid, preferably it is D, E; P2 can be any amino acid, preferably it is A, H, S or V, K, I, T; P3 can be any amino acid, preferably it is V, I or E, F, Y, G and P4 can be any amino acid, preferably it is D, E or W H, Q.
  • P1’ can be any amino acid, preferably it is G, A, L, M, Q, E, S, P, V, I, C, R, N, D, T
  • P1 can be any amino acid, preferably it is D, E
  • P2 can be any amino acid, preferably it is A,
  • protease recognition site is composed of foreign amino acids and/or a linker and/or a tag sequence are further comprised, further foreign amino acids may optionally be introduced at the N-terminus.
  • the protease recognition site is a sequence naturally constituting the C-terminus of the cp bacterial enzyme, one or more further foreign amino acids are introduced at the terminus to inactivate the enzyme, which foreign amino acids can represent a linker, a tag or a sequence of amino acid residues.
  • linker refers to any amino acid sequence that does not interfere with the function of elements being linked.
  • Linkers may connect e.g., nucleotide sequences, or amino acid sequences.
  • the linkers may be used to engineer appropriate amounts of flexibility.
  • the linkers are short, e.g., 2-20 nucleotides or amino acids, and are typically flexible.
  • Amino acid linkers commonly used consist of a number of glycine, serine, and optionally alanine, in any order. Such linkers usually have a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or up to 20 amino acids, as required.
  • the linker comprises 2 to 12 amino acid residues, preferably it is a short linker.
  • the linker is a GS, (GS)n, GSG or G4S linker or any combination thereof.
  • the linker comprises one or more units, repeats or copies of a motif, such as for example GS, GSG or G4S (SEQ ID NO:62).
  • tag or “tag sequence” refers to protein tags which are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes.
  • the cp enzyme described herein comprises one or more tag sequences, specifically the tag is of about 6 amino acids, specifically it is a histidine tag, more specifically it is a hexahistidine tag.
  • the target-specific protease described herein comprises one or more N-terminal and/or C-terminal tag sequences.
  • tag sequence may comprise any number of amino acids of more than 2, 5 or 10 amino acids and up to 20, 50, 100, 200 or more amino acids.
  • tag sequences used herein may be any tag sequence known to the person skilled in the art.
  • tag sequences used herein are selected from affinity tags, solubility enhancement tags or monitoring tags.
  • affinity tag sequences used herein are selected from histidine (His) tag, specifically a poly-histidine tag, arginine-tag, specifically a poly-arginine tag, peptide substrate for antibodies, chitin binding domain, RNAse S peptide, protein A, b-galactosidase, FLAG tag, Strep II tag, streptavidin- binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, or c-Myc tag.
  • the tag is a His tag comprising one or more H, specifically a hexahistidine tag.
  • solubility enhancement tag sequences used herein are selected from T7A3 tag, T7AC tag, calmodulin-binding peptide (CBP), poly Arg, poly Lys, G B1 domain, protein D, Z domain of Staphylococcal protein A, and thioredoxin.
  • CBP calmodulin-binding peptide
  • poly Arg poly Arg
  • poly Lys poly Lys
  • G B1 domain protein D
  • protein D Z domain of Staphylococcal protein A
  • thioredoxin thioredoxin.
  • T7A3 SEQ ID NO:32
  • T7AC SEQ ID NO:34
  • the monitoring tag sequence used herein is m-Cherry, GFP or f-Actin.
  • target specific variants of natural proteases may be selected by screening for variants capable of efficiently cleaving substrates comprising amino residues at their PT site which are not well tolerated by the respective wild type protease. Therefore, essential enzymes are circularly permuted and modified by introducing protease recognition and cleavage sites useful for selecting proteases specifically targeting said recognition sites and exactly cleaving the foreign amino acids from the N-terminus of the cp enzyme thus reactivating its enzymatic activity.
  • the cp bacterial enzyme can be stably integrated into the bacterial host cell or may be introduced simultaneously or sequentially together with proteases to be screened for target specific proteolytic cleavage.
  • target specific protease and the cp enzyme described herein can be introduced into the host cell by any means of transforming bacterial cells such as, but not limited to transformation by electroporation, transformation by heat shock etc..
  • the bacterial host cells may comprise one cp enzyme with one specific protease recognition site or 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cp enzymes each of them differing in at least one amino acid residue, specifically differing in the protease recognition site, specifically differing in the P1’ amino acid residue.
  • bacterial cell culture comprising a plurality of cp enzymes having different protease recognition sites thus enabling isolation of a plurality of proteases having different specificities for recognition sites and/or having different proteolytic cleavage sites.
  • a library of proteases is screened using the herein described method.
  • said library comprises 10, 100, 500, 1000 or 5000 or more variants of a protease.
  • said library comprises 10, 100, 500, 1000 or 5000 or more variants of different proteases.
  • the library comprises variants of caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13 and/or caspase-14.
  • proteases can be produced by methods well known in the art, such as but not limited to in silico methods and random mutagenesis. Mutations can be introduced in the gene encoding the protease either by random mutagenesis or at a specific region by rational design. The advantage of random mutagenesis is that no knowledge of the protein’s structure or function is needed. Mutations can be introduced with several techniques, such as error prone PCR and overlap extension PCR. The amount of mutations can be modified by changing the Mg(ll) and Mn(ll) ion concentrations in the PCR buffer.
  • the cp enzyme used for the method as described herein is a cpATCase.
  • ATCase refers to an enzyme (EC 2.1.3.2), specifically required for pyrimidine synthesis.
  • ATCase catalyzes the first step in the pyrimidine de novo biosynthesis wherein carbamoylphosphate and L-aspartate are condensed to N-carbamoylaspartate and orthophosphate.
  • Pyrimidines and purines are the nucleotide building blocks in DNA and RNA, and form parts of coenzymes. Purines and pyrimidines can be synthesized de novo or in a salvage pathway by recycling of metabolites.
  • ATCase was first purified from E. coli and crystallized by Shepherdson and Pardee in 1960. It has a molecular size of 310 kDa and comprises regulatory (R, r-chain) and catalytic (C, c-chain) polypeptide chains that can be separated from each other and show very different properties and functions (Gerhart JC. and Schachman HK., Biochemistry 4(6): 1054-62).
  • the dodecameric holoenzyme C6R6 consists of three regulatory dimers, 17 kDa each, and two catalytic trimers, 34 kDa each.
  • the catalytic trimers are stacked on top of each other, linked by three dimers of the regulatory chains (Ke HM., et al., J Mol Biol., 204(3): 725-747, 1988).
  • the regulatory chain comprises a Zn domain that binds the zinc cofactor and an allosteric domain that binds allosteric regulators.
  • ATCase’s catalytic chains consist of two folding domains. The N-terminal domain binds aspartate and consists of residues 1-134, and the second domain comprises residues 149-284 to bind carbamoylphosphate and is located at the C- terminus. Both folding domains are composed of a core of b-sheets among several a- helices.
  • Helix 5 (residues 135-149) and helix 12 (residues 285-305) connect the two folding domains (Peterson CB., et al., Proc Natl Acad Sci., 88(2):458-462, 1992).
  • Helix 12 is a long structure in close proximity to the C-terminus. It crosses over from the C- terminal domain to the N-terminal domain. Helix 12 plays an important role in folding of the catalytic chains and their ability to form stable and active ATCase. It was found that shortening of the peptide chain after residue 305 had no influence on the activity and assembly of ATCase. The last five residues are not essential for the formation of intact enzyme and can therefore be deleted.
  • the circularly permuted catalytic subunit of cpATCase harboring its modified N-terminus in a beta strand located in the interior of the protein, is used for the selection of variants of the proteases described herein comprising desired characteristics such as for example increased PT tolerance or different or improved recognition site specificity.
  • cp ATCase c227 e.g. as described by Zhang and Schachman (Zhang and Schachman, Protein Science, 5(7): 1290-1300, 1996), is used in the method described herein.
  • the respective E. coli gene is named pyrB, its gene product forms a complex quaternary structure with the regulatory subunit pyrl in a stoichiometry of 3 regulatory subunit dimers and 2 catalytic subunit trimers.
  • This cp enzyme is used to detect specific proteases via the growth of £ coli, because fusion of any stretch of amino acids towards this N-terminus renders the enzyme inactive as it can no longer fold properly due to space limitations in the interior of the protein. If a protease is provided that can exactly cleave off this additional stretch of amino acids, the enzyme gets reactivated. As this is an essential enzyme of the pyrimidine synthesis in £. coli, it is possible to use this reactivation for applying a strong selection pressure.
  • An £ coli mutant is provided that lacks the original ATCase (e.g. by deleting pyrB and pyrl) and carries a plasmid encoding a cpATCase, e.g. cp-pyrB and pyrl provided on a single vector, that is inhibited by a N- terminal fusion sequence harboring a protease recognition site.
  • the £ coli mutant becomes a pyrimidine auxotroph strain which can only survive in media supplemented with pyrimidines or when the cells are complemented with a vector encoding ATCase.
  • the cpATCase can be activated by catalytic cleavage of the N-terminal fusion sequence. If a respective protease is provided via an additional plasmid, the £ coli can grow. Thereby, proteases can be selected that specifically recognize the recognition sites in the N-terminal fusion and/or that have increased tolerance for specific PT residues.
  • a cpATCase can be used comprising an inhibiting N-terminal fusion of 1 , 2, 3, 4, 5 or 6 amino acid residues, specifically histidine residues, optionally a short linker of 1 , 2, 3, 4, or 5 amino acid residues, more specifically of 3 amino acid residues, more specifically of serine and glycine residues and the amino acid recognition site for a protease.
  • a cpATCase can be used for the method described herein, comprising an inhibiting N-terminal fusion of a His-tag consisting of 6 histidines, a short linker (GSG) followed by an amino acid recognition site for a caspase.
  • GSG short linker
  • the cells are cultivated under selection conditions as described herein.
  • Isolating the target specific protease from the host cells can be performed by any means known for protein isolation and purification.
  • the protease may comprise a C- terminal or N-terminal tag.
  • tag sequences used herein may be any tag sequence known to the person skilled in the art.
  • tag sequences used herein are selected from affinity tags, solubility enhancement tags or monitoring tags.
  • allelic variant or “functionally active variant” as used herein also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants.
  • an allelic variant is an alternate form of a nucleic acid or peptide that is characterized as having a substitution, deletion, or addition of one or more nucleotides or one or more amino acids that does essentially not alter the biological function of the nucleic acid or polypeptide.
  • Functional variants may be obtained by sequence alterations in the polypeptide or the nucleotide sequence, e.g. by one or more point mutations, wherein the sequence alterations retains or improves a function of the unaltered polypeptide or the nucleotide sequence, when used in combination of the invention.
  • sequence alterations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions.
  • Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.
  • a point mutation is particularly understood as the engineering of a poly-nucleotide that results in the expression of an amino acid sequence that differs from the non-engineered amino acid sequence in the substitution or exchange, deletion or insertion of one or more single (non-consecutive) or doublets of amino acids for different amino acids.
  • sequence identity is understood as the relatedness between two amino acid sequences or between two nucleotide sequences and described by the degree of sequence identity or sequence complementarity.
  • sequence identity of a variant, homologue or orthologue as compared to a parent nucleotide or amino acid sequence indicates the degree of identity of two or more sequences.
  • Two or more amino acid sequences may have the same or conserved amino acid residues at a corresponding position, to a certain degree, up to 100%.
  • Two or more nucleotide sequences may have the same or conserved base pairs at a corresponding position, to a certain degree, up to 100%.
  • Sequence similarity searching is an effective and reliable strategy for identifying homologs with excess (e.g., at least 50%) sequence identity. Sequence similarity search tools frequently used are e.g., BLAST, FASTA, and HMMER.
  • Sequence similarity searches can identify such homologous proteins or polynucleotides by detecting excess similarity, and statistically significant similarity that reflects common ancestry.
  • Homologues may encompass orthologues, which are herein understood as the same protein in different organisms, e.g., variants of such protein in different organisms or species.
  • one of the two sequences needs to be converted to its complementary sequence before the % complementarity can then be calculated as the % identity between the first sequence and the second converted sequences using the above-mentioned algorithm.
  • Percent (%) identity with respect to an amino acid sequence, homologs and orthologues described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • sequence identity between two amino acid sequences can be determined using the NCBI, specifically NCBI BLAST + 2 9.0 program version (Apr-02-2019) or any version thereof appropriate for the purposes described herein.
  • Percent (%) identity with respect to a nucleotide sequence e.g., of a nucleic acid molecule or a part thereof, in particular a coding DNA sequence, is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Optimal alignment may be determined with the use of any suitable algorithm tor aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (lllumina, San Diego, CA), SOAP (available at soap.genomies.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Transform e.g., the Burrows Wheeler Aligner
  • ClustalW Clustal X
  • BLAT Novoalign
  • ELAND lllumina, San Diego, CA
  • SOAP available at soap.genomies.org.cn
  • Maq available at maq.sourceforge.net.
  • heterologous compounds refers to a compound which is either foreign, i.e. “exogenous”, such as not found in nature, to a given host cell; or that is naturally found in a given host cell, e.g., is “endogenous”, however, in the context of a heterologous construct, e.g., employing a heterologous nucleic acid, thus “not naturally-occurring”.
  • the heterologous nucleotide sequence as found endogenously may also be produced in an unnatural, e.g., greater than expected or greater than naturally found, amount in the cell.
  • heterologous nucleotide sequence or a nucleic acid comprising the heterologous nucleotide sequence, possibly differs in sequence from the endogenous nucleotide sequence but encodes the same protein as found endogenously.
  • heterologous nucleotide sequences are those not found in the same relationship to a host cell in nature (i.e., “not natively associated”). Any recombinant or artificial nucleotide sequence is understood to be heterologous.
  • nucleic acid molecules containing a desired coding sequence of an expression product such as e.g., a fusion protein as described herein or a cp caspase-2 as described herein may be used for expression purposes. Hosts transformed or transfected with these sequences are capable of producing the encoded proteins.
  • the expression system may be included in a vector; however, the relevant DNA may also be integrated into the host chromosome.
  • the term refers to a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
  • Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular polypeptide or protein.
  • Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA.
  • Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms.
  • Recombinant cloning vectors often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, one or more nuclear localization signals (NLS) and one or more expression cassettes.
  • “Expression vectors” or “vectors” as used herein are defined as DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e. of recombinant genes and the translation of their mRNA in a suitable host organism.
  • a sequence encoding a desired expression product such as e.g. the fusion protein described herein or the cp caspase-2 described herein, is typically cloned into an expression vector that contains a promoter to direct transcription.
  • Suitable bacterial and eukaryotic promoters are well known in the art. The promoter used to direct expression of a nucleic acid depends on the particular application.
  • a strong constitutive promoter is typically used for expression and purification of fusion proteins.
  • either a constitutive or an inducible promoter can be used, depending on the particular use of the expression product.
  • a preferred promoter for administration can be a weak promoter.
  • the promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements and lac repressor response elements.
  • Expression vectors comprise the expression cassette and additionally usually comprise an origin for autonomous replication in the host cells or a genome integration site, one or more selectable markers (e.g., an amino acid synthesis gene or a gene conferring resistance to antibiotics such as zeocin, kanamycin, G418 or hygromycin), a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, which components are operably linked together.
  • selectable markers e.g., an amino acid synthesis gene or a gene conferring resistance to antibiotics such as zeocin, kanamycin, G418 or hygromycin
  • An “expression cassette” refers to a DNA coding sequence or segment of DNA coding for an expression product that can be inserted into a vector at defined restriction sites.
  • the cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame.
  • foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA.
  • a segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a “DNA construct”.
  • vector includes autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences.
  • a common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily be introduced into a suitable host cell.
  • a plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA.
  • vector refers to a vehicle by which a DNA or RNA sequence (e.g ., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • a DNA or RNA sequence e.g ., a foreign gene
  • promote expression e.g., transcription and translation
  • Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al.).
  • the circular permuted enzymes or the proteases described herein are expressed as inclusion body.
  • Methods for the purification of recombinant proteins expressed as inclusion bodies are well known in the art. Typically, 70 to 80% of recombinant proteins expressed in bacteria, such as e.g. E. coli, are contained in inclusion bodies.
  • the purification of the expressed proteins from the inclusion bodies requires two main steps: extraction of inclusion bodies from the bacteria, for example via cell lysis followed by affinity purification, followed by solubilization and optionally refolding of the purified inclusion bodies.
  • a method for the selection of a target-specific protease comprising the sequential steps of i. providing bacterial host cells comprising at least one circularly permuted (cp) bacterial enzyme essential for growth of said host cells under selection conditions, comprising at its N- or C-terminus one or more additional amino acids inactivating its enzymatic activity and a protease recognition site for target-specific proteolytic cleavage, wherein upon target-specific proteolytic cleavage and removal of the foreign amino acids its enzymatic activity is restored, and wherein said host cells do not comprise said bacterial enzyme in enzymatically active form, ii. introducing at least one protease into the host cells, and iii.
  • the cp bacterial enzyme comprises at its N- terminus a sequence of foreign amino acids comprising the following structure from C- to N-terminus i. a recognition site for proteolytic cleavage, comprising at least 5 amino acids of the sequence PT-P1-P2-P3-P4, wherein PT is the wild-type N-terminal amino acid of the enzyme, and ii. optionally a linker, and iii. optionally a tag sequence.
  • cpATCase circularly permuted aspartate transcarbamoylase
  • the bacterial host cells are £ coli cells comprising a mutation or deletion in the endogenous pyrB gene, specifically comprising the sequence of SEQ ID NO:1, and wherein the circularly permuted bacterial enzyme is circularly permuted pyrB, preferably comprising SEQ ID NO:2 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:2, comprising at its N- terminus one or more foreign amino acids inactivating its enzymatic activity and a recognition site for proteolytic cleavage.
  • circularly permuted bacterial enzyme is circularly permuted pyrB comprising at its N-terminus a caspase-14 recognition site, a GSG linker and a His-tag, specifically comprising SEQ ID NO:4 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:4.
  • circularly permuted bacterial enzyme is circularly permuted pyrB comprising at its N-terminus a recognition site comprising the amino acid sequence VEID, DEVD or VDVAD, a GSG linker and a His-tag, specifically comprising SEQ ID NO:6 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:6.
  • protease is a caspase selected from the group consisting of caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14 and any chimera comprising a small subunit and a large subunit of any caspase, specifically a caspase-6/14 chimera, comprising a small caspase-6 subunit and a large caspase-14 subunit or a circularly permuted caspase (cp caspase) selected from the group consisting of cp caspase-1, cp caspase-2, cp caspase-3, cp caspase-4, cp caspase-5, cp caspase-6, cp caspase- 7, cp caspase-8, cp caspsa
  • a circularly permuted (cp) bacterial enzyme essential for growth under selection conditions, comprising at its N-terminus one or more foreign amino acids inactivating its enzymatic activity and a recognition site for proteolytic cleavage, wherein upon proteolytic cleavage of the one or more foreign amino acids its enzymatic activity is restored.
  • the cp enzyme of item 21 or 22, comprising at its N-terminus a sequence of foreign amino acids comprising the following structure from C- to N-terminus i. a recognition site for proteolytic cleavage, comprising at least 5 amino acids of the sequence PT-P1-P2-P3-P4, wherein PT is the wild-type N-terminal amino acid of the enzyme, ii. optionally a linker, and iii. optionally a tag sequence.
  • the cp enzyme of item 23, wherein the recognition site for proteolytic cleavage comprises at least 5 amino acids of the sequence P4-P3-P2-P1-PT, wherein
  • P1 ' can be any amino acid, preferably it is G, A, L, M, Q, E, S, P, V, I, C, R, N, D,
  • P1 can be any amino acid, preferably it is D, E;
  • P2 can be any amino acid, preferably it is A, H, S or V, K, I, T;
  • P3 can be any amino acid, preferably it is V, I or E, F, Y, G; and
  • P4 can be any amino acid, preferably it is D, E or W H, Q.
  • cpATCase cp aspartate transcarbamoylase
  • 26 The cp enzyme of item 25, wherein the enzyme is a cpATCase derived from £ coli comprising SEQ ID NO:2 or a variant thereof having at least 80%, 85%, 90%, specifically at least 95%, specifically at least 99% sequence identity with SEQ ID NO:2.
  • a bacterial host cell comprising the cp bacterial enzyme essential for growth under selection conditions comprising at its N-terminus one or more foreign amino acids inactivating its enzymatic activity and representing a recognition site for proteolytic cleavage according to any one of items 21 to 26, wherein said cell does not comprise said bacterial enzyme endogenously in enzymatically active form.
  • the host cell of item 27 comprising a vector expressing said circularly permuted bacterial enzyme or wherein said circularly permuted bacterial enzyme is stably integrated into the genome of said cell.
  • bacterial cell is an £. coli cell comprising a mutation or deletion of its endogenous pyrB gene, comprising SEQ ID NO:1 and wherein said cp bacterial enzyme is a cp aspartate carbamoyltransferase catalytic subunit enzyme (cp-pyrB), specifically comprising SEQ ID NO:2, comprising at its N-terminus one or more foreign amino acids inactivating its enzymatic activity and a recognition site for proteolytic cleavage.
  • cp-pyrB carbamoyltransferase catalytic subunit enzyme
  • Example 1 Establishment of a selection system with the circular permuted essential bacterial enzyme ATCase
  • a circularly permuted catalytic subunit of aspartate transcarbamoylase (cpATCase) is used which harbors its new N-terminus in a beta strand located in the interior of the protein.
  • the respective E. coli gene is named pyrB and the gene product forms a complex quaternary structure with the regulatory subunit pyrl in a stoichiometry of 3 regulatory subunit dimers and 2 catalytic subunit trimers.
  • This cp enzyme is used to detect specific proteases via the growth of E. coli, because fusion of any stretch of amino acids towards this N-terminus renders the enzyme inactive as it can no longer fold properly due to space limitations in the interior of the protein.
  • An E. coli mutant is provided that lacks the original ATCase (e.g. by deleting pyrB and pyrl) and carries a plasmid encoding a cpATCase, e.g. cp-pyrB and pyrl on a single vector, that is inhibited by an N-terminal fusion sequence harboring a protease recognition site.
  • coli mutant becomes a pyrimidine auxotroph strain which can only survive in media supplemented with pyrimidines or when the cells are complemented with a vector encoding ATCase.
  • the cpATCase can be activated by catalytic cleavage of the N-terminal fusion sequence. If a respective soluble protease is provided via an additional plasmid, the E. coli can grow. Thereby proteases can be selected that specifically recognize the recognition sites in the N-terminal fusion.
  • E. coli BL21 (DE3) with pyrBI operon exchanged to a kanamycin resistance
  • Circularly permuted caspase-2 A circularly permuted caspase-2 variant (cp caspase-2, SEQ ID NO:21) was designed. Based on the sequence of human caspase- 2 (UniProtKB14 ID P42575) the N-terminal CARD was removed and the order of large and small subunit exchanged to create a constitutively active caspase. To ensure expression as a single chain protein, an aspartate (Asp 343 in the wild-type sequence, Asp 21 in the cp protein) was mutated to alanine, to avoid cleavage of the small subunit from a p14 to a p12 chain.
  • Asp 343 in the wild-type sequence, Asp 21 in the cp protein was mutated to alanine, to avoid cleavage of the small subunit from a p14 to a p12 chain.
  • the protein sequence was codon optimized for £ coli with the GeneArtTM online tool (Thermo Fisher Scientific). Between the small and the large subunit, a glycine-serine linker was added which also forms a Bam HI restriction site. This enables the separate cloning of the subunits and facilitates the creation of chimera consisting of subunits from different caspases.
  • the N-terminal His tag enabled IMAC- purification.
  • Circularly permuted caspase-7 The circularly permuted variant of caspase-7 was constructed from wild type caspase-7 (UniProtKB P55210). The propeptide separating the subunits was deleted, and an aspartate in the propeptide N-terminal of the large subunit was mutated to alanine to remove an internal cleavage site (residue 23 in the wild type, residue 129 in the cp caspase). A 6His tag was added N-terminal for IMAC purification.
  • caspases were codon-optimized for expression in £. coli.
  • pETDuetTM-1 has an ampicillin-resistance as selection marker, while the pACYCDuetTM- 1 vector has a chloramphenicol resistance marker.
  • Mutant gene libraries of cp caspase-2 were generated by error prone (ep) PCR and overlap extension (oe) PCR.
  • the linear DNA fragments were ligated using T4 DNA ligase.
  • the amount of mutations can be modified by changing the Mg(ll) and Mn(ll) ion concentrations in the PCR buffer. The used concentrations caused in average one to three amino acid exchanges in the caspase.
  • the selection protocol was performed with respective cotransfections with simultaneous use of ampicillin, kanamycin and chloramphenicol in the selection medium described below.
  • proteases the highly specific human caspases were chosen, which required the introduction of a mutation into the regulatory pyrl subunit of cpATCase, because it harbors the amino acid sequence DQVD (position 69-72), a bona fide recognition site for several caspases.
  • DQVD amino acid sequence DQVD
  • the crucial aspartate at position 72 was mutated to glutamate (DQVE) to be sure that pyrl is not cut and inactivated at this site in the presence of caspases.
  • the pyrB used is circularly permuted and its new N-terminus is located in the interior of the protein.
  • a 6His tag, followed by a GSG linker and a caspase recognition site were fused to this N-terminus. This renders the inactive as it can no longer fold properly.
  • a caspase is provided that can exactly cleave off this additional stretch of amino acids, the enzyme gets reactivated.
  • ATCase is an essential enzyme of the pyrimidine synthesis in £ coli, it is possible to use this reactivation for applying a strong selection pressure.
  • Cp pyrB with different recognition sites were used.
  • DEVD SEQ ID NO:31
  • VEID SEQ ID NO:6
  • VDVAD SEQ ID NO:19
  • WEHD WEHD
  • the method was further improved by using a selection medium that consists of a minimal medium with special additives (such as M9 minimal medium with additives as shown below) so that an easy interpretable readout was received, namely a significant growth of the strain with a non-inhibited or reactivated cpATCase.
  • a selection medium that consists of a minimal medium with special additives (such as M9 minimal medium with additives as shown below) so that an easy interpretable readout was received, namely a significant growth of the strain with a non-inhibited or reactivated cpATCase.
  • M9 minimal medium based on the recipe described by Sambrook et al. was used as a medium that contains no nucleotides to select autotroph cells (Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2 nd edition, 1989). It was also used to express caspase variants. Addition of vitamins, iron(ll) ions and casamino acids had a positive effect on cell growth.
  • the final composition used was: 50 mM Na2HP04, 20 mM KH2PO4, 10 mM NaCI, 1 mM MgS04, 0.1 mM CaCb, 0.4 % glucose, 20 mM NH4CI, 0.5 g/100 ml casamino acids, 10 pg/ml FeS04, 0.025 mM IPTG, Vitamins: each 0.001 mg/ml of biotin, thiamine, riboflavin, pyridoxin, niacinamide Selection of Caspase Libraries
  • the caspase mutant library was transformed into £ coli BL21(DE3) ApyrBI cells.
  • competent cells were prepared that already contained the ATCase plasmid with the desired protease cleavage site and PT residue. After recovery in SOC medium the cells were either diluted in M9 medium in a baffled Erlenmeyer shaking flask or plated on M9 agar plates and incubated at 30 °C for 24-48 h. Liquid cultures were used to enrich mutants with improved growth. After incubation for about 24 h of the transformed cells in M9 medium at 30 °C they were plated on M9 agar, incubated at 30 °C and single colonies analyzed by sequencing.
  • the construct of the caspase to be tested was transformed into £. coli BL21 (DE3) ApyrBI cells harboring an ATCase substrate with a specific cleavage site. After recovery in SOC medium the cell suspension was plated on TY agar plates containing the appropriate antibiotics and incubated at 37 °C, overnight. A TY culture was inoculated with a single colony and shaken at 37 °C, 220 rpm, overnight. On the next day the overnight culture was diluted 1 :20 in fresh TY medium in a culture tube and grown at 37 °C, 220 rpm for 60-90 min to an OD600 of 1.0. The cells were washed with M9 medium to remove TY medium and its pyrimidines.
  • the washed cell pellet was suspended in M9 medium. An aliquot was diluted in fresh M9 medium containing IPTG. The optical density was measured at the starting point of incubation. The cultures were shaken for 6-48 h at 220 rpm, 30 °C and OD was measured to determine the increase in optical density. To control cell viability of the cultures several dilutions of the cultures were plated on M9 agar plates.
  • Figure 2 shows the multiplication of optical density of ATCase/pyrB deficient E. coli cells during 21 hour growth in minimal medium with special supplements transformed with cp caspase-6 and different constructs harboring recognition sites of caspase-3, -6 and -14, respectively (6H: His-tag, DEVD (SEQ ID NO:31), VEID (SEQ ID NO:6) and WEHD (SEQ ID NO:4).
  • cleavage by cp caspase-6 results in the full activation of the inhibited cpATCase - there is no difference in growth to a strain that harbors the non-inhibited cpATCase.
  • Cp caspase-6 was also able to partially reactivate a cpATCase inhibited with an N-terminal fusion containing the caspase-3 site DEVD (SEQ ID NO:31) - this was expected as caspase-6 is known to partially recognize this sequence.
  • no growth was detectable with the recognition site for caspase-14, as it is known that caspase-6 cannot recognize this site. This shows, that the assay is highly specific and correlates with the known substrate preferences of caspase-6.
  • Figure 3 shows the number of colonies harboring active cp caspase-6-S per transformation containing different amounts of cp caspase-6-S DNA. As background 25 ng of DNA containing inactive chimera were used.
  • FIG. 4 A scheme of the selection by co-expression of two proteins is shown in Figure 4. To ensure that the cells contain both plasmids, pETDuet-1 (pyrB and pyrl, ampicillin resistance) and pACYCDuet-1 (caspase, chloramphenicol resistance) vectors were used.
  • Figure 4 provides a scheme of the selection system for caspases co-expressed with tagged cp ATCase in £ coli BL21 (DE3) ApyrBI knock-out cells.
  • Active caspase cleaves the XXXXD tag off from the ATCase and the knock-out cells can produce pyrimidines to survive.
  • Inactive caspase does not cleave the tag and the ATCase is blocked. The cell cannot survive the selection process in M9 minimal medium without pyrimidines.
  • Figure 5 shows the growth behavior of the knock-out strain, expressing inhibited cpATCase containing the caspase-6 recognition site VEID (SEQ ID NO:6) in its N- terminal fusion, supplemented with original chimera 6/14, mutated chimera 6/14 or mutated chimera which had been inactivated by substitution of its catalytic Cys to Ala.
  • VEID caspase-6 recognition site
  • the PT tolerance was especially improved for amino acids only weakly accepted by the original variant. These are branched, hydrophobic, and polar residues. Up to 55-fold increased PT cleavage activity could be observed after two rounds of mutation and selection. The potency of the selection system was demonstrated by the selection of significantly improved, highly efficient caspase variants.
  • the standard substrate for in vitro testing of the cleavage activity of selected caspases was a modified Ubiquitin-conjugating enzyme E2 (EC: 2.3.2.23, UniProt: P63146). Its molecular weight is 21.33 kDa and it contains an N-terminal 6His tag followed by a short linker (GSG) and the respective caspase recognition sites.
  • E2 Ubiquitin-conjugating enzyme
  • GSG short linker
  • the human enzyme superoxide dismutase (SOD, E.C: 1.15.1.1.) was used.
  • the enzyme contains an N-terminal 6His tag, followed by a GSG linker and the respective caspase recognition sites, and two C-terminal tags.
  • the PT for SOD cleavage reactions is a glycine residue.
  • the VDVAD-SOD protein has sequence SEQ ID NO:17, the enzyme has a molecular weight of 20.38 kDa.
  • the substrates were expressed in BL21 (DE3) at 0.5 mM IPTG induction, at 37 °C for 4 h.
  • the caspase plasmid was transformed into BL21 (DE3). A single colony was used to inoculate TY medium and grown shaking at 220 rpm at 37 °C overnight. The following day the culture was diluted 1 :200 in TY medium in a baffled shaking flask, incubated shaking at 37 °C for about 3-4 h. 10 ml of the culture were diluted into 500 ml optimized selective M9 minimal medium in a baffled 2 I Erlenmeyer flask.
  • the culture was incubated at 37 °C, 220 rpm until OD600 reached 1.0, expression was induced with 0.1 mM IPTG and incubated shaking overnight at 25 °C, 220 rpm. Cell pellets were harvested and stored at -20 °C until purification.
  • Substrates and caspases were purified using Immobilized Metal Affinity Purification (IMAC).
  • IMAC Immobilized Metal Affinity Purification
  • the eluted fractions were pooled and concentration determined by a BCA assay.
  • the purified substrate aliquots were stored in S-Buffer (buffer recipe from Bozhkov and Salvesen, 2014) with 2 mM DTT at -80 °C. Testing of Catalytic Activity of Selected Variants in vitro
  • the standard reaction condition for testing of caspase cleavage activity was 1 mg/ml E2 substrate incubated with 0.01 mg caspase in S-Buffer (buffer recipe from Bozhkov and Salvesen, 2014) in a total volume of 50 pi in a 1.5 ml microcentrifuge tube. The reaction was incubated in a digital dry bath at 25 °C. For very slow reactions the caspase concentration was adapted to 0.1 mg/ml. After specific time intervals samples were taken from the reaction and inactivated by suspending them in SDS sample buffer.
  • the cleavage reactions were analyzed with SDS-PAGE to separate cleaved and uncut E2-substrate from the caspase. Band intensities of cleaved substrate were determined to evaluate the percentage of cleavage at a specific time point.
  • E2 substrates with all 20 amino acids at the PT position were used to analyze the PT tolerance of the protease.
  • VDVAD-G-E2 Cleavage of VDVAD-G-E2 (1 mg/ml) to 50 % by caspases (0.01 mg/ml) within 1 min was used as the standard value and was defined as 100 % (mass ratio 1 :100, molar ratio 1 :170). All results of the other cleavages were normed to the respective PT Gly value. If, for example, 1 mg/ml VDVAD-N-E2 was cleaved to 50 % by 0.01 mg/ml caspase in 50 min, the cleavage activity would be 2 % of the VDVAD-G-E2 value.
  • Example 1 The selection system as described in Example 1 was implemented, but in addition a cp pyrB containing VDVAD (SEQ ID NO:41) as cleavage site after the tag was used, as this is the caspase-2 recognition site.
  • VDVAD SEQ ID NO:41
  • VDVAD-cp pyrB A deletion variant of VDVAD-cp pyrB was cloned.
  • the construct VDVAD-AM-cp pyrB (SEQ ID NO:38) was tested and the activity was improved by the deletion. Incubation times of the deletion variant could be reduced to 6 h compared to 24-48 h with the original construct. The deletion variant was then used as the standard substrate for cp caspase-2 selections.
  • cpATCase variants with all 19 possible amino acids at the c-chain’s N-terminus were cloned. It was found that the N-terminal amino acid greatly influenced the activity of the ATCase and the growth of the cells. Except for cpATCase starting with amino acids His, Lys, Phe, Tyr, and Trp all variants were active.
  • Negative controls contained inactive VDVAD-cpATCase and an empty pACYC- Duet-1 vector.
  • Positive controls contained the active cpATCase without tag and either an empty pACYCDuet vector or a cp caspase-2 construct to test the influence of caspases on cell viability.
  • substrates comprising a 6His tag followed by a GSG linker and the respective caspase recognition site was used.
  • the PT residue was a glycine.
  • Figure 6 shows the in vivo activity of Growth of cells containing cp caspase-2 co expressed with cpATCase substrates containing the caspase-3 recognition site DEVD, the caspase-6 recognition site VEID or the caspase-14 recognition site WEHD. Multiplication of starting OD600 in M9 medium after cultivation at 30 °C for 22 h.
  • the positive control contained empty pACYCDuet-1 vector and cpATCase, the negative control empty pACYCDuet-1 vector and VDVAD-ATCase.
  • Figure 7 shows the in vitro cleavage of cp caspase-2 with E2 fusion protein containing a VDVAD or DEVD cleavage site respectively.
  • FIG. 6 shows that cp caspase-2 is able to cleave the VDVAD-tag and reactivate VDVAD-ATCase (SEQ ID NO:39). It is however not able to efficiently remove the tag from DEVD-ATCase (SEQ ID NO:31), nor the VEID-ATCase (SEQ ID NO:6), or WEHD- ATCase (SEQ ID NO:4) leading to cell death due to lack of pyrimidines. This is consistent with results from in vitro cleavage experiments. An E2 substrate with DEVD-recognition site is cleaved more than a hundred times slower than with VDVAD-recognition site.
  • the cp caspase-2 cannot or only weakly process the cleavage sites preferred by caspases-3, - 6, or -14, as indicated by the in vivo experiment.
  • the results correlate, which proves that the cleavage activity of a caspase in vivo can indicate its behavior in the in vitro cleavage reactions.
  • Mutant libraries in E. coli BL21(DE3) ApyrBI cells were selected with VDVAD- cpATCase with different PT residues. Selections were executed with Pro, Met, Thr, and Val. Selections with P1’ Met were executed with cp ATCase without deletion of the native methionine, all other selections were executed with constructs comprising SEQ ID No. 41. Selections were executed with several IPTG concentrations to adapt the sensitivity of the system. Selection was carried out either in liquid cultures or on agar plates. In order to obtain single colonies from liquid cultures, they were streaked on agar plates after turbidity was clearly visible. Selection with Met, Thr, and Val as P1 ’ lead to hundreds of positive variants, thus only the largest colonies were analyzed.
  • variants were analyzed and several were selected for expression and characterization by in vitro cleavages. Variants were chosen when they had been enriched in liquid culture or contained mutations that were found several times independently. Before expression the pETDuet-1 plasmid with ATCase had to be eliminated from the selected cells.
  • the E105V mutation was found repeatedly in variants selected with cpATCase that contained a methionine as a PT and 0.025 mM IPTG.
  • a variant containing this mutation was purified and tested (S9 variant, SEQ ID NO:14). It had the same activity under standard conditions as cp caspase-2 (50 % of VDVAD-E2-Gly cleaved in 1 min) but showed slightly higher solubility and a significantly improved PT tolerance.
  • the S9 variant was used for a new round of mutation because of its improved characteristics and PT tolerance.
  • a mutated gene library of S9 was generated.
  • the cp caspase-2 S9 mutant library was transformed into E. coli BL21(DE3) ApyrBI cells containing VDVAD-AM-P-cpATCase (SEQ ID NO:18), to select for caspases with a higher tolerance towards a PT Pro residue.
  • the mutant library contained about 10,000 variants. After 48 h a variant was enriched in liquid culture which had three mutations additional to the E105V exchange originating from the S9 variant.
  • the so-called mS9 Pro variant has the mutations E105V, G171 D, V225G, and D282E (SEQ ID NO:15).
  • the mutant was tested in liquid culture with the selection substrate VDVAD-AM-P-cpATCase.
  • the mS9 Pro variant had an increased activity, as shown in Figure 8.
  • the original cp caspase-2 only doubled once in 24 h, whereas the S9 variant showed a 7-fold increase in OD600.
  • the highest rise was observed for variant mS9 Pro.
  • the increase of the starting OD600 was nearly 120- fold which indicated a significantly improved PT tolerance for proline.
  • Figure 8 provides a comparison of in vivo activity of variants S9, mS9 Pro, and cp caspase-2 co-expressed with VDVAD-AM-P-cpATCase (SEQ ID NO: 18). Multiplication of starting OD600 in optimized M9 medium after cultivation at 30 °C for 24 h.
  • Example 5 Characterization of the new caspase variants of Example 4
  • the caspases were expressed and purified as described herein.
  • the cleavage activity under standard conditions was the same as for cp caspase-2, 50 % of 1 mg/ml VDVAD-G-E2 were cleaved by 0.01 mg/ml caspase in 1 min.
  • the PT tolerance was significantly improved compared to the S9 variant.
  • the cleavage activity of the S9 variant (E105V) was improved for all 19 P1’ substrates.
  • the values for most residues were at least twice as high as with standard cp caspase-2.
  • the S9 variant was nearly 5 times more tolerant towards Leu as PT compared to the standard variant.
  • Figure 9 shows the PT tolerance of mS9 Pro and variants with single mutations E105V (variant S9), G171 D, V225G, and D282E in % compared to cp caspase-2. All VDVAD-E2 (1 mg/ml) substrates except for VDVAD-P-E2 were incubated with 0.01 mg/ml caspase at 25 °C. All values are normed to the cleavage rate of cp caspase-2. Level of significance: no asterisk (not significant, p > 0.5), * (p ⁇ 0.05), ** (p £ 0.01),
  • the caspase variant’s Vmax, KM, and kcat were determined. The results were comparable to the analyses of E2 protein cleavages. Overall, the order of acceptance of PT residues measured with a FRET assay using peptide substrates correlates with the order measured with the densitometric methods in this work using the protein PT substrate E2. The improved properties of the mutated variants were confirmed with FRET measurements, significantly higher tolerances were found for all PT residues.
  • FRET Forster resonance energy transfer
  • the substrates were obtained from Bachem AG and were of the general structure of Abz-VDVAD-XA-Dap(Dnp), where all 20 amino acids were substituted for X (the PT position). All substrates were dissolved in 10 mM HEPES, pH 7.5 to a concentration of 750 mM.
  • the buffer for the assay was 50 mM HEPES, 150 mM NaCI, pH 7.2.
  • the initial slope was measured by measuring the fluorescence for 3-15 minutes (or 3 to 20 hours for proline as PT) and calculating the slope of the initial measurement in mM product generated per second. Fluorescence was measured in black 96 well plates on a Tecan Infinite M200 Pro plate reader. Excitation wavelength was 320 nm, emission wavelength 420 nm. In the FRET assay all substrates, except for proline as PT showed excellent linearity for at least a few minutes.
  • Vmax is the maximum rate
  • KM is the Michaelis constant
  • [S] is the substrate concentration.
  • Vmax and KM were fitted kcatwas calculated by dividing Vmax by the enzyme concentration [E].
  • Figure 12 shows an example Michaelis-Menten kinetic measured by FRET assay.
  • the measured substrate was Abz-VDVADHA-Dap(Dnp) at concentrations given on the x-axis.
  • the y-axis gives the measured initial slope values. Shaded circles represent measured data points, the full line represents the model fit and the dashed lines represent upper and lower 95 % confidence intervals of the model fit.
  • Caspase variant mS9 Pro has four amino acid exchanges compared to cp caspase-2. To get insights of their individual influence on the properties of mS9 Pro the mutations were inserted in cp caspase-2 separately.
  • constructs were transformed into BL21 (DE3) cells, caspases were expressed and purified and PT tolerance, stability, and specificity were tested as described herein.
  • Tables 4/5 Mean values for PT tolerance in % of VDVAD-G-E2 cleavage for cp caspase-2 variants with single mutations.
  • Tables 4/5 show the values for the PT tolerance of cp caspase-2 variants with single mutations.
  • the tolerance of variants V225G and D282E was nearly identical compared to cp caspase-2.
  • Figure 9 shows that the mutation G171 D had the highest influence on PT tolerance compared to mutations E105V, V225G and D282E, 11 of the 19 PT activities were significantly higher than the S9 values, only the tolerance for Tyr was significantly lower. Branched amino acids were especially well tolerated, as well as proline, threonine, and glutamic acid. The amino acid exchanges V225G and D282E did not alter the PT tolerance significantly compared to the original cp caspase-2.
  • the mutation and selection protocol was also executed with a cp caspase-2 variant containing a mutation of the active site cysteine (C279S).
  • This active site mutant is 4,800-fold less active than the original variant.
  • One round of mutation and selection created a variant with two mutations (H185R, Y290R) and improved the activity to about 400 % of the original variant.
  • the mutation and selection protocol described herein was used to optimize circularly permuted proteases for anti-cancer therapy, in which cp proteases specifically bind tumor-relevant proteins for proteolytic cleavage or degradation.
  • caspase-7 A circular permuted variant of caspase-7 was created.
  • the canonical recognition site of caspase-7 is DEVD, to alter this site EFKD/S, HYID/G, EISD/G, and QGTD/A were fused to cpATCase. These sites are contained in caspase accessible regions EGFR and EGFL6, to potential targets for anti-cancer therapy.
  • a library of cp caspase-7 was created, as described above.
  • the above selection system was slightly altered: After transformation of 10 ng of the gene bank (pACYCDuet- 1) into BL21(DE3) ApyrBI cells (carrying a pETDuet-1 vector with 6-His-XXXX-pyrB and pyrl in the cloning sites) via electroporation the cells were recovered in 360 pi SOC medium. The rich medium was removed and the cells suspended in 10 ml of liquid M9 minimal medium. While cp caspase-2 cultures were incubated for only two days, changing the recognition site required a longer protocol.
  • the cells were incubated at 30 °C and 220 rpm for 3 days before repeating the dilution step 3 more times using 50 mI of the previous culture. As final step, 100 mI of the culture were plated onto optimized M9 minimal plates and incubated for 3 days at 30 °C before further characterizing the colonies.
  • a cp caspase-7 variant By selection with EISD/G cp ATCase a cp caspase-7 variant was found that shows a significantly reduced activity towards its canonical cutting site DEVD/X. This can be achieved by changing the selection protocol from colony selection to liquid culture competition selection with multiple dilution steps, thus strongly promoting the best growing strain. In the case of cp caspase-7 this resulted in the identification of a variant (cp caspase-7A, Figures 10 and 11) with dramatically (1000-fold) reduced activity towards its canonical DEVD/G site while retaining significant cutting activity for the site used for selection (EISD).
  • Figure 10 shows that the removal of N-terminal tag fused to the 25 kDa protein E2 via the cutting site DEVD proceeds slower with cp caspase-7A than with cp caspase-7, as can be seen by the shift of the protein to a lower molecular weight.
  • Figure 11 shows that the activity of cp caspase-7A towards EISD is retained while the recognition of HYID is reduced compared to cp caspase-7.
  • cp caspase- 7A is highly specific for EISD. Both variants (cp caspase-7 and cp caspase-7A) are unable to cut EFKD.
  • cp caspase-7A SEQ ID NO:36
  • EISD EISD
  • HYID EISD
  • cp caspase-7B SEQ ID NO:12
  • cp caspase-7C (SEQ ID NO:13): improved activity (about 300 % improved) towards the QGTD site of EGFL6 and can cut all 3 EGFR-sites.
  • Example 8 Selection of target-specific proteases (e.q. Npro, SARS-CoV-2
  • the mutation and selection protocol described herein can be used to optimize autoproteases.
  • An autoprotease is fused to the N- or C-terminus of cpATCase, thereby inactivating it.
  • the cpATCase is activated upon autocatalytic removal of the fused autoprotease. This method can be used to improve the activity of the autoprotease or to render the autoprotease tolerant towards specific PT residues.
  • the coding sequence of the pestiviral autoprotease N pro was cloned N-terminally of the circularly permuted catalytic subunit of the cpATCase construct in the pETDuet-1 vector, resulting in N pro -cpATCase (SEQ ID NO:44, comprising the active N pro variant).
  • N pro -cpATCase SEQ ID NO:44, comprising the active N pro variant
  • a catalytically inactive variant of N pro was fused to the N-terminus of the cpATCase, resulting in an inactive N pro -cpATCase construct (SEQ ID NO:46, comprising the inactive N pro variant).
  • N pro -cpATCase constructs were used to transform £ coli ApyrBI cells, the washed cell suspension was plated on M9 minimal agar with 0.1 mM IPTG. As a positive control in parallel cpATCase without a tag (SEQ ID NO:2) was transformed and plated.
  • the SARS-CoV-2 main protease M pro (also 3C-like proteinase, 3CL pro ) was used to block cpATCase. By autocatalytic cleavage of M pro the cpATCase is released from the protein chain and reactivated.
  • M pro is a cysteine autoprotease encoded in the viral RNA and catalyzes cleavage at 11 sites of the replicase polyprotein chain into several proteins essential for viral replication, thus making it an attractive target for drug design.
  • the coding sequence of the SARS-CoV-2 main protease M pro was codon optimized for £. coli with the GeneArtTM online tool, synthesized (Thermo Fisher Scientific) and cloned to the N-terminus of the cpATCase construct in a pETDuet-1 vector with a Ser as PT (SEQ ID No:48), as this represents the PT amino acid of the natural C-terminal self-cleavage site of M pro in the viral replicase protein.
  • the M pro -cpATCase construct was used to transform £ coli ApyrBI cells, the washed cell suspension was plated on M9 minimal agar with 0.1 mM IPTG. After 48 hours incubation at 30 °C colonies were visible on the agar plates.
  • the mutation and selection protocol described herein can be used to optimize carboxypeptidases.
  • carboxypeptidase B which removes arginine or lysine from the C-terminus of proteins is used in many industrial biotechnological processes. Optimization of CPB with the herein described protocol could have a major impact on the economics of such processes like the production of insulins. To achieve such an optimization the selection is performed with a cpATCase with an additional arginine or lysine fused to its C-terminus. Introduction of a library of mutated carboxypeptidases into the selection strain would then allow for the selection of the most efficient variant via the bacterial growth.
  • E. coli BL21(DE3) ApyrBI cells were transformed with the constructs, and the washed cell suspension was plated on M9 minimal agar containing 0.1 mM IPTG.
  • the mutation and selection protocol described herein can be used to optimize aminopeptidases by adding the respective N-terminal target residue to the natural N- terminus of the cpATCase described herein. Optimization of aminopeptidase is of importance for industrial biotechnological productions - e.g. for expression of biopharmaceuticals in E. coli.
  • the N-termini of the respective products often show undesired variations, namely the presence or absence of the N-terminal methionine which is only partly removed by the bacterial methionine aminopeptidase. As this difference is only a minor change given the size of the biopharmaceutical product, respective purification schemes to obtain a pure product (which is an important prerequisite for approval) are tedious.
  • the methionine aminopeptidase could be optimized with the mutation and selection protocol described herein by adding one or more additional methionines to the N-terminus of the catalytic subunit of cpATCase. This would lead to a selection of methionine aminopeptidases variants that can efficiently remove one or even more methionines from the N-terminus of proteins. This method could even be expanded to also include an additional mutation selection of the used strains to obtain £ coli variants that not only harbor an efficient methionine aminopeptidase, but also provide the biological background (e.g.
  • the mutation and selection protocol described herein can be used to optimize expression tags for production and solubility improvement.
  • An N-terminal tag that contains a region of random mutations (produced by PCR using partly degenerate primers) is fused to a caspase variant that shows low solubility or expression rate to obtain a library of potential solubility- and expression-enhancing tags.
  • the selection is performed using a cpATCase that is inhibited by a weakly accepted recognition site and a strong negatively influencing PT residue (e.g. proline).
  • a tag-variant promoting the expression of large amounts of soluble caspase enables the reactivation of enough cpATCase to promote the growth of the host bacteria in selective media. Sequencing the respective tags of the selected caspases reveals optimal expression tags to enhance production of soluble proteins.
  • protease that render the protease resistant against a certain inhibitory drug.
  • Proteases are a valuable drug target and several approved drugs against viral infections are protease inhibitors.
  • the development of drug resistance poses serious problems and the knowledge which protease variant is resistant against which drug is of high importance for efficient therapeutic interventions.
  • the respective target protease is mutated and expressed in an E. coli strain that harbors a cpATCase which has an N-terminal fusion at the catalytic subunit that can be removed via a specific recognition and cleavage by the respective protease.
  • the E. coli strain can grow.
  • treatment of the bacteria with a drug inhibitory for the protease will stop the growth of bacteria harboring sensitive proteases. Only proteases that, due to certain mutations, become resistant towards the drug can still promote bacterial growth in this selection system. Sequencing of the respective proteases provides the information necessary to predict which mutations confer resistance to a certain drug.
  • the selection system described here can be adapted for the selection of protease inhibitors, like small peptide sequences.
  • a region in cpATCase is identified that is easily accessible for a protease (preferably a loop or helix on the surface of the enzyme, preferably where there is interaction with other subunits or domains) that tolerates the insertion of a protease recognition sequence, without interfering with ATCase activity.
  • a protease preferably a loop or helix on the surface of the enzyme, preferably where there is interaction with other subunits or domains
  • This inhibitor selection can also be adapted for other essential enzymes in the selection host.
  • Exemplary adenylate cyclase catalytic subunits can be separated (as used in bacterial Two-Hybrid systems). If the subunits are connected via a recognition sequence for a protease, the cell will not survive under selective conditions if a protease is active and cleaves the tag. If the protease is inhibited by a drug or small peptide sequence the adenylate cyclase remains intact and the cell survives the selection.
  • the inhibitory peptide can be provided as an auto-inhibitory domain - e.g. as a peptide-library at the C-terminus of a protease. If this C-terminus is close to the catalytic site, respective inhibitory peptides can be identified by growth of the respective bacterial strain expressing this peptide as C-terminal fusion to the protease.
  • the selection system as described in Examples 1 and 2 above can be used to identify mutations in SARS-CoV-2 main protease M pro (also 3C-like proteinase, 3CL pro , as described in Example 8.2) that are responsible for drug resistance to inhibitors.
  • the N-terminal self-cleavage site AVLQ-S represents the recognition site (P4P3P2P1-PT) of the protease and AVLQ was inserted to the N-terminus of cp-pyrB with a serine as PT (SEQ ID NO:57).
  • the cDNA of SARS-CoV-2 main protease M pro (SEQ ID NO:58) was codon optimized for E. coli with the GeneArtTM online tool, synthesized (Thermo Fisher Scientific) and cloned into the MCSII of a pACYCDuet-1 vector with restriction sites A/c/el and Xho ⁇ .
  • an M pro library can be generated to select for proteases with a mutation-mediated drug resistance in the presence of an inhibitor during selection.

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Abstract

La présente invention concerne un procédé de sélection de protéases spécifiques à une cible à l'aide de cellules bactériennes comprenant au moins une enzyme bactérienne à permutation circulaire essentielle pour la croissance desdites cellules dans des conditions de sélection, comprenant à son extrémité N ou C terminale un ou plusieurs acides aminés étrangers inactivant son activité enzymatique et un site de reconnaissance de protéase pour un clivage protéolytique spécifique à une cible. Lors du clivage protéolytique spécifique à la cible et l'élimination des acides aminés étrangers, son activité enzymatique est rétablie, lesdites cellules ne comprenant pas ladite enzyme bactérienne sous une forme enzymatiquement active, en introduisant au moins une protéase dans les cellules hôtes, le maintien desdites cellules hôtes dans des conditions permettant le clivage spécifique à une cible de l'enzyme bactérienne à permutation circulaire par la protéase, la sélection de cellules hôtes capables de croître dans des conditions de sélection, et l'isolement de la protéase spécifique à une cible à partir desdites cellules hôtes. La présente invention concerne également des enzymes bactériennes permutées circulaires et des cellules hôtes bactériennes, spécifiquement destinées à être utilisées dans le procédé de sélection.
PCT/EP2020/072893 2019-08-14 2020-08-14 Système de sélection bactérienne pour des protéases spécifiques à une cible WO2021028577A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066615A1 (fr) 1999-05-04 2000-11-09 Rutgers, The State University Of New Jersey Compositions et procedes de detection de proteases actives
US6846628B1 (en) 1994-04-29 2005-01-25 Jacob Nathaniel Wohlstadter Selection methods
WO2008045148A2 (fr) 2006-07-05 2008-04-17 Catalyst Biosciences, Inc. Procédés de criblage de protéases et protéases identifiées par lesdits procédés

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Publication number Priority date Publication date Assignee Title
US6846628B1 (en) 1994-04-29 2005-01-25 Jacob Nathaniel Wohlstadter Selection methods
WO2000066615A1 (fr) 1999-05-04 2000-11-09 Rutgers, The State University Of New Jersey Compositions et procedes de detection de proteases actives
WO2008045148A2 (fr) 2006-07-05 2008-04-17 Catalyst Biosciences, Inc. Procédés de criblage de protéases et protéases identifiées par lesdits procédés

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