RU2801120C2 - Sortase-labeled clostridia neurotoxins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of obtaining a labeled polypeptide, a labeled polypeptide for its visualization, and a method of determining at least the internalization and/or intracellular transport of the labeled polypeptide are proposed. A polypeptide containing an acceptor site for sortase or a donor site for sortase, a non-cytotoxic protease containing the L-chain of clostridial neurotoxin, or its proteolytically inactive mutant, a targeting part (TP) that is capable of binding to a binding site on a target cell, and a translocation domain are obtained. The polypeptide is incubated with sortase and a labeled substrate containing a sortase donor site or an acceptor site for sortase, respectively, and a conjugated detectable label. A labeled polypeptide that does not have reduced potency compared to the equivalent unlabeled polypeptide and/or exhibits similar cell binding, translocation, and cleavage of the SNARE protein to the equivalent unlabeled polypeptid are obtained.

EFFECT: visualization of cell binding, translocation and/or cleavage of the SNARE protein using a cell-based assay or in vivo.

15 cl, 6 dwg, 5 ex

Description

The present invention relates to labeled polypeptides and methods for their preparation and use.

Bacteria of the genus Clostridia produce highly potent and specific protein toxins that can poison neurons and other cells to which they are delivered. Examples of such clostridial neurotoxins include those produced by C. tetani (TeNT) and C. botulinum (BoNT) serotypes A-G and X (see WO 2018/009903 A2) as well as neurotoxins produced by C. baratii and C. butyricum.

Clostridial neurotoxins are some of the most potent toxins known. As an example, botulinum neurotoxins have median lethal dose (LD ₅₀ ) values in mice ranging from 0.5 to 5 ng/kg depending on serotype. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, in particular the release of neurotransmitters. While botulinum toxin acts on neuromuscular junctions and inhibits cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central nervous system.

Clostridial neurotoxins are expressed in Clostridium as single chain polypeptides. Each clostridial neurotoxin has a catalytic light chain separated from the heavy chain (spanning the N-terminal translocation domain and the C-terminal receptor-binding domain) by a surface region called the activation loop. During protein maturation, proteolytic cleavage of the activation loop separates the light and heavy chains of the clostridial neurotoxin, which are held together by a disulfide bridge, to form a fully active double-stranded toxin.

Also known in the art are retargeted clostridial neurotoxins that can be modified to include an exogenous ligand known as the targeting moiety (TM). The TM is chosen to provide binding specificity for the desired target cell, and as part of the retargeting process, the native binding portion of the clostridial neurotoxin (eg, _HC domain or H _CC domain) can be removed. Retargeting technology is described, for example, in: EP-B-0689459; WO 1994/021300; EP-B-0939818; US 6461617; US 7192596; WO 1998/007864; EP-B-0826051; US 5989545; US 6395513; US 6962703; WO 1996/033273; EP-B-0996468; US 7,052,702; WO 1999/017806; EP-B-1107794; US 6,632,440; WO2000/010598; WO 2001/21213; W02006/059093; WO 2000/62814; WO2000/04926; WO 1993/15766; WO2000/61192; and WO 1999/58571; all of which are incorporated by reference in their entirety.

A further variant includes polypeptides derived from one or more of a non-cytotoxic protease, clostridial neurotoxin translocation or binding domains, or from polypeptides of equivalent/similar functionality.

Binding, translocation, and proteolytic cleavage of SNARE proteins by clostridial neurotoxins (or other polypeptides described herein) remain poorly understood. Thus, there remains a need for an assay method that allows visualization of each of these steps, in particular in real time and/or in living cells. Such an analysis may facilitate the development and characterization of clostridial neurotoxin therapeutics, especially the characterization of novel BoNT therapeutics, hybrid toxins, and retargeted clostridial neurotoxins (and variants thereof).

Moreover, the amount of antibodies (eg, fluorescent antibodies) used in conventional methods to visualize clostridial neurotoxins and other such polypeptides is low, with limited specificity and/or sensitivity. Moreover, such conventional methods are generally based on cell fixation, which can have an adverse effect on cell architecture, and are unsuitable for in vivo/real-time imaging, particularly in complex biological systems, e.g., in in vivo animals. . Thus, there remains a need for improved/alternative methods.

The present invention solves one or more of the above problems.

The present inventors have surprisingly found that sortase can be used to conjugate a detectable label to the polypeptides of the invention (comprising a non-cytotoxic protease or a proteolytically inactive mutant thereof; a targeting moiety (TM) that binds to a binding site on a target cell; and a translocation domain) without reducing the effectiveness of the labeled polypeptide. In other words, labeled polypeptides show similar (or improved) cell binding, translocation, and cleavage of the SNARE protein to an equivalent unlabeled polypeptide. This was completely unexpected given that polypeptides labeled using alternative technologies (eg, non-site-specific labeling and SNAP labeling) showed reduced efficacy.

Moreover, the polypeptides of the invention containing the sortase acceptor or donor site can be readily purified and expressed, again unexpected given that GFP labeling has been associated with expression/purification difficulties, indicating that the inclusion of acceptor or donor sites for sortase did not adversely affect the structure or folding of the polypeptide.

In addition, methods involving the use of sortase allowed the production of a double-labeled polypeptide, which also allowed the visualization of translocation events occurring in cellular endosomes, one of the least understood aspects of clostridial neurotoxin (and retargeted clostridial neurotoxin) movement. Advantageously, the present invention provides visualization of translocation using live imaging microscopy and will make a significant contribution to understanding the mechanisms of translocation in several cell models and tissues.

The labeled polypeptides of the invention open up new avenues for in vivo and/or real time monitoring of the mechanism of action of said polypeptides and obviate the need for fixative products that have an adverse effect on cellular architecture. Thus, the present invention allows the imaging of toxins in more complex biological systems such as ex vivo tissue preparations (e.g. brain sections), histopathological specimens, and in vivo animals, and is not limited to simple cell systems such as immortalized cell lines. and neurons, as in the case of conventional methods. Thus, the polypeptides of the present invention can be used (for example) to detect the distribution of the polypeptide from the site of administration.

In one aspect, the invention relates to a method for producing a labeled polypeptide, comprising:

a. obtaining a polypeptide containing:

i. an acceptor or donor site for sortase;

ii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iv. translocation domain;

b. incubation of the polypeptide with:

sortase; And

a labeled substrate containing a sortase donor and acceptor site and a conjugated detectable label;

where the sortase catalyzes the conjugation between the amino acid of the acceptor site for sortase and the amino acid of the donor site for sortase, thereby labeling the polypeptide; And

c. obtaining a labeled polypeptide.

When the method of the invention includes the use of a polypeptide containing a sortase acceptor site, the labeled substrate containing a conjugated detectable label (eg, as indicated in b.) contains a sortase donor site. Similarly, when the method of the invention includes the use of a polypeptide containing a sortase donor site, a labeled substrate containing a conjugated detectable label (eg, as indicated in b.) contains a sortase acceptor site.

Thus, the invention relates to the use of a sortase acceptor site and a corresponding sortase donor site, wherein the sortase is capable of catalyzing the conjugation of an amino acid of the sortase acceptor site and an amino acid of the sortase donor site. Thus, the appropriate acceptor and donor sites for sortase to be used within the scope of the invention are chosen so that conjugation by sortase can be carried out.

Thus, in one embodiment, the method of the invention comprises:

a. obtaining a polypeptide containing:

i. acceptor site for sortase;

ii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iv. translocation domain;

b. incubation of the polypeptide with:

sortase; And

a labeled substrate containing a sortase donor site and a conjugated detectable label;

where the sortase catalyzes the conjugation between the amino acid of the acceptor site for sortase and the amino acid of the donor site for sortase, thereby labeling the polypeptide; And

c. obtaining a labeled polypeptide.

In another embodiment, the method of the invention comprises:

a. obtaining a polypeptide containing:

i. donor site for sortase;

ii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iv. translocation domain;

b. incubation of the polypeptide with:

sortase; And

a labeled substrate containing an acceptor site for sortase and a conjugated detectable label;

where the sortase catalyzes the conjugation between the amino acid of the acceptor site for sortase and the amino acid of the donor site for sortase, thereby labeling the polypeptide; And

c. obtaining a labeled polypeptide.

Also, the present invention relates to a labeled polypeptide, which can be obtained by the method according to the invention.

In one embodiment, the detectable label is conjugated at or near an acceptor or donor site for sortase in a polypeptide comprising a non-cytotoxic protease or a proteolytically inactive mutant thereof; a targeting moiety (TM) and a translocation domain.

In one embodiment, the detectable label is conjugated to a sortase acceptor or donor site, for example conjugated directly to an amino acid of the sortase acceptor or donor site. Alternatively, the detectable label can be conjugated C-terminally from the sortase acceptor or donor site, for example 1-50, eg 1-25 or 1-10 amino acids from the C-terminus of the sortase acceptor or donor site.

In another embodiment, the detectable label is conjugated N-terminally from the sortase acceptor or donor site, e.g., 1-50, e.g., 1-25 or 1-10 amino acids from the N-terminus of the sortase acceptor or donor site.

The term "obtainable" as used herein also encompasses the term "obtained". In one embodiment, the term "obtainable" means "obtained".

In a related aspect, the invention relates to a polypeptide for labeling using sortase, and the polypeptide contains:

i. an acceptor or donor site for sortase;

ii. a non-cytotoxic protease that is capable of cleaving a protein of the exocytic fusion apparatus in the target cell, or a proteolytically inactive mutant thereof;

iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iv. a translocation domain that is capable of translocating a non-cytotoxic protease from the endosome across the endosomal membrane and into the cytosol of the target cell;

where, when the polypeptide contains a sortase donor site, the sortase donor site is at the N-terminus of the polypeptide, and where, when the sortase donor site contains G _n or A _n , n is at least 2; And

where the N-terminal residue of the donor site is the N-terminal residue of the polypeptide; or

wherein the polypeptide comprises one or more amino acid residues N-terminally from the sortase donor site and a cleavage site which, when cleaved, exposes the N-terminus of the sortase donor site.

In one embodiment, the sortase labeling polypeptide comprises:

i. donor site for sortase;

ii. a non-cytotoxic protease that is capable of cleaving a protein of the exocytic fusion apparatus in the target cell, or a proteolytically inactive mutant thereof;

iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iv. a translocation domain that is capable of translocating a non-cytotoxic protease from the endosome across the endosomal membrane and into the cytosol of the target cell;

where the sortase donor site is at the N-terminus of the polypeptide, and where, when the sortase donor site contains G _n or A _n , n is at least 2; And

where the N-terminal residue of the donor site is the N-terminal residue of the polypeptide.

In one embodiment, the sortase labeling polypeptide comprises:

i. donor site for sortase;

ii. a non-cytotoxic protease that is capable of cleaving a protein of the exocytic fusion apparatus in the target cell, or a proteolytically inactive mutant thereof;

iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iv. a translocation domain that is capable of translocating a non-cytotoxic protease from the endosome across the endosomal membrane and into the cytosol of the target cell;

where the sortase donor site is at the N-terminus of the polypeptide, and where, when the sortase donor site contains G _n or A _n , n is at least 2; And

wherein the polypeptide comprises one or more amino acid residues N-terminally from the sortase donor site and a cleavage site which, when cleaved, exposes the N-terminus of the sortase donor site.

In one embodiment, the sortase labeling polypeptide comprises:

i. acceptor site for sortase;

ii. a non-cytotoxic protease that is capable of cleaving a protein of the exocytic fusion apparatus in the target cell, or a proteolytically inactive mutant thereof;

iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iv. a translocation domain that is capable of translocating a non-cytotoxic protease from the endosome across the endosomal membrane and into the cytosol of the target cell.

The polypeptide is suitably used in the method of the invention.

The polypeptide of the invention may contain an acceptor site for sortase. Alternatively, said polypeptide may contain a sortase donor site.

In a preferred embodiment, said polypeptide comprises a sortase acceptor site and a sortase donor site.

The polypeptide of the present invention may contain a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 2. In one embodiment, the polypeptide of the invention contains a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: : 2. Preferably, the polypeptide of the invention comprises (more preferably consists of) the polypeptide shown as SEQ ID NO: 2.

The polypeptide of the present invention may contain a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 4. In one embodiment, the polypeptide of the invention contains a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: : 4. Preferably, the polypeptide of the invention comprises (more preferably consists of) the polypeptide shown as SEQ ID NO: 4.

The polypeptide of the present invention may contain a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 40. In one embodiment, the polypeptide of the invention contains a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: : 40. Preferably, the polypeptide of the invention comprises (more preferably consists of) the polypeptide shown as SEQ ID NO: 40.

The polypeptide may be encoded by the nucleic acid of the invention.

The invention also relates to a labeled polypeptide, wherein the polypeptide contains:

i. a detectable label conjugated to the polypeptide;

ii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iv. translocation domain.

The invention also relates to a labeled polypeptide, wherein the polypeptide contains:

i. a detectable label conjugated to the polypeptide;

ii. amino acid sequence that contains L(A/P/S)X(T/S/A/C)G_n(SEQ ID NO: 59)_, where X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A_n(SEQ ID NO: 60), where X is any amino acid and n is at least 1, NPQTN (SEQ ID NO: 61), YPRTG (SEQ ID NO: 62), IPQTG (SEQ ID NO: 63), VPDTG (SEQ ID NO: 64), LPXTGS (SEQ ID NO: 65) where X is any amino acid, NPKTG (SEQ ID NO: 46), XPETG (SEQ ID NO: 47), LGATG (SEQ ID NO: 48) , IPNTG (SEQ ID NO: 49), IPETG (SEQ ID NO: 50), NSKTA (SEQ ID NO: 51), NPQTG (SEQ ID NO: 52), NAKTN (SEQ ID NO: 53), NPQSS (SEQ ID NO: 54), LPXTX (SEQ ID NO: 55), where X is any amino acid, NPX₁TX₂(SEQ ID NO: 56), where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G (SEQ ID NO: 57), where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G (SEQ ID NO: 58), where X₁ is Ala, Cys or Ser, LPXS (SEQ ID NO: 66), LAXT (SEQ ID NO: 67), MPXT (SEQ ID NO: 68), MPXTG (SEQ ID NO: 69), LAXS (SEQ ID NO: 70), NPXT (SEQ ID NO: 71), NPXTG (SEQ ID NO: 72), NAXT (SEQ ID NO: 73), NAXTG (SEQ ID NO: 74), NAXS (SEQ ID NO: 75), NAXSG ( SEQ ID NO: 76), LPXP (SEQ ID NO: 77), LPXPG (SEQ ID NO: 78), where X is any amino acid, LRXTG_n(SEQ ID NO: 111) or LPAXG_n(SEQ ID NO: 106), where X is any amino acid and n is at least 1;

iii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iv. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

v. translocation domain.

The invention also relates to a labeled polypeptide, wherein the polypeptide contains:

i. a detectable label conjugated to the polypeptide;

ii. an amino acid sequence that contains L(A/P/S)X(T/S/A/C)G _n, where X is any amino acid and n is at least 1, L(A/P/S)X( T/S/A/C)A _n, where X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, where X is any amino acid;

iii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iv. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

v. translocation domain.

In one embodiment, the labeled polypeptide contains:

i. a detectable label conjugated to the polypeptide;

ii. amino acid sequence that contains L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, where X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1;

iii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iv. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

v. translocation domain.

In one embodiment, the labeled polypeptide contains:

i. a detectable label conjugated to the polypeptide;

ii. an amino acid sequence that contains L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS where X is any amino acid;

iii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iv. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

v. translocation domain.

In one embodiment, a labeled polypeptide of the invention exhibits similar cell binding, translocation, and SNARE protein cleavage to an equivalent unlabeled polypeptide. In another embodiment, the labeled polypeptide exhibits improved cell binding, translocation and/or cleavage of the SNARE protein compared to an equivalent unlabeled polypeptide. In a particularly preferred embodiment, the labeled polypeptide exhibits improved cell binding, translocation, and cleavage of the SNARE protein compared to an equivalent unlabeled polypeptide. Cell binding, translocation and/or cleavage of the SNARE protein can be determined using any method known in the art and/or described herein. In one embodiment, cell binding, translocation, and/or cleavage of the SNARE protein can be determined using a cell-based or in vivo assay. Suitable assays may include abduction score score (DAS), dorsal root ganglion (DRG) assay, spinal cord neuron (SCN) assay, and dome phrenic nerve (PNHD) assay, which are standard in the art. A suitable assay may be the assay described in Donald et al. (2018), Pharmacol Res Perspect, e00446, 1-14, which is incorporated herein by reference. Preferably, a suitable assay is a SNAP25 cleavage assay as described in Fonfria, E., S. Donald and V.A. Cadd (2016), "Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments." J Recept Signal Transduct Res 36(1): 79-88, which is incorporated herein by reference.

In one embodiment, the detectable label is conjugated to or near a sequence containing L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1. In one embodiment, the detectable label is conjugated to or near a sequence containing L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG or LPXTGS.

In one embodiment, an amino acid sequence containing L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1, may be on the C-terminal side of the TM polypeptide. In one embodiment, an amino acid sequence containing L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG or LPXTGS, may be C-terminal of the TM polypeptide. In another embodiment, an amino acid sequence containing L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1, may be N-terminal to a non-cytotoxic protease or proteolytically inactive mutant thereof in the polypeptide. In another embodiment, an amino acid sequence containing L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, may be N-terminal to a non-cytotoxic protease or proteolytically inactive mutant thereof in a polypeptide.

In one embodiment, the labeled polypeptide contains two or more detectable labels, preferably the labeled polypeptide contains two detectable labels. In a preferred embodiment, the detectable labels are different, such as different colored fluorophores.

The first and second (or more) detectable labels can be conjugated to or near an amino acid sequence containing L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1, where the first and second (or more) detectable labels are conjugated to different regions of the labeled polypeptide. The first and second (or more) detectable labels can be conjugated to or near an amino acid sequence containing L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, wherein the first and second (or more) detectable labels are conjugated to different regions of the labeled polypeptide. For example, the first detectable label may be conjugated to the amino acid sequence N-terminal of the non-cytotoxic protease or its proteolytically inactive mutant, and the second detectable label may be conjugated to the amino acid sequence C-terminal of the TM (or vice versa). Preferably, the amino acid sequences to which the first and second (or more) detectable tags are conjugated are different.

In one embodiment, the detectable label is conjugated to L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1. Alternatively, a detectable label can be conjugated at the C-terminus of L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1, such as 1-50, such as 1-25 or 1-10 amino acids C-terminal from L(A/P/S)X(T/ S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1.

In one embodiment, the detectable label is conjugated to L(A/P/S)X(T/S/A/C)G _n , L(A/P/S)X(T/S/A/C)A _n , NPQTN, YPRTG, IPQTG, VPDTG or LPXTGS. Alternatively, the detectable label may be C-terminally conjugated from L(A/P/S)X(T/S/A/C)G _n , L(A/P/S)X(T/S/A/ C)A _n , NPQTN, YPRTG, IPQTG, VPDTG or LPXTGS, e.g. 1-50, e.g. 1-25 or 1-10 amino acids C-terminal from L(A/P/S)X(T /S/A/C)G _n , L(A/P/S)X(T/S/A/C)A _n , NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS.

In another embodiment, the detectable label is N-terminally conjugated from L(A/P/S)X(T/S/A/C)G_n, L(A/P/S)X(T/S/A/C)A_n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1, such as 1-50, such as 1-25 or 1-10 amino acids N-terminal from L(A/P/S)X(T/ S/A/C)G_n.

In another embodiment, the detectable label is N-terminally conjugated from L(A/P/S)X(T/S/A/C)G _n , L(A/P/S)X(T/S/A /C)A _n , NPQTN, YPRTG, IPQTG, VPDTG or LPXTGS, e.g. 1-50, e.g. 1-25 or 1-10 amino acids N-terminal from L(A/P/S)X( T/S/A/C)G _n .

In embodiments where the amino acid sequence contains L(A/P/S)X(T/S/A/C)A _n , X is any amino acid and n can be at least 2, 3, 4, 5, 6 , 7, 8, 9 or 10, for example, the amino acid sequence may contain LPXTA _n (SEQ ID NO: 102). Preferably n is 1-10, more preferably 1-4. In such embodiments, a conjugated detectable label and an amino acid sequence that contains L(A/P/S)X(T/S/A/C)A _n , where X is any amino acid and n is at least 1, indicate that the polypeptide has been successfully labeled with a sortase (eg, from Streptococcus pyogenes).

In a particularly preferred embodiment, the amino acid sequence comprises L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1. Such amino acid sequence may contain LPXSG _n (SEQ ID NO: 103), LAXTG _n (SEQ ID NO: 104), LPXTG _n (SEQ ID NO: 105), LPXCG _n (SEQ ID NO: 107), LAXSG _n (SEQ ID NO: 108), LPXAG _n (SEQ ID NO: 109), or LSXTG _n (SEQ ID NO: 110). Preferably the amino acid sequence may contain LPXSG _n , LAXTG _n , LPXTG _n or LAXSG _n .

In one embodiment, the amino acid sequence contains LRXTG _n , where X is any amino acid and n is at least 1.

In one embodiment, the amino acid sequence contains LPAXG _n , where X is any amino acid and n is at least 1.

A conjugated detectable label and an amino acid sequence that contains L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1, indicates that the polypeptide was successfully labeled with sortase. In one embodiment, n may be at least 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably n is 1-10, more preferably 1-4.

In one embodiment, the detectable label is conjugated to or near L(A/P/S)X(T/S/A/C)G _n .

In one embodiment, the detectable label is conjugated to L(A/P/S)X(T/S/A/C)G _n , such as its amino acid residue G. Alternatively, the detectable label can be conjugated at the C-terminal side from L(A/P/S)X(T/S/A/C)G _n , for example, at a distance of 1-50, for example, 1-25 or 1-10 amino acids from the C-terminal side of L(A/ P/S)X(T/S/A/C)G _n .

In another embodiment, the detectable label is N-terminally conjugated to L(A/P/S)X(T/S/A/C)G _n , such as 1-50, such as 1-25 or 1 -10 amino acids N-terminal from L(A/P/S)X(T/S/A/C)G _n .

In one embodiment, the detectable label is conjugated to or near the amino acid sequence LPXSG _n , where n is at least 1, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, n is 1 -10, more preferably 1-5. The detectable label is preferably conjugated at the C-terminal side of LPXSG _n , for example, with a lysine residue at the C-terminal side of LPXSG _n . X is any amino acid such as E.

In one embodiment, the detectable label is conjugated to or near the amino acid sequence LAXTG _n , where n is at least 1, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, n is 1-10, more preferably 1-4. The detectable label is preferably conjugated at the N-terminal side of LAXTG _n , for example, with a histidine residue at the N-terminal side of LAXTG _n . X is any amino acid such as E.

In one embodiment, the first detectable label is conjugated to or near the amino acid sequence LPXSG _n (where n is at least 1, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably where n is 1-10, more preferably 1-5) and the second detectable label is conjugated to or near the amino acid sequence LAXTG _n (where n is at least 1, e.g. at least 2, 3, 4, 5, 6, 7, 8 , 9 or 10, preferably where n is 1-10, more preferably 1-4). The first detectable label is preferably conjugated at the C-terminal side of LPXSG _n , for example, with a lysine residue at the C-terminal side of LPXSG _n and the second detectable label is preferably conjugated at the N-terminal side of LAXTG _n , for example, with a histidine residue with N-terminal side from LAXTG _n . X is any amino acid, such as E. In one embodiment, the first detectable label is C-terminal of the TM polypeptide and the second detectable label is N-terminal of the non-cytotoxic protease or its proteolytically inactive mutant (preferably the non-cytotoxic protease) in the polypeptide.

The labeled polypeptide of the present invention may comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 26. In one embodiment, the labeled polypeptide of the invention comprises a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 26. Preferably, the labeled polypeptide of the invention comprises (more preferably consists of) the polypeptide shown as SEQ ID NO: 26.

The sortase described herein may be sortase A, sortase B, sortase C, or sortase D. For a review of the biological properties of sortases, see Mazmanian, S.K., G. Liu, H. Ton-That and O. Schneewind (1999). "Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall." Science 285(5428): 760-763 and Paterson, G.K. and T.J. Mitchell (2004). "The biology of Gram-positive sortase enzymes." Trends Microbiol 12(2): 89-95, both of which are incorporated herein by reference.

Also, the present invention covers sortase variants. Sortase variants suitably have altered specificity so that they recognize alternative sortase sites (eg, acceptor sites). Sortase variants are described in Dorr, B.M., H.O. Ham, C. An, E.L. Chaikof and D.R. Liu (2014). "Reprogramming the specificity of sortase enzymes." Proc Natl Acad Sci U S A 111(37): 13343-13348, Chen, I., B.M. Dorr and D.R. Liu (2011). "A general strategy for the evolution of bond-forming enzymes using yeast display." Proc Natl Acad Sci U S A 108(28): 11399-11404, Dorr, B.M., H.O. Ham, C. An, E.L. Chaikof and D.R. Liu (2014). "Reprogramming the specificity of sortase enzymes." Proc Natl Acad Sci U S A 111(37): 13343-13348, and Chen, L., J. Cohen, X. Song, A. Zhao, Z. Ye, C. J. Feulner, P. Doonan, W. Somers, L. Lin and P. . Chen (2016). "Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency." Sci Rep 6: 31899, each of which is incorporated herein by reference. Specialized variants of sortases can be obtained using the methodology described in the references indicated. The person skilled in the art will select appropriate sortase donor and/or acceptor sites recognized by the sortase variant when using said variant within the scope of the present invention. In addition, a person skilled in the art will understand that these donor and/or acceptor sites for sortase may differ from the sites described in the present description.

In one embodiment, the sortase variant may include a modified Staphylococcus aureus sortase A. The modified sortase A may include one or more mutations relative to the sequence of SEQ ID NO: 31 described in the present description. For example, modified sortase A may contain one or more of the following mutations relative to the sequence of SEQ ID NO: 31: P86L, P94S, P94R, N98S, A104T, E106G, A118T, F122S, F122Y, D124G, N127S, K134R, F154R, D160N, D165A , K173E, G174S, K177E, I182V, K190E, K196T or a combination. In some embodiments, the present invention provides a modified sortase that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of these mutations. The above amino acid substitutions can provide a modified sortase that efficiently utilizes acceptor and/or donor sites that the corresponding wild-type parent sortase does not bind to. For example, in some embodiments, the modified sortase uses a sortase acceptor site having an LPXTG sequence and a donor site having an N-terminal polyglycine motif. In some embodiments, the modified sortase uses an acceptor and/or donor site that is different from the acceptor and/or donor site (respectively) used by the original sortase, e.g., an acceptor site for sortase comprising an LPXS, LAXT, LAXTG motif (SEQ ID NO: 116), MPXT, MPXTG, LAXS, LAXSG (SEQ ID NO: 120), NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG or LPXTA (SEQ ID NO: 114).

Preferably, the sortase is sortase A or a variant thereof. Sortase A is a transpeptidase that recognizes the (preferably C-terminal) L(A/P/S)X(T/S/A/C)(G/A) motif of proteins to cleave between (T/S/A/C ) and G/A, and then transfers the acyl moiety to a nucleophile containing (preferably N-terminal) (oligo)glycine (where the motif is L(A/P/S)X(T/S/A/C)G) or (oligo)alanine (wherein the motif is L(A/P/S)X(T/S/A/C)A). In one embodiment, sortase A may be a sortase derived from Streptococcus pyogenes (e.g., SEQ ID NO: 37), wherein said sortase recognizes (among others) a sortase acceptor site having the sequence LPXTA, in such cases, preferably a sortase acceptor site is A _n , where n is at least 1. The use of S. pyogenes sortase is described in Antos et al (2009), J Am Chem Soc, 131, 10800-10801, which is incorporated herein by reference.

Preferably, sortase A may be a sortase derived from Staphylococcus aureus, or a variant thereof.

In one embodiment, the sortase acceptor site may comprise (or consist of) L(A/P/S)X(T/S/A/C)(G/A), NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, where X is any amino acid. For example, a sortase acceptor site may comprise (or consist of) L(A/P/S)X(T/S/A/C)G, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, where X is any amino acid.

In one embodiment, the sortase acceptor site may comprise (or consist of) NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG (SEQ ID NO: 123) or LPAXG (SEQ ID NO: 118), where X is any amino acid.

The acceptor site for sortase X ₁ PX ₂ X ₃ G may be recognized by sortase A. In some embodiments, where the acceptor site for sortase contains (or consists of) X ₁ PX ₂ X ₃ G, X ₂ may be Asp, Glu, Ala , Gln, Lys or Met. in some embodiments, said sortase acceptor site comprises (or consists of) LPX ₁ TG, where X ₁ is any amino acid. In other embodiments, the sortase acceptor site comprises (or consists of): LPKTG, LPATG, LPNTG, LPETG, LPNAG, LPNTA, LGATG, IPNTG, or IPETG.

The NPX ₁ TX ₂ sortase acceptor site can be recognized by sortase B. In some embodiments, the sortase acceptor site comprises (or consists of): NPQTN, NPKTG, NSKTA, NPQTG, NAKTN, or NPQSS.

Acceptor site for sortase LPXTX can be recognized by sortase C.

In one embodiment, the sortase acceptor site does not contain (or does not consist of) NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, where X is any amino acid, NPX ₁ TX ₂ , where X ₁ is Lys or Gln and X ₂ is Asn, Asp or Gly, X ₁ PX ₂ X ₃ G where X ₁ is Leu, Ile, Val or Met, X ₂ is any amino acid and X ₃ is Ser , Thr or Ala, LPEX ₁ G, where X ₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG or LPAXG, where X is any amino acid.

In embodiments where sortase A is used, the sortase A region (e.g., acceptor or donor region) is the sortase A region.

In a preferred embodiment, the sortase A acceptor site described herein may be the sortase A site. The sortase A consensus acceptor site may be L(A/P/S)X(T/S/A/C)(G/ A), where X is any amino acid such as E. However, it is preferred that the consensus acceptor site of sortase A is L(A/P/S)X(T/S/A/C)G.

In one embodiment, the acceptor site of sortase A comprises or is selected from LPXSG (SEQ ID NO: 115), LAXTG, LPXTG (SEQ ID NO: 117), LPAXG, LPXCG (SEQ ID NO: 119), LAXSG, LPXAG (SEQ ID NO: 119), LAXSG, LPXAG (SEQ ID NO: 117). : 121), LSXTG (SEQ ID NO: 122), LRXTG and LPXTA. Preferably, the acceptor site of sortase A can be selected from LPXSG, LAXTG, LPXTG and LAXSG, more preferably LPXSG or LAXTG. For example, the acceptor site of sortase A can be LPESG (SEQ ID NO: 112) or LAETG (SEQ ID NO: 113) as illustrated herein.

In some embodiments, the sortase acceptor site described herein is followed by one or more C-terminal amino acid residues, such as 1-50, 1-10, or preferably 1-5 (eg, 2) amino acid residues. In some embodiments, the sortase acceptor site is followed by one or more acidic amino acid residues. The acidic amino acid residue may be aspartate or glutamate.

The sortase donor site may comprise (or consist of) G _n , where n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, n is at least 2 Preferably n is 2-10, such as 2-5. More preferably, n is 4. Such a donor site may preferably be a sortase A site, preferably for use with an acceptor site for sortase AL(A/P/S)X(T/S/A/C)G.

In some embodiments, the sortase donor site may be G _n K, where n is at least 1 (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, in one embodiment implementation n is at least 2, and preferably n is 2-10, such as 2-5).

In one embodiment, a sortase acceptor site for use in the present invention comprises (or consists of) L(A/P/S)X(T/S/A/C)G, where X is any amino acid, and a donor site for sortase for use within the scope of the invention contains (or consists of) G _n where n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

The sortase donor site may comprise (or consist of) A _n , where n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, n is at least 2 Preferably n is 2-10, such as 2-5. More preferably, n is 4. Such a donor site may preferably be a sortase A site, preferably for use with an acceptor site for sortase AL(A/P/S)X(T/S/A/C)A.

In one embodiment, a sortase acceptor site for use in the invention comprises (or consists of) L(A/P/S)X(T/S/A/C)A, where X is any amino acid, and a donor site for sortase for use within the scope of the invention contains (or consists of) A _n where n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In the context of acceptor or donor sites for sortases, X can be any amino acid, for example, selected from the standard amino acids: aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine, serine, threonine, tyrosine, methionine, tryptophan, cysteine , alanine, glycine, valine, leucine, isoleucine, proline and phenylalanine. In some embodiments, X may be any amino acid except for proline.

When a non-sortase A acceptor site is used, such as:

Sortase B region of Staphylococcus aureus: NPQTN;

Streptococcus pneumoniae sortase B region: YPRTG, IPQTG, or VPDTG;

Streptococcus pyogenes sortase B region: LPXTGS;

Streptococcus pneumoniae sortase C region: YPRTG, IPQTG, or VPDTG; And

Streptococcus pneumoniae sortase D region: YPRTG, IPQTG, or VPDTG;

one of skill in the art will select an appropriate donor site for use with said non-sortase A acceptor site based on knowledge in the art.

Sortase B can be a catalytically active polypeptide having at least 70% sequence identity with SEQ ID NO: 32 or 34. In one embodiment, sortase B can be a catalytically active polypeptide having at least 80% or 90% sequence identity with SEQ ID NO: 32 or 34. Preferably, sortase B may be a catalytically active polypeptide comprising (more preferably consisting of) SEQ ID NO: 32 or 34.

Sortase C can be a catalytically active polypeptide having at least 70% sequence identity with SEQ ID NO: 35. In one embodiment, sortase C can be a catalytically active polypeptide having at least 80% or 90% sequence identity with SEQ ID NO: 35. Preferably, sortase C may be a catalytically active polypeptide comprising (more preferably consisting of) SEQ ID NO: 35.

Sortase D may be a catalytically active polypeptide having at least 70% sequence identity with SEQ ID NO: 36. In one embodiment, sortase D may be a catalytically active polypeptide having at least 80% or 90% sequence identity with SEQ ID NO: 36. Preferably, sortase D may be a catalytically active polypeptide comprising (more preferably consisting of) SEQ ID NO: 36.

The acceptor site for sortase is preferably located at the C-terminus of the polypeptide. The sortase donor site is preferably located at the N-terminus of the polypeptide.

The term "located at the C-terminus", as used in this context, may mean that the C-terminal residue of the acceptor region is up to 50 amino acid residues N-terminal from the C-terminal residue of the polypeptide, for example, that C- the terminal residue of the acceptor site is at a distance of 1-50, preferably 10-40 amino acid residues from the N-terminal side of the C-terminal residue of the polypeptide. In particularly preferred embodiments, the C-terminal residue of the acceptor region may be the C-terminal residue of the polypeptide.

In embodiments where there is one or more residues C-terminal from the acceptor site for sortase of the polypeptide, it is preferred that said one or more residues be removed prior to using the polypeptide in the labeling method described herein.

The term "located at the N-terminus", as used in this context, may mean that the C-terminal residue of the donor site is up to 50 amino acid residues from the C-terminal side of the N-terminal residue of the polypeptide, for example, that N- the end residue of the donor site is at a distance of 1-50, preferably 1-25 amino acid residues from the C-terminal side of the N-terminal residue of the polypeptide. In particularly preferred embodiments, the N-terminal residue of the donor site may be the N-terminal residue of the polypeptide.

In embodiments where one or more residues are present on the N-terminal side of the sortase donor site in the polypeptide, it is preferred that said one or more residues be removed before the polypeptide is used in the labeling method described herein.

In one embodiment, the sortase acceptor or donor site is C-terminal to the TM polypeptide. In one embodiment, the sortase acceptor or donor site is N-terminal to a non-cytotoxic protease or a proteolytically inactive mutant thereof.

In one embodiment, the polypeptide of the invention comprises at least two sortase acceptor sites, at least two sortase donor sites, or at least one sortase acceptor site and at least one sortase donor site. Preferably, the polypeptide of the invention contains one sortase acceptor site and one sortase donor site. When labeled in the method of the invention, polypeptides containing at least two (preferably two) sites, as described herein, contain at least two (preferably two) detectable labels. For such polypeptides, at least two sites are preferably different, for example, one site may be a donor site and one may be an acceptor site, or alternatively, when at least two sites are the same (for example, both donor sites or both acceptor sites ), preferably the regions have different amino acid sequences. This allows different sortases to be used to mediate labeling, such as sortases that recognize different acceptor sites.

In one embodiment, the polypeptide of the invention comprises a sortase acceptor site located C-terminally from the TM polypeptide and a sortase donor site located N-terminally from a non-cytotoxic protease or a proteolytically inactive mutant thereof (preferably a non-cytotoxic protease).

In one embodiment, a method for labeling a polypeptide comprises a two step labeling process. In one embodiment, one of the steps includes using a sortase that recognizes a first sortase acceptor site in the polypeptide or labeled substrate, and a second step includes using a different sortase that recognizes a different acceptor site for the polypeptide or labeled substrate. One skilled in the art will appreciate that if more than two different sortase sites are used, the method may include more than two labeling steps and may use two different sortases, where each sortase recognizes one of the different sortase acceptor sites.

Preferably the polypeptide contains an acceptor site containing (or consisting of) LPXSG, and a donor site containing (or consisting of) G _n where n is 2-5. In a particularly preferred embodiment, the polypeptide contains an acceptor site containing (or consisting of) LPESG, and a donor site containing (or consisting of) G ₃ .

In one embodiment, the method of the invention comprises:

a. obtaining a polypeptide containing an acceptor site for sortase and a donor site for sortase;

b. incubation of the polypeptide with:

a first sortase that recognizes an acceptor site for sortase; And

a first labeled substrate containing a sortase donor site and a conjugated detectable label;

where the first sortase catalyzes the connection of the amino acid of the acceptor site for the sortase and the amino acid of the donor site for the sortase, thereby labeling the polypeptide;

c. further incubation of the polypeptide with:

a second labeled substrate comprising a different sortase acceptor site and a conjugated detectable label, wherein the sortase acceptor site is different from the sortase acceptor site in the polypeptide; And

a second sortase that recognizes a different sortase acceptor site (and preferably does not recognize a sortase acceptor site in the polypeptide);

where the second sortase catalyzes the connection between the amino acid of another acceptor site for sortase and the amino acid of the donor site for sortase, thereby further labeling the polypeptide; And

d. obtaining a labeled polypeptide.

One skilled in the art will appreciate that the order of steps b. and c. the above method can be any.

In another embodiment, the method of the invention comprises:

a. obtaining a polypeptide containing a first acceptor site for sortase and a second acceptor site for sortase, where the first and second acceptor sites for sortase differ;

b. incubation of the polypeptide with:

a first sortase that recognizes a first sortase acceptor site (and preferably does not recognize a second sortase acceptor site); And

a labeled substrate containing a sortase donor site and a conjugated detectable label;

where the first sortase catalyzes the connection of the amino acid of the first acceptor site for sortase and the donor site amino acid for sortase, thereby carrying out labeling of the polypeptide;

c. further incubation of the polypeptide with:

a second sortase that recognizes the second sortase acceptor site (and preferably does not recognize the first sortase acceptor site); And

a labeled substrate containing a sortase donor site and a conjugated detectable label;

where the second sortase catalyzes the connection of the amino acid of the second acceptor site for sortase with the amino acid donor site for sortase, thereby further labeling the polypeptide; And

d. obtaining a labeled polypeptide.

One skilled in the art will appreciate that the order of steps b. and c. the above method can be any.

At stage c. the labeled substrate preferably contains a different detectable label from the labeled substrate of step b., for example different colored fluorophores.

In another embodiment, the method of the invention comprises:

a. obtaining a polypeptide comprising a first sortase donor site and a second sortase donor site;

b. incubation of the polypeptide with:

a first labeled substrate containing a first sortase acceptor site and a conjugated detectable label; And

a first sortase that recognizes a first sortase acceptor site (and preferably does not recognize a second sortase acceptor site);

where the first sortase catalyzes the connection of the amino acid of the first acceptor site for sortase with the amino acid of the first or second donor site for sortase, thereby implementing labeling of the polypeptide;

c. further incubation of the polypeptide with:

a second labeled substrate comprising a second sortase acceptor site and a conjugated detectable label, wherein the second sortase acceptor site is different from the first sortase acceptor site; And

a second sortase that recognizes the second sortase acceptor site (and does not recognize the first sortase acceptor site); And

where the second sortase catalyzes the connection of the amino acid of the second acceptor site for sortase with the amino acid of the first or second donor site for sortase, thereby further labeling the polypeptide; And

d. obtaining a labeled polypeptide.

One skilled in the art will appreciate that the order of steps b. and c. the above method can be any.

At stage c. the labeled substrate preferably contains a different detectable label from the labeled substrate of step b., for example different colored fluorophores.

In a preferred embodiment, the method of the invention comprises:

a. obtaining a polypeptide containing a sortase acceptor site containing LPXSG, where X is any amino acid, and a sortase donor site containing G _n where n is 2-5;

b. incubation of the polypeptide with:

a first sortase that recognizes a sortase acceptor site containing LPXSG (and preferably does not recognize a sortase acceptor site containing LAXTG); And

a first labeled substrate containing a sortase donor site containing G _n where n is 2-10 (preferably 2-5) and a conjugated detectable label;

where the first sortase catalyzes the connection of the amino acid of the acceptor site for the sortase of the polypeptide with the amino acid of the donor site for the sortase of the first labeled substrate, thereby labeling the polypeptide;

c. incubation of the polypeptide with:

a second labeled substrate containing a sortase acceptor site containing LAXTG, where X is any amino acid, and a conjugated detectable label; And

a second sortase that recognizes a sortase acceptor site containing LAXTG (and preferably does not recognize a sortase acceptor site containing LPXSG);

wherein the second sortase catalyzes the association of the amino acid of the sortase acceptor site of the second labeled substrate with the amino acid of the sortase donor site in the polypeptide, thereby further labeling the polypeptide; And

d. obtaining a labeled polypeptide.

One skilled in the art will appreciate that the order of steps b. and c. the above method can be any.

The detectable labels conjugated to the first and second labeled substrates preferably differ, such as different colored fluorophores.

One skilled in the art will appreciate that when more than two detectable labels are intended to be added to a polypeptide, the polypeptide may contain more than two sites (eg, donor or acceptor sites) and that the method may be carried out repeatedly.

The term "does not recognize the sortase acceptor site" (or permutations thereof) may mean that the sortase has lower activity (e.g., cleavage or conjugation) for the polypeptide containing the sortase acceptor site than it does for the sortase polypeptide, which recognizes the specified region. In one embodiment, the term "does not recognize a sortase acceptor site" may mean that sortase has essentially no or no activity (e.g., cleavage or conjugation) on a polypeptide containing that sortase acceptor site, as compared to activity on a sortase polypeptide that recognizes the specified site. In one embodiment, the term "does not recognize the sortase acceptor site" (or permutations thereof) may mean that the sortase has a lower activity (e.g., cleavage or conjugation) on the polypeptide containing the sortase acceptor site, compared to the activity of the specified sortase with respect to a polypeptide containing a sortase acceptor site recognized by sortase. In one embodiment, the term "does not recognize a sortase acceptor site" may mean that the sortase has essentially no or no activity (e.g., cleavage or conjugation) on the polypeptide containing the sortase acceptor site, as compared to the activity of said sortase. with respect to a polypeptide containing an acceptor site for sortase recognized by sortase. A sortase acceptor site recognized by a sortase may be a sortase site known in the art to be recognized by said sortase.

The incubation step of the method of the invention can be carried out under any conditions that allow successful labeling of the polypeptide using sortase. Such conditions can be determined by a person skilled in the art using standard techniques/optimization.

The amounts of polypeptide, sortase, and labeled substrate for use in the incubation step of the method as described herein can be determined by one of skill in the art using standard methods. In one embodiment, the method comprises using an excess of labeled substrate for the polypeptide relative to sortase, and optionally an excess of sortase relative to the polypeptide. In one embodiment, the method comprises using a 1:2:20 mass ratio of polypeptide, sortase, and labeled substrate. In another embodiment, the method includes using a 1:2:20 molar ratio of polypeptide, sortase, and labeled substrate.

The reaction conditions for the process incubation step as described herein can also be determined by one of ordinary skill in the art using standard methods. For example, the reaction can be carried out for at least 2, 4, 6, 8, 10 or 12 hours. Preferably, the reaction can be carried out for at least 10 hours. The reaction can be carried out at 1-40°C, for example 1-37°C. In one embodiment, the reaction can be carried out at 1-10°C, preferably 3-5°C, for example, approximately 4°C. The reaction time can be adjusted depending on the temperature used, for example, lower temperatures may require longer incubation times.

After the incubation step of the method of the invention, any free labeled substrate and/or sortase and/or unlabeled polypeptide can be separated from the labeled polypeptide. In one embodiment, resolution is achieved by a label on the sortase or labeled polypeptide, preferably a label (eg, His tag) on the labeled polypeptide. The label may be present on the labeled polypeptide but not on the unlabeled polypeptide, for example where the label is present on a labeled substrate conjugated to the labeled polypeptide.

In one embodiment, a separation step can be used when the polypeptide contains two or more regions and the method includes two or more incubation/labeling steps. A separation step can be used after each incubation/labeling step.

In one embodiment, the method of the invention includes a first incubation and a second incubation (eg, as detailed herein) where, after the first incubation, a first label is used to separate the labeled polypeptide from the unlabeled polypeptide. Preferably, the first tag is absent from the labeled polypeptide but is present from the unlabeled polypeptide, and the unlabeled polypeptide can be removed by immunodepletion. The first label may be a Strep label. In one embodiment, after the second incubation, a second label is used to separate the doubly labeled polypeptide from any single labeled (or unlabeled) polypeptide. Preferably, the second label is present in the doubly labeled polypeptide but not in the singly labeled (or unlabeled) polypeptide, and the doubly labeled polypeptide can be separated by immunoaffinity chromatography. The second tag may be a His tag.

In embodiments where the sortase labeling polypeptide comprises a sortase donor site, the N-terminus of said site may be protected, for example, by one or more amino acid residues N-terminally therefrom. Advantageously, this may prevent the circularization of a polypeptide further containing an acceptor site for sortase. Said one or more amino acids can be removed by a cleavable site, such as a TEV cleavage site, thereby exposing the N-terminus of said donor site to sortase. Thus, the method of the invention may include the step of deprotecting the N-terminus of the sortase donor, for example by removing one or more amino acids N-terminally therefrom. The deprotection step can be carried out between the first and second incubation steps.

In one embodiment, when a polypeptide of the invention contains a cleavage site (eg, a cleavage site on the N-terminal side of a sortase donor site), said cleavage site can be any cleavage site. In one embodiment, the cleavage site may be a site that is non-native (ie, exogenous) to the clostridial neurotoxin. In some embodiments, the cleavage site is a protease recognition site, or a variant thereof, provided that the variant is cleaved by the appropriate protease. The cleavage site may be one cleaved by enterokinase, factor Xa, tobacco etch virus (TEV), thrombin, PreScission, ADAM17, human airway trypsin-like protease (HAT), elastase, furin, granzyme, or caspase 2, 3, 4, 7, 9, or 10. The cleavage site may comprise a polypeptide sequence having at least 70% sequence identity with any of SEQ ID NOs: 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. In one embodiment, the cleavage site may contain a polypeptide sequence having at least 80% or 90% sequence identity with any of SEQ ID NOs: 84, 85, 86, 87, 88 , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. In another embodiment, the cleavage site comprises (preferably consists of) a non-clostridial cleavage site with a polypeptide sequence represented as any of the SEQs. ID NO: 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. Preferably, the cleavage site comprises (more preferably consists of) a cleavage site TEV presented as SEQ ID NO: 87.

A sortase for use in the present invention may contain a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 14. In one embodiment, a sortase for use in the present invention may contain a polypeptide having at least 80% or 90 % sequence identity with SEQ ID NO: 14. Preferably, sortase for use in the present invention may contain (more preferably consist of) the polypeptide sequence shown as SEQ ID NO: 14.

A sortase for use within the scope of the invention may be encoded by a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 13. In one embodiment, a sortase for use within the scope of the present invention may be encoded by a nucleic acid sequence having at least 80% or 90% sequence identity with SEQ ID NO: 13. Preferably, sortase for use in the present invention may be encoded by a nucleic acid sequence containing (more preferably consisting of) the nucleic acid sequence shown as SEQ ID NO: 13.

A sortase for use in the present invention may contain a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 16. In one embodiment, a sortase for use in the present invention may contain a polypeptide having at least 80% or 90 % sequence identity with SEQ ID NO: 16. Preferably, sortase for use in the present invention may contain (more preferably consist of) the polypeptide sequence shown as SEQ ID NO: 16.

A sortase for use within the scope of the invention may be encoded by a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 15. In one embodiment, a sortase for use within the scope of the present invention may be encoded by a nucleic acid sequence having at least 80% or 90% sequence identity with SEQ ID NO: 15. Preferably, sortase for use in the present invention may be encoded by a nucleic acid sequence containing (more preferably consisting of) the nucleic acid sequence shown as SEQ ID NO: 15.

Sortase A can be a catalytically active polypeptide having at least 70% sequence identity with SEQ ID NO: 31, 33 or 37. In one embodiment, sortase A can be a catalytically active polypeptide having at least 80% or 90% sequence identity with SEQ ID NO: 31, 33, or 37. Preferably, sortase A may be a catalytically active polypeptide comprising (more preferably consisting of) SEQ ID NO: 31, 33, or 37.

The present invention may encompass the use of at least two sortases (more preferably two), for example, wherein said sortases comprise polypeptides having at least 70% sequence identity with SEQ ID NOs: 14 and 16, respectively. In one embodiment, the present invention may encompass the use of at least two sortases, wherein said sortases comprise polypeptides having at least 80% or 90% sequence identity with SEQ ID NOs: 14 and 16, respectively. Preferably, the present invention may involve the use of at least two sortases, wherein said sortases comprise (more preferably consist of) polypeptides having SEQ ID NOs: 14 and 16, respectively.

A labeled substrate for use in methods involving the use of sortase is a sortase substrate, and contains a sortase donor or acceptor site and is conjugated to a detectable label. Where the labeled substrate is intended to label a polypeptide containing a sortase acceptor site, the labeled substrate contains a sortase donor site and vice versa. The labeled substrate may be a peptide or polypeptide, preferably a peptide.

The labeled substrate may contain any of the sortase donor or acceptor sites described herein. The labeled substrate may also contain one or more labels, such as purification labels (eg, a His tag) to facilitate its purification or separation from the labeled polypeptide.

In one embodiment, the labeled substrate contains a sortase donor site. An example of a labeled substrate containing a sortase donor site is given under SEQ ID NO: 29. Thus, in one embodiment, a labeled substrate is provided containing a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 29. Labeled substrate may contain a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 29. Preferably, the labeled substrate contains (more preferably consists of) the polypeptide sequence shown as SEQ ID NO: 29.

In one embodiment, the labeled substrate contains an acceptor site for sortase. An example of a labeled substrate containing an acceptor site for sortase is provided under SEQ ID NO: 30. Thus, in one embodiment, a labeled substrate is provided containing a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 30. Labeled substrate may contain a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 30. Preferably, the labeled substrate contains (more preferably consists of) the polypeptide sequence shown as SEQ ID NO: 30.

The acceptor site for sortase is preferably located at the C-terminus of the labeled substrate. The sortase donor site is preferably located at the N-terminus of the labeled substrate.

The polypeptide of the invention is preferably intended for use as a double chain polypeptide, where the two chains are joined together via a disulfide bond. In such embodiments, the polypeptide may comprise a sortase donor site located at the N-terminus of one of the two polypeptide chains. For example, a double-stranded polypeptide may contain a sortase donor site on the N-terminal side of a non-cytotoxic protease (or a proteolytically inactive mutant thereof) and/or its translocation domain. In embodiments where the sortase donor site is N-terminal to the translocation domain of the polypeptide, the sortase donor site may only be available for use in the method of the invention after the polypeptide has been converted to a double-stranded form (e.g., via proteolytic activation).

The term "located at the C-terminus", as used in this context, may mean that the C-terminal residue of the acceptor site is up to 50 amino acid residues N-terminal from the C-terminal residue of the labeled substrate, for example that C -terminal residue of the acceptor site is located at a distance of 1-50, preferably 10-40 amino acid residues from the N-terminal side of the C-terminal residue of the labeled substrate. In particularly preferred embodiments, the C-terminal residue of the acceptor site may be the C-terminal residue of a labeled substrate.

In embodiments where there is one or more residues C-terminal from the acceptor site for sortase of the labeled substrate, it is preferred that these one or more residues be removed prior to using the labeled substrate in the labeling method described herein.

The term "located at the N-terminus", as used in this context, may mean that the C-terminal residue of the donor site is up to 50 amino acid residues on the C-terminal side of the N-terminal residue of the labeled substrate, for example, that N -terminal residue of the donor site is located at a distance of 1-50, preferably 1-25 amino acid residues from the C-terminal side of the N-terminal residue of the labeled substrate. In particularly preferred embodiments, the N-terminal residue of the donor site may be the N-terminal residue of a labeled substrate.

In embodiments where there is one or more residues N-terminal from the sortase donor site in the labeled substrate, it is preferred that these one or more residues be removed prior to using the labeled substrate in the labeling method described herein.

Through validation and proof of principle data, the present inventors have demonstrated that any labeling method similar to sortase-mediated labeling can be used within the scope of the present invention without adversely affecting the potency (e.g., binding, translocation, and/or catalytic activity) of the polypeptide of the invention. . Thus, the present invention encompasses the use of alternative enzymes that are capable of conjugating a labeled polypeptide to a polypeptide of the invention. They can be used in place of or in addition to sortase (preferably in addition, for example when labeling is carried out in an additional site). Enzymes that are also applicable within the scope of the present invention may include alternative transpeptidases or ligases. Thus, the embodiments described herein with respect to sortases can be applied to alternative transpeptidases or ligases.

In one embodiment, the present invention may cover the use of a ligase such as butelase 1 (or a variant thereof), which is a ligase derived from plants of the species Clitoria ternatea and is described in Nguyen, G.K., Y. Cao, W. Wang, C.F. Liu and J.P. Tam (2015). "Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide." Angew Chem Int Ed Engl 54(52): 15694-15698 and Nguyen et al. (2016), Nature Protocols, 11, 10, 1977-1988, which are incorporated herein by reference. When the invention involves the use of a transpeptidase or ligase in an alternative sortase, the labeled substrate is the substrate of said transpeptidase or ligase, respectively.

In embodiments where butelase 1 is used, the polypeptide contains an acceptor or donor site for butelase 1, and a labeled substrate containing a donor or acceptor site for butelase 1 and a conjugated detectable label is used. Similar to methods involving the use of sortase, where the polypeptide contains an acceptor site for butelase, a labeled substrate containing a conjugated detectable label contains a donor site for butelase (and vice versa). In such embodiments, the labeled substrate is a butelase (eg, butelase 1) substrate.

Butelase cleaves between Asn/Asp and His the C-terminal consensus sequence Asn/Asp-His-Val, and it can ligate a polypeptide containing the N-terminal amino acid sequence Xaa-(Ile/Leu/Val/Cys), where Xaa is any amino acid other than proline to form a bond between Asn/Asp-Xaa-(Ile/Leu/Val/Cys). In one embodiment, the butelase acceptor site contains (or consists of) Asn/Asp-His-Val. In one embodiment, the butelase donor site contains (or consists of) Xaa-(Ile/Leu/Val/Cys), where Xaa is any amino acid other than proline.

In the context of butelase sites, Xaa can be selected from (for example) standard amino acids: aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine, serine, threonine, tyrosine, methionine, tryptophan, cysteine, alanine, glycine, valine, leucine , isoleucine and phenylalanine.

Thus, a method for producing a labeled polypeptide is provided, the method comprising:

a. obtaining a polypeptide containing:

i. an acceptor or donor site for butelase;

ii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iv. translocation domain;

b. incubation of the polypeptide with:

butelase (for example, butelase 1); And

a labeled substrate containing a butelase donor or acceptor site and a conjugated detectable label;

where butelase catalyzes the conjugation between the amino acid of the butelase acceptor site and the amino acid of the butelase donor site, thereby labeling the polypeptide; And

c. obtaining a labeled polypeptide.

In another aspect, the invention relates to a polypeptide for labeling with butelase, containing:

an acceptor or donor site for butelase;

a non-cytotoxic protease that is capable of cleaving a protein of the exocytic fusion apparatus in the target cell, or a proteolytically inactive mutant thereof;

a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

a translocation domain that is capable of translocating a non-cytotoxic protease from the endosome across the endosomal membrane and into the cytosol of the target cell;

where, when the polypeptide contains a butelase donor site, the butelase donor site is at the N-terminus of the polypeptide; And

where the N-terminal residue of the donor site is the N-terminal residue of the polypeptide; or

wherein the polypeptide comprises one or more amino acid residues N-terminally from the butelase donor site and a cleavage site which, when cleaved, exposes the N-terminus of the butelase donor site.

The invention also relates to a labeled polypeptide, wherein the polypeptide contains:

i. a detectable label conjugated to the polypeptide;

ii. an amino acid sequence that contains Asn/Asp-Xaa-(Ile/Leu/Val/Cys), where Xaa is any amino acid other than proline;

iii. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

iv. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

v. translocation domain.

Thus, a labeled polypeptide may contain a detectable label conjugated to or near an amino acid sequence that contains (or consists of) Asn/Asp-Xaa-(Ile/Leu/Val/Cys), where Xaa is any amino acid other than proline.

In one embodiment, a transpeptidase or ligase, such as butelase 1, is used in combination with sortase to produce a polypeptide having two or more tags. Thus, in one embodiment, a polypeptide of the invention may comprise at least one sortase acceptor or donor site as described herein and at least one butelase (eg, butelase 1) acceptor or donor site.

Butelase 1 may be a catalytically active polypeptide containing a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 27 or 28 (preferably SEQ ID NO: 28). In one embodiment, butelase 1 may contain a polypeptide sequence having at least 80%, 90% or 95% sequence identity with SEQ ID NO: 27 or 28 (preferably SEQ ID NO: 28). Preferably, butelase 1 may comprise (more preferably consist of) the polypeptide sequence shown as SEQ ID NO: 27 or 28 (preferably SEQ ID NO: 28).

Other ligases may include PATG (SEQ ID NO: 41), PCY1 (SEQ ID NO: 42), POPB (SEQ ID NO: 43), or the butelase homologue OaAEP1b SEQ ID NO: 44 and 45) (Harris et al (2015), Nat Commun, 6, 10199). When said ligases have a signal peptide or other N-terminal leader, said signal peptide or leader is preferably removed prior to use in the present invention.

POPB, as well as suitable methods of using it, are described in the art. For example, they are described in Luo H (2014), Chemistry and Biology 21: 1610-1617, which is incorporated herein by reference.

Thus, a ligase for use in the present invention may contain a polypeptide sequence having at least 70% sequence identity with any of SEQ ID NOs: 41-44. In one embodiment, the ligase may contain a polypeptide sequence having at least 80%, 90% or 95% sequence identity with any of SEQ ID NOs: 41-44. Preferably, the ligase may contain (more preferably consist of) the polypeptide sequence shown as any of SEQ ID NOs: 41-44.

The present invention encompasses the use of any suitable detectable label known to a person skilled in the art. A detectable label may be a label that can be detected visually due to the optical properties of the label. The detection of such a label can be carried out using fluorescence methods, for example, fluorescence microscopy. Thus, in a particularly preferred embodiment, the detectable label is a fluorophore. Preferably, the detectable label is (or contains) a fluorescent dye, such as the fluorescent dyes HiLyte (commercially available from AnaSpec), AlexaFluor (commercially available from Thermo Fisher), Atto (commercially available from Sigma-Aldrich), Quantum Dots, commercially available from Sigma-Aldrich), Janelia Fluor dyes (available from Janelia, US), among others. In a preferred embodiment, the detectable label does not contain a polysaccharide and/or a polyhydric alcohol and/or a bacterial or viral polymer (eg a polysaccharide or a polypeptide).

In one aspect, the invention also relates to a method for analyzing a polypeptide of the present invention, the method comprising:

a. bringing the target cell into contact with the labeled polypeptide of the invention; And

b. detecting a detectable label.

Such methods can be carried out in vitro or in vivo (eg, in a mammal, such as a non-human mammal, eg, a mouse). Preferably, the methods are carried out in vitro. When performed in vivo, the method may include removing a tissue sample for ex vivo analysis.

The methods of the invention are preferably carried out using living cells/tissues, preferably in real time. Advantageously, these methods allow determination of the binding, movement and translocation of a polypeptide of the invention.

The method may be a pulse-chase experiment or include a pulse step (eg, involving the use of a labeled polypeptide) and a tracking step (eg, not including the use of a labeled polypeptide and optionally including the use of an unlabeled polypeptide).

Detection of a detectable label allows detection of the polypeptide or a portion thereof. For example, when the polypeptide contains a first detectable label conjugated to a non-cytotoxic protease or a proteolytically inactive mutant thereof and a second detectable label conjugated to a translocation or TM domain, the method may include detecting both of said detectable labels.

The method of the invention may include detecting the presence or absence of co-localization of two or more detectable labels. Detection can be performed using any method known to the person skilled in the art (eg, FRET and similar methods). In one embodiment, the method of the invention includes detecting a change in co-localization of two or more detectable labels, eg, over time. In embodiments where the polypeptide comprises a first detectable label conjugated to a non-cytotoxic protease or a proteolytically inactive mutant thereof and a second detectable label conjugated to a translocation domain or TM, detecting a decrease in co-localization of the first and second detectable labels (e.g., over time). time) may allow detection of translocation of a non-cytotoxic protease or its proteolytically inactive mutant from the endosome. The time taken by such a change in co-localization can be used to determine the rate of translocation. Detection of no change (eg, essentially no change) of co-localization may indicate that no translocation has occurred.

The method may include detecting the presence of a first detectable label in the cytosol of the cell and/or a second detectable label in the endosome of the cell, which can be performed by translocation analysis. Similarly, detection of the first and second detectable marks (co-localization) in the endosome may indicate that successful endocytosis of the polypeptide has occurred.

In some embodiments, the implementation of the method according to the invention may include quantitative determination of a detectable label, for example, at a specific position in the cell and/or over a specific period of time. Such a quantification can be carried out by detecting the intensity of a detectable label at a particular position in the cell (eg, over time). Alternatively or additionally, the quantification can be carried out by determining the number or size of agglomerates containing the specified detectable label present to the cell.

In one embodiment, the method of the invention comprises:

i) contacting a target cell with a labeled polypeptide of the invention to be evaluated for release from an endosome, wherein said target cell contains a cell membrane comprising a binding site present on the outer surface of the cell membrane of said cell;

ii) incubating the labeled polypeptide with said target cell, thereby allowing

a) the labeled polypeptide bind and form an associated complex with the binding site present on the target cell, thereby allowing said associated complex to enter the target cell via endocytosis;

b) be formed by one or more endosomes in the specified cell, where one or more endosomes contain a labeled polypeptide; And

c) enter the specified labeled polypeptide in the cytosol of the target cell by passing through the endosomal membrane of one or more endosomes;

iii) removing excess labeled polypeptide that has not bound to binding sites present on the target cells;

iv) detecting, after a certain period of time, the amount of labeled polypeptide present in one or more endosomes, or detecting the amount of labeled polypeptide present in the cytosol of said target cell;

v) comparing the amount of labeled polypeptide detected in step iv) with a control value, where said control value is the amount of labeled polypeptide present in aqueous or multiple endosomes or the amount of labeled polypeptide present in the cytosol prior to step iv);

vi) calculating the amount of release from endosomes for the labeled polypeptide by determining the relative change in the amount of labeled polypeptide that is present in one or more endosomes, or by determining the relative change in the amount of labeled polypeptide present in the cytosol of said target cell.

The target cell may be a eukaryotic cell such as a mammalian cell, for example the target cell described herein.

The incubation step ii) can be carried out for any given period of time, for example, for a period of 5 minutes to 5 days. A typical time period is 1-12 hours, such as 2-10 hours, 4-8 hours, or 6-8 hours. During this period, the target cell (i.e., the outer surface of the cell membrane) may be exposed to the labeled polypeptide (typically an excess of labeled polypeptide) resulting in a "steady state" in which the labeled polypeptide enters and exits intracellular endosomes. at approximately the same speed. This point in time corresponds to the optimal point in time for carrying out steps iii) and/or iv).

Step iii) may involve reducing or eliminating the source of the labeled polypeptide from outside the target cell, thereby reducing (or substantially preventing) the amount of labeled polypeptide entering the cell. This decrease in the amount of labeled polypeptide entering the target cell, in turn, provides a change in the amount of labeled polypeptide entering the endosome, which in turn leads to a change in the amount (or rate) of the labeled polypeptide leaving the endosomes and/or entering the cytosol of the cell -targets. It represents the amount (or rate) of labeled polypeptide leaving endosome structures, which in one embodiment can be the basis of the analysis - the amount (or rate) of labeled polypeptide leaving endosome structures can be measured by the change in the amount of labeled polypeptide present in endosomes and / or by changing the amount of labeled polypeptide present in the cytosol. When measuring the amount of labeled polypeptide present in endosomes, a decrease in the amount of labeled polypeptide present is typically observed. By measuring the amount of labeled polypeptide present in the cytosol, an increase or decrease in the amount of labeled polypeptide present in the cytosol can be observed. As an example, an increase in the amount of labeled polypeptide in the cytosol can be observed when step iii) is started before stationary endosomal transport of the labeled polypeptide is established. Alternatively, a decrease in the amount of labeled polypeptide in the cytosol can be observed when the rate of cellular secretion of the labeled polypeptide from the target cell exceeds the rate of endosomal transport of the labeled polypeptide from endosomes to the cytosol.

The target cells used in the assay may be immobilized on the surface. Cell immobilization can be done as a pre-analysis step (ie, prior to immobilization), or it can be done as part of the assay protocol. Thus, in one embodiment, the cells in the assay are pre-immobilized. Immobilization of target cells can be carried out by any conventional means. As an example, cells are seeded in high density assay plates and allowed to adhere prior to assay. Alternatively, cells are plated on assay dishes and cultured for several days prior to use to obtain a contiguous monolayer. Cell attachment can be enhanced using conventional coatings such as poly-D-lysine coated dishes.

In one embodiment, immobilization of target cells can be performed prior to or during step iii), thereby providing a simple means for separating said cells from cell-free (eg, unbound or exogenous) labeled polypeptide. Alternatively, immobilization can be carried out after step iii), for example, to simplify the detection step iv).

Step iii) may include a filtration step or an affinity ligand step in which cells are separated from excess (eg, unbound or exogenous) labeled polypeptide. Step iii) may include a washing step in which excess (eg, unbound or exogenous) labeled polypeptide is washed out of the target cells, eg using a conventional buffer. An excess labeled polypeptide is meant to mean a labeled polypeptide that is present in the assay medium on the outside of the target cells and that has not yet bound to a binding site present on the surface of the target cells.

Detection of the labeled polypeptide in step iv) is generally carried out shortly after step iii). As an example, a typical time period for step iv) is 5 minutes to 5 hours after step iii). In one embodiment, step iv) is carried out within 15-240 minutes, or 30-180 minutes, or 45-150 minutes after step iii). The detection step iv) can be repeated over several time intervals, for example at intervals of 10 minutes, or 15 minutes, or 30 minutes - this will allow the calculation of the rate of endosomal release.

The detection step iv) can be carried out by any conventional means. The detection of a labeled polypeptide may be based on the intracellular localization of said labeled polypeptide.

In comparison step v) a reference value is used which corresponds to the amount of labeled polypeptide present in the endosomes and/or cytosol prior to detection step iv). The control value is generally determined by the same means/method by which the amount of labeled polypeptide is determined in the detection step iv). The control value generally corresponds to the amount of labeled polypeptide present in the endosomes and/or cytosol at or before step iii). As an example, the control value may correspond to the amount of labeled polypeptide present in the endosomes and/or cytosol during or at the end of step ii) - in one embodiment, the control value corresponds to the amount of labeled polypeptide that is present in the endosomes and/or cytosol after establishing " "stationary" rate of translocation, namely, when the labeled polypeptide enters and leaves intracellular endosomes at approximately the same rate.

In the above embodiments, the term "labeled polypeptide" may also encompass a portion thereof, such as a non-cytotoxic protease domain, a translocation domain, or TM (eg, a translocation domain and TM). The methods may also include detecting two or more labels, such as a label on one part of the polypeptide and a label on the second part of the polypeptide.

In one embodiment, the method of the invention may also include an assay for cleavage of an exocytic fusion apparatus protein (eg, SNARE protein).

The detection of a detectable label can be carried out using any suitable methods known to the person skilled in the art. In one embodiment, microscopy is used to detect a detectable label. Methods for detecting a detectable label may include any suitable light, confocal (preferably in vivo confocal 3D microscopy) imaging technique, super resolution imaging technique, or single molecule imaging technique (e.g., light microscopy, confocal microscopy, super resolution microscopy, or single molecule microscopy). Microscopes such as STED, PALM, STORM and TIRF can be used in the methods of the invention. Such microscopy methods are well developed and have high resolution.

The term "proteolytically inactive mutant" is meant to encompass a non-cytotoxic protease mutant that exhibits significantly reduced cleavage of the proteins of the exocytic fusion apparatus in the target cell compared to its wild form. Preferably, the proteolytically inactive mutant comprises a proteolytically inactive Clostridial neurotoxin L chain. In one embodiment, the proteolytically inactive mutant may comprise the L chain of SEQ ID NO: 38 or 40.

In one embodiment, the "proteolytically inactive mutant" is substantially devoid of non-cytotoxic protease activity, preferably devoid of non-cytotoxic protease activity. The term "substantially no non-cytotoxic protease activity" means that the proteolytically active mutant has less than 5% of the non-cytotoxic protease activity of its wild-type (i.e., proteolytically active) form, e.g., less than 2%, 1%, or preferably 0.1 % of the activity of the non-cytotoxic protease of its wild-type form. Non-cytotoxic protease activity can be determined in vitro by incubating the non-cytotoxic protease mutant with the SNARE protein and comparing the amount of SNARE protein cleaved by the non-cytotoxic protease under test with the amount of SNARE protein cleaved by its non-mutant (i.e., proteolytically active) form under the same conditions. To quantify the cleaved SNARE protein, standard methods such as SDS-PAGE and Western blotting can be used. Suitable in vitro assay methods are described in WO 2019/145577 A1, which is incorporated herein by reference. Alternatively or additionally, you can use the cellular analysis described in the present description.

In one embodiment, the proteolytically inactive mutant may have one or more mutations that inactivate the protease. For example, a proteolytically inactive non-cytotoxic protease mutant may comprise a BoNT/A L chain containing an active site residue mutation such as His223, Glu224, His227, Glu262 and/or Tyr366. The position numbering corresponds to the amino acid positions of SEQ ID NO: 17 and can be established by aligning the polypeptide to SEQ ID NO: 17.

The polypeptide of the invention preferably has one or more activities associated with a clostridial neurotoxin (eg, botulinum neurotoxin). In other words, the polypeptide of the invention may be an active neurotoxin. For example, a polypeptide of the invention may cleave a protein from an exocytic fusion apparatus of a target cell, may be capable of binding to a binding site on a target cell, and/or may have translocation activity. Preferably, the polypeptide of the invention may cleave a protein of the exocytic fusion apparatus in the target cell, may be capable of binding a binding site on the target cell, and may have translocation activity. Thus, preferably the polypeptide is not (and has not been) detoxified. For example, the polypeptide may not be (and might not be) chemically inactivated and/or heat inactivated. In one embodiment, the polypeptide is not (and has not been) contacted with a crosslinker, more preferably the polypeptide is not (and has not been) contacted with formaldehyde.

The polypeptide described herein preferably contains a non-cytotoxic protease that is capable of cleaving the protein of the exocytic fusion apparatus in the target cell.

The targeting portion (TM) of a polypeptide of the invention is preferably capable of binding to a binding site on a target cell that is capable of undergoing endocytosis for incorporation into an endosome in the target cell.

The translocation domain is preferably capable of translocating a non-cytotoxic protease from the endosome across the endosome membrane and into the cytosol of the target cell.

In a preferred embodiment, the non-cytotoxic protease of the polypeptide described herein comprises the L chain of a clostridial neurotoxin. More preferably, the Clostridial neurotoxin L chain is a botulinum neurotoxin L chain.

In a preferred embodiment, the translocation domain of the polypeptide described herein comprises a clostridial neurotoxin translocation domain. More preferably, the clostridial neurotoxin translocation domain is a botulinum neurotoxin translocation domain.

In one embodiment, the polypeptide described herein lacks a functional clostridial neurotoxin H _C domain.

In an alternative embodiment, the polypeptide described herein contains the TM binding domain ( _HC domain) of a clostridial neurotoxin. More preferably, the TM binding domain ( _HC domain) of clostridial neurotoxin is the TM binding domain ( _HC domain) of botulinum neurotoxin.

Thus, in a preferred embodiment, the polypeptide described herein comprises a clostridial neurotoxin L chain, a clostridial neurotoxin translocation domain, and a non-clostridial TM.

An equally preferred alternative embodiment, the polypeptide described herein comprises a clostridial neurotoxin L chain and a clostridial neurotoxin H chain (having a clostridial neurotoxin translocation domain [H _N ] and an _HC domain). In such embodiments, the polypeptide described herein is a clostridial neurotoxin.

More preferably, the polypeptide described herein comprises a botulinum neurotoxin L chain, a botulinum neurotoxin translocation domain, and non-clostridial TM.

An equally preferred alternative embodiment, the polypeptide described herein comprises a botulinum neurotoxin L chain and a botulinum neurotoxin H chain (having a botulinum neurotoxin translocation domain [H _N ] and an _HC domain). In such embodiments, the polypeptide described herein is a botulinum neurotoxin.

Preferably, the polypeptide is a botulinum neurotoxin (BoNT) further comprising an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/S /A/C)G_nwhere X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A_nwhere X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, where X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1 (more preferably L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1). The BoNT may be one or more selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, or BoNT/X. Variants thereof are also contemplated, including a proteolytically inactive non-cytotoxic protease mutant.

Preferably, the polypeptide is a botulinum neurotoxin (BoNT) further comprising an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/S /A/C)G _n where X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A _n where X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, where X is any amino acid (more preferably L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1). The BoNT may be one or more selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, or BoNT/X. Variants thereof are also contemplated, including a proteolytically inactive non-cytotoxic protease mutant.

Alternatively, the polypeptide may be a tetanus neurotoxin (TeNT) further comprising an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/ S/A/C)G_nwhere X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A_nwhere X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, where X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1 (more preferably L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1). Variants containing a proteolytically inactive non-cytotoxic protease mutant are also contemplated.

Alternatively, the polypeptide may be a tetanus neurotoxin (TeNT) further comprising an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/ S/A/C)G _n where X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A _n where X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, where X is any amino acid (more preferably L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1). Variants containing a proteolytically inactive non-cytotoxic protease mutant are also contemplated.

Representative polypeptide sequences for BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X and TeNT are described herein as SEQ ID NOs: 17-25 , respectively. These polypeptide sequences may be modified to include an acceptor or donor site for sortase for use in the present invention.

The polypeptide of the invention may be a polypeptide containing an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/S/A/ C)G_nwhere X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A_nwhere X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, where X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1 (more preferably L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1) and wherein the polypeptide further comprises a polypeptide sequence having at least 70% sequence identity with any of SEQ ID NOs: 17-25. In one embodiment, the polypeptide of the invention may be a polypeptide comprising an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/ S/A/C)G_nwhere X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A_nwhere X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, where X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1 (more preferably L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1) and wherein the polypeptide further comprises a polypeptide sequence having at least 80% or 90% sequence identity with any of SEQ ID NOs: 17-25. Preferably, the polypeptide of the invention may be a polypeptide containing an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/S/A /C)G_nwhere X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A_nwhere X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, where X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1 (more preferably L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1) and wherein the polypeptide further comprises a polypeptide comprising (more preferably consisting of) any of SEQ ID NOs: 17-25.

The polypeptide of the invention may be a polypeptide containing an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/S/A/ C)G _n where X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A _n where X is any amino acid and n is equal to at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, where X is any amino acid (more preferably L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1) and wherein the polypeptide further comprises a polypeptide sequence having at least 70% sequence identity with any of SEQ ID NOs: 17-25. In one embodiment, the polypeptide of the invention may be a polypeptide comprising an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/ S/A/C)G _n where X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A _n where X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, where X is any amino acid (more preferably L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1) and wherein the polypeptide further comprises a polypeptide sequence having at least 80% or 90% sequence identity with any of SEQ ID NOs: 17-25. Preferably, the polypeptide of the invention may be a polypeptide containing an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/S/A /C)G _n where X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A _n where X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, where X is any amino acid (more preferably L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1) and wherein the polypeptide further comprises a polypeptide comprising (more preferably consisting of) any of SEQ ID NOs 17-25.

Alternatively, the polypeptide of the invention may be a polypeptide comprising an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/S/A /C)G_nwhere X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A_nwhere X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, where X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1 (more preferably L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1) and where the polypeptide further comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 38. In one embodiment, the polypeptide of the invention may be a polypeptide, containing an acceptor and/or donor site for sortase and/or a detectable label conjugated to it, and an amino acid sequence that contains L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A_nwhere X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, where X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1 (more preferably L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1) and wherein the polypeptide further comprises a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 38. Preferably, the polypeptide of the invention may be a polypeptide containing an acceptor and/or donor site for sortase and/or a detectable label conjugated to it, and an amino acid sequence that contains L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A_nwhere X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, where X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX where X is any amino acid, NPX₁TX₂, where X₁ is Lys or Gln and X₂ is Asn, Asp or Gly, X₁PX₂X₃G, where X₁is Leu, Ile, Val or Met, X₂is any amino acid and X₃is Ser, Thr or Ala, LPEX₁G, where X₁ is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, where X is any amino acid, LRXTG_n or LPAXG_n, where X is any amino acid and n is at least 1 (more preferably L(A/P/S)X(T/S/A/C)G_nwhere X is any amino acid and n is at least 1) and wherein the polypeptide further comprises a polypeptide comprising (more preferably consisting of) SEQ ID NO: 38.

Alternatively, the polypeptide of the invention may be a polypeptide comprising an acceptor and/or donor site for sortase and/or a detectable label conjugated thereto and an amino acid sequence that contains L(A/P/S)X(T/S/A /C)G _n where X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A _n where X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, where X is any amino acid (more preferably L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1) and wherein the polypeptide further comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 38. In one embodiment, the polypeptide of the invention may be a polypeptide containing an acceptor and/or a sortase donor site and/or a detectable label conjugated thereto, and an amino acid sequence that contains L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A _n , where X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS where X is any amino acid (more preferably L(A/P/S)X(T/S/A/C)G _n where X is any amino acid and n is at least 1) and where the polypeptide is further contains a polypeptide sequence having at least 80% or 90% sequence identity with SEQ ID NO: 38. Preferably, the polypeptide of the invention may be a polypeptide containing an acceptor and/or donor site for sortase and/or a detectable label conjugated to it , and an amino acid sequence that contains L(A/P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1, L(A/P/S) X(T/S/A/C)A _n where X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS where X is any amino acid (more preferably L(A /P/S)X(T/S/A/C)G _n , where X is any amino acid and n is at least 1) and where the polypeptide further comprises a polypeptide comprising (more preferably consisting of) SEQ ID NO: 38 .

The polypeptides described herein (or the nucleotide sequences encoding them) may contain one or more tags (eg, purification tags), such as a His tag or a Strep tag. The present invention is also intended to cover polypeptide sequences (and the nucleotide sequences encoding them) that have been delabeled, for example, prior to their use. The polypeptide may also contain one or more cleavage sites, such as a TEV cleavage site, to facilitate label removal.

The present invention is applicable to many different types of clostridial neurotoxin. Thus, in the context of the present invention, the term "clostridial neurotoxin" encompasses toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H and X), C. tetani (tetanus neurotoxin) , C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F), as well as modified clostridial neurotoxins or derivatives derived from any of the above. The term "clostridial neurotoxin" also encompasses botulinum neurotoxin serotype H. Preferably, the clostridial neurotoxin is not BoNT/C1.

Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex consisting of BoNT itself in complex with a number of accessory proteins. There are currently nine different classes of botulinum neurotoxin, namely botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X, all of which have similar structures and modes of action. Different BoNT serotypes can differ based on inactivation by a specific neutralizing antiserum, with such serotype classification correlated with percent sequence identity at the amino acid level. BoNT proteins of a given serotype are further subdivided into different serotypes based on percent amino acid sequence identity.

BoNTs are absorbed in the gastrointestinal tract and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve endings and prevent the release of their neurotransmitter acetylcholine. BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave synaptobrevin/vesicle-associated protein (VAMP); BoNT/C1, BoNT/A, and BoNT/E cleave a 25 kDa synaptosome-associated protein (SNAP-25); and BoNT/C1 cleaves the syntaxin. BoNT/X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

Tetanus toxin is produced in one serotype of C. tetani. C. butyricum produce BoNT/E while C. baratii produce BoNT/F.

The term "clostridial neurotoxin" is also intended to encompass modified clostridial neurotoxins and their derivatives, including, but not limited to, the neurotoxins described below. The modified clostridial neurotoxin or derivative may contain one or more amino acids that are modified from the native (unmodified) form of the clostridial neurotoxin, or may contain one or more built-in amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin. As an example, a modified clostridial neurotoxin may have modified amino acid sequences in one or more domains relative to a native (unmodified) clostridial neurotoxin sequence. Such modifications may modify the functional aspects of the toxin, such as biological activity or stability. Thus, in one embodiment, the polypeptide of the invention is a modified clostridial neurotoxin, or a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative.

The modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified _HC domain) wherein said modified heavy chain binds to target nerve cells with higher or lower affinity than native (unmodified) clostridial neurotoxin. Such modifications in the _HC domain may include modification of residues in the ganglioside-binding region of the _HC domain or protein-binding (SV2 or synaptotagmin) region that alters binding to the ganglioside receptor and/or protein receptor of the target nerve cell. Examples of such modified clostridial neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are incorporated herein by reference in their entirety.

The modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the light chain, such as modifications in the substrate binding or catalytic domain, which may alter or modify the specificity of the modified L chain for the SNARE protein. Examples of such modified clostridial neurotoxins are described in WO 2010/120766 and US 2011/0318385, both of which are incorporated herein by reference in their entirety.

The modified clostridial neurotoxin may contain one or more modifications that increase or decrease the biological activity and/or biological stability of the modified clostridial neurotoxin. For example, the modified clostridial neurotoxin may comprise a leucine or tyrosine-based motif, wherein said motif increases or decreases the biological activity and/or biological stability of the modified clostridial neurotoxin. Suitable leucine-based motifs include xDxxxLL (SEQ ID NO: 79), xExxxLL (SEQ ID NO: 80), xExxxIL (SEQ ID NO: 81) and xExxxLM (SEQ ID NO: 82) (where x is any amino acid). Suitable tyrosine-based motifs include Y-x-x-Hy (SEQ ID NO: 83) (where Hy is a hydrophobic amino acid). Examples of modified clostridial neurotoxins containing leucine and tyrosine motifs are described in WO 2002/08268, which is incorporated herein by reference in its entirety.

The term "clostridial neurotoxin" is intended to encompass hybrid and chimeric clostridial neurotoxins. The hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or subtype thereof and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment, the hybrid clostridial neurotoxin may comprise an entire light chain from one clostridial neurotoxin subtype and a heavy chain from another clostridial neurotoxin subtype. In another embodiment, the chimeric clostridial neurotoxin may comprise a portion (eg, a binding domain) of the heavy chain of one clostridial neurotoxin subtype, with the other portion of the heavy chain derived from a different clostridial neurotoxin subtype. Similarly or alternatively, the therapeutic element may comprise light chain portions from various clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means to provide therapeutic benefit of such clostridial neurotoxins in patients who are immunologically resistant to a given clostridial neurotoxin subtype, in patients who may have a lower than average concentration of receptors for a given heavy binding chain domain of a clostridial neurotoxin, or in patients who may have a protease-resistant variant of the toxin's membrane or vesicular substrate (eg, SNAP-25, VAMP, and syntaxin). Hybrid and chimeric clostridial neurotoxins are described in US 8071110, which is incorporated herein by reference in its entirety. Thus, in one embodiment, the engineered clostridial neurotoxin of the invention is an engineered hybrid clostridial neurotoxin or an engineered chimeric clostridial neurotoxin.

The term "clostridial neurotoxin" is also intended to cover newly discovered members of the botulinum neurotoxin protein family expressed by non-clostridial microorganisms, such as the Enterococcus encoded toxin which has the highest sequence identity with BoNT/X, the Weissella oryzae encoded toxin referred to as BoNT/Wo (NCBI Ref Seq: WP_027699549.1) which cleaves VAMP2 in W89-W90, Enterococcus faecium encoded toxin (GenBank: OTO22244.1) which cleaves VAMP2 and SNAP25, and Chryseobacterium pipero encoded toxin (NCBI Ref. Seq: WP_034687872.1).

The "bioactive" component of the polypeptides of the present invention is provided by a non-cytotoxic protease. This distinct group of proteases act through the proteolytic cleavage of intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP or syntaxin) - see Gerald K (2002) "Cell and Molecular Biology" ( ^4th edition) John Wiley & Sons , Inc. The abbreviated name SNARE comes from the term "Soluble NSF Attachment Receptor", where NSF stands for N-ethylmaleimide-sensitive factor (N-ethylmaleimide-Sensitive Factor). SNARE proteins are required for the formation of intracellular vesicles and thus for secretion of the molecule via vesicular transport out of the cell. Thus, once delivered to the desired target cell, the non-cytotoxic protease is able to inhibit cellular secretion from the target cell.

Non-cytotoxic proteases are a distinct class of molecules that do not kill cells; instead, they act by inhibiting cellular processes other than protein synthesis. Non-cytotoxic proteases are produced as part of a larger toxin molecule by various plants and various microorganisms such as Clostridium sp. and Neisseria sp.

Clostridial neurotoxins are a large group of non-cytotoxic toxin molecules and they contain two polypeptide chains linked together by a disulfide bond. The two chains are called the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. It is an L chain that has protease function and exhibits high substrate specificity for vesicle and/or plasma membrane associated proteins (SNARE) involved in the exocytic process (eg synaptobrevin, syntaxin or SNAP-25). These substrates are important components of the neurosecretory apparatus.

Neisseria sp., especially N. gonorrhoeae species, and Streptococcus sp., especially S. pneumoniae species, produce functionally similar non-cytotoxic toxin molecules. An example of such a non-cytotoxic protease is the IgA protease (see WO99/58571, which is incorporated herein by reference in its entirety). Thus, the non-cytotoxic protease of the present invention is preferably a clostridial neurotoxin protease or an IgA protease.

Referring further to the targeting portion (TM) component of the present invention, it is this component that binds the polypeptide of the present invention to the target cell.

Thus, the TM of the present invention binds to a receptor on the target cell. As an example, the TM of the present invention can bind to a receptor on a neuronal cell, such as a receptor on a sensory or motor neuron. Alternatively, the TM of the present invention may bind to the EGF receptor. In one embodiment, the target cell is a neuronal cell, such as a motor or sensory neuron. In another embodiment, the target cell is a cell that expresses the EGF receptor. However, one of skill in the art can select a TM peptide to target a selected target cell based on the presence of a binding site (eg, cell surface receptor) for said peptide on the target cell.

In one embodiment, the polypeptide of the invention may comprise a TM comprising one or more of the following peptides: growth hormone releasing factor (GHRH) peptide, somatostatin peptide, cortistatin peptide, ghrelin peptide, bombesin peptide, urotensin peptide, melanin concentrating hormone peptide, KISS-1 peptide, gonadotropin-releasing hormone (GnRH) peptide, or prolactin-releasing peptide. These TMs and polypeptides containing them are described in WO 2009/150469, which is incorporated herein by reference.

In one embodiment, a polypeptide of the invention may comprise a TM comprising one or more of the following peptides: leptin peptide, insulin like growth factor (IGF) peptide, transforming growth factor (TGF) peptide, VIP-glucagon-GRF-secretin superfamily peptide, PACAP peptide , vasoactive intestinal peptide (VIP), orexin peptide, interleukin peptide, nerve growth factor (NGF) peptide, vascular endothelial growth factor (VEGF) peptide, thyroid hormone peptide, estrogen peptide, ErbB peptide, epidermal growth factor (EGF) peptide , EGF-TGF-α chimeric peptide, amphiregulin peptide, betacellulin peptide, epigen peptide, epiregulin peptide, heparin-binding EGF (HB-EGF) peptide, bombesin peptide, urotensin peptide, melanin-concentrating hormone (MCH) peptide, Kisspeptin- 10, Kisspeptin-54 peptide, corticotropin-releasing hormone peptide, urocortin 1 peptide, or urocortin 2 peptide. These TMs and polypeptides containing them are described in WO2009/150470, which is incorporated herein by reference.

In another embodiment, the polypeptide of the invention may comprise a TM containing one or more of the following: thyroid stimulating hormone (TSH); antibodies to the TSH receptor; antibodies to islet-specific monosialoganglioside, GM2-1; insulin, insulin-like growth factor and antibodies to the receptors of both of them; releasing hormone TSH (protirelin) and antibodies to its receptor; releasing hormone FSH/LH (gonadorelin) and antibodies to its receptor; corticotrophin-releasing hormone (CRH) and antibodies to its receptor; and ACTH and antibodies to its receptor. These TMs and polypeptides containing them are described in WO 01/21213, which is incorporated herein by reference.

The polypeptides of the present invention may contain 3 main components: a non-cytotoxic protease or a proteolytically inactive mutant thereof; TM and translocation domain. The core technology associated with making such fusion proteins is also referred to as retargeted toxin technology. For an example, see: WO94/21300; WO96/33273; WO98/07864; WO00/10598; WO01/21213; WO06/059093; WO00/62814; WO00/04926; WO93/15766; WO00/61192 and WO99/58571. All of these publications are incorporated herein by reference.

In more detail, the TM component of the present invention may be fused to either a protease component or a translocation component of the present invention. Said fusion is preferably carried out via a covalent bond, for example either a direct covalent bond or via a spacer/linker molecule. The protease component and the translocation component are preferably linked together via a covalent bond, eg either a direct covalent bond or a spacer/linker molecule. Suitable spacer/linker molecules are well known in the art and typically contain a sequence based on amino acids 5-40, preferably 10-30 amino acid residues in length.

The polypeptides used have a double chain conformation wherein the protease component and the translocation component are linked together, preferably via a disulfide bond.

Thus, the polypeptides and labeled polypeptides of the invention may be in single chain form or double chain form, preferably in double chain form.

The polypeptides of the present invention can be obtained by conventional methods of chemical conjugation, which are well known to the person skilled in the art. By way of example, Hermanson, G.T. (1996), Bioconjugate techniques, Academic Press, and to Wong, S.S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press, Nagy et al., PNAS 95 p1794-99 (1998). Additional detailed methodologies for linking synthetic TMs to the polypeptide of the present invention are given, for example, in EP0257742. The aforementioned publications relating to conjugation are incorporated herein by reference.

Alternatively, polypeptides can be obtained by recombinant production of a single protein from fused polypeptides (see, for example, WO98/07864). This method is based on an in vivo bacterial mechanism whereby native clostridial neurotoxin (i.e. holotoxin) is produced and results in a fusion protein having the following "simplified" structural organization:

NH ₂ - [protease component] - [translocation component] - [TM] - COOH

According to WO98/07864, TM is placed on the C-terminal side of the fusion protein. The fusion protein is then activated by treatment with a protease which cleaves at the site between the protease component and the translocation component. Thus, a double-stranded protein is obtained containing the protease component as a single polypeptide chain, covalently linked (through a disulfide bridge) to another single polypeptide chain containing the translocation component plus TM.

Alternatively, according to WO06/059093, the TM component of the fusion protein is located in the middle of the linear fusion protein sequence between the protease cleavage site and the translocation component. This results in the TM being associated with the translocation domain (ie, as in the case of the native clostridial holotoxin), although in this case the order of the two components is reversed with respect to the native holotoxin. Subsequent cleavage at the protease cleavage site exposes the N-terminal portion of TM and provides a double-stranded polypeptide fusion protein.

The above protease cleavage sequence(s) can be introduced (and/or any native cleavage sequence removed) at the DNA level by conventional methods such as site-directed mutagenesis. Screening to confirm the presence of cleavage sequences can be performed manually or using computer software (eg, MapDraw program from DNASTAR, Inc.). While any protease cleavage site (i.e. clostridial or non-clostridial) may be used, the following are preferred:

Enterokinase (DDDDK↓, SEQ ID NO: 84)

Factor Xa (IEGR↓ / IDGR↓, SEQ ID NOs: 85 and 86)

TEV (tobacco etch virus) (ENLYFQ↓G, SEQ ID NO: 87)

Thrombin (LVPR↓GS, SEQ ID NO: 88)

PreScission (LEVLFQ↓GP, SEQ ID NO: 89).

Additional protease cleavage sites include recognition sequences that are cleaved by a non-cytotoxic protease, such as clostridial neurotoxin. These include SNARE protein recognition sequences (eg, SNAP-25, syntaxin, VAMP) that are cleaved by non-cytotoxic proteases such as clostridial neurotoxins. Specific examples are given in US2007/0166332, which is incorporated herein by reference in its entirety.

Also, the term "protease cleavage site" refers to intein, which is a self-cleaving sequence. The self-splicing reaction is controlled, for example by varying the concentration of the reducing agent present. The above "activation" cleavage sites can also be used as a "destructive" cleavage site (discussed below), if it is intended to be included in the polypeptide of the present invention.

In a preferred embodiment, the fusion protein of the present invention may contain one or more N-terminal and/or C-terminal purification tags. While any label can be used for cleaning, the following are preferred:

His tag (e.g. 6xhistidine), preferably as C-terminal and/or N-terminal tag,

MBP tag (maltose binding protein), preferably as N-terminal tag,

GST tag (glutathione-S-transferase), preferably as an N-terminal tag,

His-MBP tag, preferably as an N-terminal tag,

GST-MBP tag, preferably as N-terminal tag,

Thioredoxin tag, preferably as N-terminal tag,

CBD tag (chitin binding domain), preferably as N-terminal tag.

One or more peptide spacer/linker molecules may be included in the fusion protein. For example, a peptide spacer may be used between the purification label and the rest of the fusion protein molecule.

In one aspect, the invention relates to a method for producing a polypeptide for labeling using sortase, the method comprising:

a. providing a nucleic acid sequence encoding a polypeptide, where the polypeptide contains:

i. a non-cytotoxic protease or a proteolytically inactive mutant thereof;

ii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And

iii. translocation domain; And

b. introducing a sortase acceptor or donor site into said nucleic acid, thereby obtaining a modified nucleic acid that encodes a polypeptide containing a sortase acceptor or donor site.

The introduction of an acceptor or donor site for sortase can be carried out by any modifications/methods known to a person skilled in the art, for example, by substitution, insertion or deletion of sequences encoding amino acid residues in the final polypeptide. As an example, modifications can be made by modifying the nucleic acid sequence using standard molecular cloning techniques, such as site-directed mutagenesis, where short DNA chains (oligonucleotides) encoding the desired amino acid(s) are used to replace the original coding sequence using an enzyme. polymerase, or by inserting/deleting portions of the gene using various enzymes (eg, ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized.

Preferably, the method further comprises expressing the modified nucleic acid in the host cell. More preferably, the method further comprises expressing the modified nucleic acid in a host cell and obtaining an expressed polypeptide. The polypeptide can be activated using the method described in the present description.

The invention also relates to a polypeptide obtainable by the method of the invention.

The term "obtaining" as used in the context of "obtaining a labeled polypeptide" or "obtaining an expressed polypeptide" may mean isolating the polypeptide. The selection can be carried out by any purification methods, such as chromatographic or immunoaffinity methods known to the person skilled in the art.

Nucleic acid for use in production methods can be a nucleic acid encoding a polypeptide described in the present description. For example, such a nucleic acid may encode a polypeptide having at least 70% sequence identity with any of SEQ ID NOs: 6, 8, 17-25, or 38. In one embodiment, the nucleic acid may encode a polypeptide having at least 80% or 90% sequence identity with any of SEQ ID NOs: 6, 8, 17-25 or 38. Preferably, the nucleic acid may encode a polypeptide containing (more preferably consisting of) any of SEQ ID NOs: 6, 8, 17-25 or 38.

A nucleic acid for use in manufacturing methods may be a nucleic acid comprising a nucleic acid sequence having at least 70% sequence identity with any of SEQ ID NO: 5 or 7. In one embodiment, a nucleic acid may be a nucleic acid containing a nucleic acid sequence having at least 80% or 90% sequence identity with any of SEQ ID NO: 5 or 7. Preferably, the nucleic acid may contain (more preferably consist of) SEQ ID NO: 5 or 7.

Thus, the present invention relates to a nucleic acid sequence (eg DNA) sequence (eg modified nucleic acid) encoding a polypeptide according to the invention. Such a nucleic acid may be included in the form of a vector, such as a plasmid, which may optionally include one or more of an origin of replication, a nucleic acid insertion site, a promoter, a terminator, and a ribosome binding site.

The nucleic acid (e.g., modified nucleic acid) of the present invention may comprise a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 1, 3, or 39. In one embodiment, the nucleic acid of the present invention may comprise a nucleic acid sequence of the present invention. acid having at least 80% or 90% sequence identity with SEQ ID NO: 1,3 or 39. Preferably, the nucleic acid of the present invention contains (more preferably consists of) the nucleic acid sequence shown as SEQ ID NO: 1 , 3 or 39.

The nucleic acid (e.g., modified nucleic acid) of the present invention may be a nucleic acid that encodes a polypeptide having at least 70% sequence identity with SEQ ID NO: 2,4, or 40. In one embodiment, the nucleic acid of the present invention may be a nucleic acid that encodes a polypeptide having at least 80% or 90% sequence identity with SEQ ID NO: 2, 4 or 40. Preferably, the nucleic acid of the present invention may be a nucleic acid that encodes a polypeptide containing (more preferably consisting of) SEQ ID NO: 2, 4 or 40.

The present invention also encompasses a host cell containing the nucleic acid or vector of the invention.

The present invention also encompasses a method for expressing a nucleic acid sequence as described above in a host cell, in particular E. coli, or via a baculovirus expression system.

The present invention also relates to a method for activating a polypeptide of the present invention, said method comprising contacting the polypeptide with a protease (eg, FXa) that cleaves the polypeptide at a recognition site (cleavage site, such as the FXa site) located between the non-cytotoxic protease component and a translocation component, thereby converting the polypeptide to a double-stranded polypeptide wherein the non-cytotoxic protease and the translocation components are joined together by a disulfide bond. In a preferred embodiment, the recognition site is not native to a naturally occurring clostridial neurotoxin and/or to a naturally occurring IgA protease.

The polypeptides of the present invention can be further modified to reduce or prevent unwanted side effects associated with distribution to non-target areas. In this embodiment, the polypeptide contains a destructive cleavage site. The destructive cleavage site is distinct from the "activation" site (ie, double strand formation) and is cleaved by a second protease, but not by a non-cytotoxic protease. Moreover, when cleaved in this manner at a destructive cleavage site by a second protease, the polypeptide has reduced potency (eg, reduced ability to bind to the intended target cell, reduced translocation activity, and/or reduced non-cytotoxic protease activity). For completeness, any of the "destructive" cleavage sites of the present invention can separately be used as an "activation" site in the polypeptide of the present invention.

Thus, according to this embodiment, the present invention relates to a polypeptide that can be inactivated and/or destroyed in a controlled manner at a position outside the target region.

In a preferred embodiment, the destructive cleavage site is recognized and cleaved by a second protease (i.e., a destructive protease) selected from a circulating protease (e.g., an extracellular protease such as a serum or coagulation cascade protease), a tissue-associated protease (e.g., a matrix a metalloproteinase (MMP) such as muscle MMP) and an intracellular protease (preferably a protease that is not present in the target cell).

Thus, if in use the polypeptide of the present invention is distributed in a different direction from the intended target cell and/or taken up by a non-target cell, the polypeptide is inactivated by cleavage of the destructive cleavage site (second protease).

In one embodiment, the destructive cleavage site is recognized and cleaved by a second protease that is present in a cell type outside the target region. In this embodiment, the cell outside the target area and the target cell are preferably different types of cells. Alternatively (or additionally), the destructive cleavage site is recognized and cleaved by a second protease that is present outside the target region (eg, away from the target cell). Thus, when destructive cleavage occurs extracellularly, the target cell and the cell outside the target region may be the same or different cell types. In this respect, a target cell and a cell outside the target area may have receptors to which the same polypeptide of the invention binds.

The destructive cleavage site of the present invention provides for the inactivation/destruction of a polypeptide when the polypeptide is in or near a non-target region. Therefore, cleavage at a destructive cleavage site minimizes the effectiveness of the polypeptide (as compared to an identical polypeptide lacking the same destructive cleavage site or having the same destructive site but in uncleaved form). By way of example, reduced efficacy includes: reduced binding (to a mammalian cell receptor) and/or reduced translocation (through the endosomal membrane of a mammalian cell line towards the cytosol) and/or reduced cleavage of the SNARE protein.

When choosing a destructive cleavage site(s) in the context of the present invention, it is preferred that the destructive cleavage site(s) is not a substrate for any proteases that can be used separately to post-translationally modify the polypeptide of the present invention as part of its manufacturing process. In this regard, the non-cytotoxic proteases of the present invention typically utilize the protease activation event (via a separate "activation" protease cleavage site that is structurally different from the destructive cleavage site of the present invention). The purpose of the cleavage activation site is to cleave the peptide bond between the non-cytotoxic protease and the tralocation or binding component in the polypeptide of the present invention, thereby providing an "activated" double-stranded polypeptide, wherein the two components are linked together via a disulfide bond.

Thus, in order to facilitate ensuring that the destructive cleavage site(s) of the polypeptides of the present invention do not adversely affect the "activation" cleavage site and subsequent disulfide bond formation, the first of these is preferably introduced into the polypeptide of the present invention at a position at a distance of at least 20, at least 30, at least 40, at least 50, and more preferably at least 60, at least 70, at least 80 (consecutive) amino acid residues from the "activation" cleavage site.

The destructive cleavage site(s) and the activation cleavage site are preferably exogenous (ie engineered/artificial) relative to the native components of the polypeptide. In other words, said cleavage sites are preferably not integral to the respective native components of the polypeptide. As an example, a protease or translocation component based on the L chain or H chain of BoNT/A (respectively) can be modified in accordance with the present invention to include a cleavage site. However, said cleavage will not occur in the corresponding native L chain or H chain of BoNT. Similarly, when a component of a targeting moiety in a polypeptide is modified to include a protease cleavage site, said cleavage site is not present in the corresponding native sequence of the corresponding targeting moiety.

In a preferred embodiment of the present invention, the destructive cleavage site(s) and the "activation" cleavage site are not cleaved by the same protease. In one embodiment, the two cleavage sites differ from each other in that at least one, more preferably at least two, particularly preferably at least three, and most preferably at least four of the allowed amino acids in the respective recognition sequences are different.

As an example, in the case of a polypeptide chimera containing a factor Xa "activation" site between the clostridial components of the L-chain and _HN , it is preferable to use a destructive cleavage site that is a site other than the factor Xa site that can be inserted anywhere in component(s) of the L-chain, and/or H _N , and/or TM. In this scenario, the polypeptide can be modified by introducing an alternative "activation" site between the L-chain and H _N components (e.g., an enterokinase cleavage site), in which case a separate factor Xa cleavage site can be included somewhere in the polypeptide as a destructive site. splitting area. Alternatively, the existing factor Xa "activation" site between the L chain and H _N components may be retained and an alternative cleavage site, such as a thrombin cleavage site, included as a destructive cleavage site.

In identifying suitable sites in the primary sequence of any of the components of the present invention to include the cleavage site(s), it is preferable to select a primary sequence that closely matches the intended cleavage site to be inserted. Through this, minimal structural changes are made to the polypeptide. By way of example, cleavage sites typically contain at least 3 consecutive amino acid residues. Thus, in a preferred embodiment, a cleavage site is selected that already has (in the correct position(s)) at least one, preferably at least two, amino acid residues that are necessary to introduce a new cleavage site. As an example, in one embodiment, a caspase 3 cleavage site (DMQD) can be introduced. In this regard, a preferred insertion position is identified which already includes a primary sequence selected from, for example, Dxxx, xMxx, xxQx, xxxD, DMxx, DxQx, DxxD, xMQx, xMxD, xxQD, DMQx, xMQD, DxQD and DMxD.

Likewise, it is preferable to introduce cleavage sites into the areas exposed to the surface. In surface-exposed regions, existing loop regions are preferred.

In a preferred embodiment of the present invention, the destructive cleavage site(s) is introduced at one or more of the following positions, which are based on the primary amino acid sequence of BoNT/A. While insertion positions are identified (for convenience) based on BoNT/A, the primary amino acid sequences of alternative protease and/or translocation domains can be easily aligned to the indicated BoNT/A positions.

For the protease component, one or more of the following positions are preferred: 194, 202-214, 237-241, 243-250, 300-304, 323-335, 375-382, 391-400 and 413-423. The above numbering preferably starts from the N-terminus of the protease component of the present invention.

In a preferred embodiment, the destructive cleavage site(s) is at a position more than 8 amino acid residues, preferably more than 10 amino acid residues, more preferably more than 25 amino acid residues, particularly preferably more than 50 amino acid residues from the N-terminus of the protease component. Similarly, in a preferred embodiment, the destructive cleavage site(s) is at a position more than 20 amino acid residues, preferably more than 30 amino acid residues, more preferably more than 40 amino acid residues, particularly preferably more than 50 amino acid residues from the C-terminus of the protease component.

With respect to the translocation component, one or more of the following positions are preferred: 823-834, 845-859. According to the above numbering, position 449 for the N-terminal component of the translocation domain of the present invention is preferably recognized as the start position, and position 871 for the C-terminal component of the translocation domain is recognized as the end position.

In a preferred embodiment, the destructive cleavage site(s) is at a position more than 10 amino acid residues, preferably more than 25 amino acid residues, more preferably more than 40 amino acid residues, particularly preferably more than 50 amino acid residues from the N-terminus of the translocation component. Similarly, in a preferred embodiment, the destructive cleavage site(s) is at a position more than 10 amino acid residues, preferably more than 25 amino acid residues, more preferably more than 40 amino acid residues, particularly preferably more than 50 amino acid residues from the C-terminus of the translocation component.

In a preferred embodiment, the destructive cleavage site(s) is more than 10 amino acid residues, preferably more than 25 amino acid residues, more preferably more than 40 amino acid residues, particularly preferably more than 50 amino acid residues from the N-terminus of the TM component. Similarly, in a preferred embodiment, the destructive cleavage site(s) is at a position more than 10 amino acid residues, preferably more than 25 amino acid residues, more preferably more than 40 amino acid residues, particularly preferably more than 50 amino acid residues from the C-terminus of the TM component.

The polypeptide of the present invention may include one or more (eg, two, three, four, five or more) destructive protease cleavage sites. When more than one destructive cleavage site is included, the cleavage sites may be the same or may be different. In this regard, the use of more than one destructive cleavage site provides improved inactivation outside the target region. Likewise, the use of two or more different destructive cleavage sites provides additional design versatility.

The destructive cleavage site(s) may be incorporated into any of the following polypeptide components: a non-cytotoxic protease component; translocation component; targeting moiety or spacer peptide (if available). In this regard, the destructive cleavage site(s) is chosen to provide a minimal adverse effect on the potency of the polypeptide (e.g., by having a minimal effect on the targeting/binding regions and/or the translocation domain and/or the non-cytotoxic protease domain) while concurrently providing for the instability of the polypeptide. away from its target area/target cell.

Preferred destructive cleavage sites (plus corresponding second proteases) are shown in the table below. The cleavage sites shown are illustrative only and are not intended to limit the present invention.

Second protease Recognition sequence in the destructive cleavage site Permissible variation in recognition sequences
P4-P3-P2-P1-▼-P1'-P2'-P3' P4 P3 P2 P1 P1' P2' P3' Thrombin LVPR▼GS (SEQ ID NO: 88) A, F, G, I, L, T, V or M A, F, G, I, L, T, V, W or A P R Not D or E Not D or E --- Thrombin GR▼G G R G Xa factor IEGR▼ (SEQ ID NO: 85) A, F, G, I, L, T, V or M D or E G R --- --- --- ADAM17 PLAQA▼VRSSS (SEQ ID NO: 90) Trypsin-like human airway protease (HAT) SKGR▼SLIGRV (SEQ ID NO: 91) ACE (peptidyl dipeptidase A) --- --- --- --- Not P Not D or E N/A Elastase (leukocyte) MEA▼VTY (SEQ ID NO: 92) M, R E A, H V, T V, T, H Y --- Furin RXR/KR▼ (SEQ ID NO: 93) R X R or K R Granzim IPPD▼ (SEQ ID NO: 94) I E P D --- --- --- Caspase 1 F, W, Y, L --- H,A,T D Not P, E.D.Q.K or R --- --- Caspase 2 DVAD▼ (SEQ ID NO: 95) D V A D Not P, E.D.Q.K or R --- --- Caspase 3 DMQD▼ (SEQ ID NO: 96) D M Q D Not P, E.D.Q.K or R --- --- Caspase 4 LEVD▼ (SEQ ID NO: 97) L E V D Not P, E.D.Q.K or R --- --- Caspase 5 L or W E H D --- --- --- Caspase 6 V E H or I D Not P, E.D.Q.K or R --- --- Caspase 7 DEVD▼ (SEQ ID NO: 98) D E V D Not P, E.D.Q.K or R --- --- Caspase 8 I or L E T D Not P, E.D.Q.K or R --- --- Caspase 9 LEHD▼ (SEQ ID NO: 99) L E H D --- --- --- Caspase 10 IEHD▼ (SEQ ID NO: 100) I E H D --- --- ---

Matrix metalloproteinases (MMPs) are a preferred group of destructive proteases in the context of the present invention. Within this group, ADAM17 (EC 3.4.24.86, also known as TACE) is preferred and cleaves many membrane-anchored cell surface proteins to shed extracellular domains. Additional preferred MMPs include the adamalysins, serralisins, and astacins.

Another group of preferred destructive proteases is the mammalian blood protease such as thrombin, coagulation factor VIIa, coagulation factor IXa, coagulation factor Xa, coagulation factor XIa, coagulation factor XIIa, kallikrein, protein C, and MBP-associated serine protease.

In one embodiment of the present invention, said destructive cleavage site comprises a recognition sequence having at least 3 or 4, preferably 5 or 6, more preferably 6 or 7, and particularly preferably at least 8 contiguous amino acid residues. In this regard, the longer (in terms of consecutive amino acid residues) is the recognition sequence, the less likely is the non-specific cleavage of the destructive site by an unintended second protease.

Preferably, the destructive cleavage site of the present invention is incorporated into the protease component and/or the targeting portion and/or the translocation component and/or the spacer peptide. Of these four components, the protease component is preferred. Thus, the polypeptide can be rapidly inactivated by direct destruction of the non-cytotoxic protease component and/or the binding component and/or the translocation component.

The polypeptides of the invention may be formulated as part of a pharmaceutical composition containing the polypeptide, together with at least one component selected from a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.

The polypeptides of the present invention can be formulated for oral, parenteral administration, continuous infusion, implantation, inhalation or topical application. Compositions suitable for injection may take the form of solutions, suspensions or emulsions, or dry powders, which are dissolved or suspended in a suitable vehicle before use.

Local delivery means may include an aerosol or other spray (eg, nebulizer). In this regard, the aerosol formulation of the polypeptide allows delivery to the lungs and/or other nasal and/or bronchial or airways.

The preferred route of administration is selected from: systemic (eg iv), laparoscopic and/or localized injection (eg transsphenoidal injection directly into the tumor).

In the case of formulations for injection, it is optional to include a pharmaceutically active agent to facilitate retention or to reduce the release of the polypeptide from the area of administration. One example of such a pharmaceutically active substance is a vasoconstrictor such as adrenaline. Such a composition provides the advantage of increasing the residence time of the polypeptide after administration and thus enhancing and/or enhancing its effect.

Dosage ranges for administering the polypeptides of the present invention are those that produce the desired therapeutic effect. It will be understood that the required dosage range depends on the precise nature of the polypeptide or composition, the route of administration, the nature of the composition, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgment of the attending physician. These dosages can be varied using standard empirical optimization techniques.

Suitable daily dosages (per kg of body weight of the patient) are in the range of 0.0001-1 mg/kg, preferably 0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularly preferably 0.004- 0.5 mg/kg. A single dosage may vary from less than 1 microgram to 30 mg, however, it is usually in the range of 0.01 to 1 mg per dose, which can be administered every day or preferably less often, for example, once a week or every six months.

A particularly preferred dosing regimen is based on 2.5 ng of the polypeptide as a 1X dose. In this regard, preferred dosages are in the range of 1X-100X (ie 2.5-250 ng).

Liquid dosage forms are typically prepared using the polypeptide and a pyrogen-free, sterile carrier. The polypeptide, depending on the carrier and concentration used, may be either dissolved or suspended in the carrier. When preparing solutions, the polypeptide can be dissolved in a vehicle, the solution can be converted to isotonic if necessary by the addition of sodium chloride, and sterilized by filtration through a sterile filter using aseptic methods before filling into sterile vials or ampoules and sealing. Alternatively, if the stability of the solution is sufficient, the solution in its closed containers can be sterilized by autoclaving. Advantageously, additives such as buffering agents, solubilizing agents, stabilizing agents, preservatives or bactericides, suspending agents or emulsifiers, and/or local anesthetics may be dissolved in the carrier.

Dry powders which are dissolved or suspended in a suitable carrier prior to use can be prepared by filling the pre-sterilized ingredients into a sterile container using aseptic technique in a sterile area. Alternatively, the ingredients may be dissolved in suitable containers using an aseptic process in a sterile area. The product is then lyophilized and the containers are sealed under aseptic conditions.

Parenteral suspensions suitable for intramuscular, subcutaneous or intradermal injection are prepared in essentially the same manner, except that the sterile components are suspended in a sterile vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The components may be isolated in a sterile state, or alternatively they may be sterilized after isolation, for example by irradiation with gamma radiation.

Preferably, a suspending agent, such as polyvinylpyrrolidone, is included in the composition(s) to facilitate uniform distribution of the components.

Targeting moiety (TM) means any chemical structure that operably interacts with the binding site to cause physical association between the polypeptide of the invention and the surface of a target cell (generally a mammalian cell, especially a human cell). The term "TM" encompasses any molecule (i.e., a naturally occurring molecule or a chemically/physically modified variant thereof) that is capable of binding to a binding site on a target cell, which is preferably capable of internalizing (e.g., forming endosomes) - also referred to as receptor-mediated endocytosis. TM may have the function of translocation across the endosomal membrane, in which case TM and the translocation domain should not be present as separate components in the agent of the present invention. Throughout the foregoing description, specific TMs have been described. Reference to these TMs is illustrative only, and the present invention encompasses all variants and derivatives thereof that have a basic ability to bind (ie, target) to a binding site on a target cell, preferably where the binding site is capable of internalization.

The TM of the present invention binds (preferably binds specifically) to the target cell in question. The term "specifically binds" preferably means that the TM binds to the target cell with a binding affinity (Ka) of 10 ⁶ M ^-1 or more, preferably 10 ⁷ M ^-1 or more, or 10 ⁸ M ^-1 or more, or 10 ⁹ M ^-1 or more. The TMs of the present invention (when in free form, namely when separated from any protease and/or translocation component) preferably exhibit a binding affinity (IC ₅₀ ) for the target receptor in question in the range of 0.05 -18 nM.

The TM of the present invention is preferably not wheat germ agglutinin (WGA).

The indication of TM in the present description covers its fragments and variants, which retain the ability to bind the target cell in question. By way of example, a variant may have at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 97 or at least 99% amino acid sequence homology with the reference TM - the latter representing any sequence TM specified in this application. Thus, a variant may include one or more amino acid analogs (eg, a non-natural amino acid), or a substituted bond. Also by way of example, the term "fragment" when used in relation to TM means a peptide having at least five, preferably at least ten, more preferably at least twenty, and most preferably at least twenty-five amino acid residues of a reference TM. The term fragment also refers to the above options. Thus, by way of example, a fragment of the present invention may comprise a peptide sequence having at least 7, 10, 14, 17, 20, 25, 28, 29, or 30 amino acids, wherein the peptide sequence shares at least 80% sequence homology. over the corresponding amino acids of the peptide sequence (consecutive) of the reference peptide.

The TM may contain a longer amino acid sequence, such as at least 30 or 35 amino acid residues, or at least 40 or 45 amino acid residues, provided that the TM is capable of binding to the target cell.

Confirmation that TM binds to the selected target cell can be done in a standard manner. For example, a simple radioactive displacement experiment can be used in which tissue or cells corresponding to target cells are exposed to labeled (eg, tritium) TM in the presence of an excess of unlabeled TM. In such an experiment, the relative ratio of non-specific to specific binding can be assessed, thereby allowing confirmation that TM binds to the target cell. Optionally, the assay may include one or more binding antagonists, and the assay may further include detection of decreased TM binding. Examples of this type of experiment can be found in Hulme, E.C. (1990), Receptor-binding studies, a brief outline, pp. 303-311, Receptor biochemistry, A Practical Approach, Ed. E.C. Hulme, Oxford University Press.

In some embodiments, the implementation of the polypeptides of the present invention lacks a functional H _C -domain of clostridial neurotoxin. Thus, these polypeptides are unable to bind rat synaptosomal membranes (via the clostridial _HC component) in binding assays as described in Shone et al. (1985) Euro. J Biochem. 151, 75-82. In a preferred embodiment, the polypeptides preferably lack the last 50 C-terminal amino acids of the clostridial neurotoxin holotoxin. In another embodiment, the polypeptides preferably lack the last 100, preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300 C-terminal amino acid residues of the clostridial neurotoxin holotoxin. Alternatively, _HC binding activity can be abolished/reduced by mutagenesis - as an example, referring to BoNT/A for convenience, modification by mutation of one or two amino acid residues (W1266 to L and Y1267 to F) in the ganglioside binding pocket results in loss of H _{The C} -region of its receptor binding function. Similar mutations can be introduced into non-serotype A clostridial peptide components, such as mutated botulinum toxin B constructs (W1262 on L and Y1263 on F) or botulinum toxin E (W1224 on L and Y1225 on F). Other active site mutations achieve the same elimination of _HC receptor binding activity, for example, Y1267S in botulinum toxin type A and the corresponding highly conserved residue in other clostridial neurotoxins. Details of these and other mutations are described in Rummel et al (2004) (Molecular Microbiol. 51:631-634), which is incorporated herein by reference.

In another embodiment, the polypeptides of the present invention lack a functional clostridial neurotoxin H _C domain and also lack any functionally equivalent TM. Thus, these polypeptides lack the natural clostridial neurotoxin binding function and are unable to bind rat synaptosomal membranes (via the clostridial _HC component or via any functionally equivalent TM) in binding assays as described in Shone et al. (1985) Euro. J Biochem. 151, 75-82.

The _HC peptide of a native clostridial neurotoxin contains approximately 400-440 amino acid residues and consists of two functionally distinct domains of approximately 25 kDa each, namely an N-terminal region (often referred to as a peptide or _HCN domain) and a C-terminal region ( often referred to as a peptide or _HCC domain). This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4:788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. Chem. 268:15, pp. 11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25:90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. microbiol. 51(3), 631-643. Moreover, it is widely known that the C-terminal region (H _CC ), which makes up the C-terminal 160-200 amino acid residues, is responsible for the binding of clostridial neurotoxin to its natural cellular receptors, namely, nerve endings at the neuromuscular junction - this fact is also confirmed by the above publications. Thus, reference throughout this specification to a clostridial heavy chain lacking a functional heavy chain _HC peptide (or domain) such that the heavy chain is unable to bind to cell surface receptors to which the native clostridial neurotoxin binds means that clostridial heavy chain the chain is devoid of a functional H _CC peptide. In other words, the H _CC region of the peptide is either partially or completely removed or otherwise modified (eg, by conventional chemical or proteolytic treatment) to inactivate its native nerve ending binding capacity at the neuromuscular junction.

Thus, in one embodiment, the H _N clostridial peptide of the present invention lacks a portion of the C-terminal peptide region (H _CC ) of the clostridial neurotoxin and thus lacks the _HC binding function of the native clostridial neurotoxin. As an example, in one embodiment, a C-terminally extended clostridial H _N -peptide lacks 40 C-terminal amino acid residues, or 60 C-terminal amino acid residues, or 80 C-terminal amino acid residues, or 100 C-terminal amino acid residues, or 120 C-terminal amino acid residues, or 140 C-terminal amino acid residues, or 150 C-terminal amino acid residues, or 160 C-terminal amino acid residues of the clostridial neurotoxin heavy chain. In another embodiment, the clostridial H _N -peptide of the present invention lacks the entire C-terminal peptide portion (H _CC ) of the clostridial neurotoxin and thus lacks the _HC binding function of the native clostridial neurotoxin. As an example, in one embodiment, the clostridial H _N -peptide lacks 165 C-terminal amino acid residues, or 170 C-terminal 170 amino acid residues, or 175 C-terminal amino acid residues, or 180 C-terminal amino acid residues, or 185 C- terminal amino acid residues, or 190 C-terminal amino acid residues, or 195 C-terminal amino acid residues of the clostridial neurotoxin heavy chain. As a further example, the clostridial H _N -peptide of the present invention lacks a clostridial H _CC reference sequence selected from the group consisting of the following:

Botulinum neurotoxin type A - amino acid residues (Y1111-L1296)

Botulinum neurotoxin type B - amino acid residues (Y1098-E1291)

Botulinum neurotoxin type C - amino acid residues (Y1112-E1291)

Botulinum neurotoxin type D - amino acid residues (Y1099-E1276)

Botulinum neurotoxin type E - amino acid residues (Y1086-K1252)

Botulinum neurotoxin type F - amino acid residues (Y1106-E1274)

Botulinum neurotoxin type G - amino acid residues (Y1106-E1297)

Tetanus neurotoxin - amino acid residues (Y1128-D1315).

The reference sequences identified above should be considered definitive as there may be little variation between subserotypes.

The protease of the present invention encompasses all non-cytotoxic proteases that are capable of cleaving one or more proteins of the exocytic fusion apparatus in eukaryotic cells.

The protease of the present invention is preferably a bacterial protease (or a fragment thereof). More preferably, the bacterial protease is selected from the genera Clostridium or Neisseria/Streptococcus (eg, Clostridial L chain, or Neisseria IgA protease, preferably from N. gonorrhoeae or S. pneumoniae).

The present invention also encompasses non-cytotoxic protease variants (ie, variants of naturally occurring protease molecules), provided that the protease variants still exhibit the desired protease activity. By way of example, a variant may share at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95 or at least 98% amino acid sequence homology with a protease reference sequence. Thus, the term variant includes non-cytotoxic proteases having increased (or decreased) endopeptidase activity - specifically, the present description refers to increased K _cat /K _m of BoNT/A mutants Q161A, E54A and K165L, see Ahmed, SA (2008) Protein J DOI 10.1007/s10930-007-9118-8, which is incorporated by reference. The term "fragment" when used in relation to a protease generally means a peptide having at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 amino acid residues of a reference protease. As with the TM "fragment" component (described above), the protease "fragments" of the present invention encompass fragments of protease variants based on a reference sequence.

The protease of the present invention preferably exhibits serine protease or metalloproteinase activity (eg, endopeptidase activity). The protease is preferably specific for a SNARE protein (eg SNAP-25, synaptobrevin/VAMP or syntaxin).

In particular, the protease domains of neurotoxins may be mentioned, for example the protease domains of bacterial neurotoxins. Thus, the present invention encompasses the use of naturally occurring neurotoxin domains as well as recombinantly produced versions of said naturally occurring neurotoxins.

Exemplary neurotoxins are produced by clostridia, and the term "clostridial neurotoxin" encompasses neurotoxins produced by C. tetani (TeNT) and C. botulinum (BoNT) serotypes A-G, as well as closely related BoNT-like neurotoxins produced by C. baratii and C. butyricum. Throughout the present description, the aforementioned abbreviations are used. For example, the nomenclature BoNT/A indicates that the source of the neurotoxin is BoNT (serotype A). The corresponding nomenclature applies to other BoNT serotypes.

BoNTs are the most potent known toxins with a median lethal dose (LD50) in mice ranging from 0.5 to 5 ng/kg depending on serotype. BoNTs are adsorbed in the gastrointestinal tract and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve endings and prevent the release of their neurotransmitter acetylcholine. BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave synaptobrevin/vesicle-associated membrane protein (VAMP); BoNT/C, BoNT/A, and BoNT/E cleave a 25 kDa synaptosome-associated protein (SNAP-25); and BoNT/C cleaves the syntaxin.

BoNTs share a common structure, being ~150 kDa double-stranded proteins consisting of a ~100 kDa heavy chain (H chain) covalently linked by a single disulfide bond to a ~50 kDa light chain (L chain). The H chain consists of two domains, each with a mass of ~50 kDa. The C-terminal domain (H _C ) is required for high affinity neuronal binding, while the N-terminal domain (H _N ) is thought to be involved in transmembrane translocation. The L chain is a zinc dependent metalloproteinase responsible for cleaving the substrate SNARE protein.

The term L chain fragment means a component of the L chain of a neurotoxin that exhibits metalloproteinase activity and is capable of proteolytically cleaving a vesicle and/or plasma membrane associated protein involved in cellular exocytosis.

Examples of suitable (reference) protease sequences include:

Botulinum neurotoxin type A - amino acid residues (1-448)

Botulinum neurotoxin type B - amino acid residues (1-440)

Botulinum neurotoxin type C - amino acid residues (1-441)

Botulinum neurotoxin type D - amino acid residues (1-445)

Botulinum neurotoxin type E - amino acid residues (1-422)

Botulinum neurotoxin type F - amino acid residues (1-439)

Botulinum neurotoxin type G - amino acid residues (1-441)

Tetanus neurotoxin - amino acid residues (1-457)

IgA protease - amino acid residues (1-959)*

*Pohlner, J. et al. (1987). Nature 325, pp. 458-462, which is incorporated herein by reference.

For the newly identified BoNT/X, the L chain has been described to correspond to its amino acids 1-439, with the L chain boundary potentially varying by about 25 amino acids (eg, 1-414 or 1-464).

The above reference sequence should be considered as a guide as there may be slight variation between subserotypes. As an example, US 2007/0166332 (incorporated herein by reference) cites slightly different clostridial sequences:

Botulinum neurotoxin type A - amino acid residues (M1-K448)

Botulinum neurotoxin type B - amino acid residues (M1-K441)

Botulinum neurotoxin type C - amino acid residues (M1-K449)

Botulinum neurotoxin type D - amino acid residues (M1-R445)

Botulinum neurotoxin type E - amino acid residues (M1-R422)

Botulinum neurotoxin type F - amino acid residues (M1-K439)

Botulinum neurotoxin type G - amino acid residues (M1-K446)

Tetanus neurotoxin - amino acid residues (M1-A457).

Various light chain fragments of clostridial toxins can be used in aspects of the present invention, provided that these light chain fragments can specifically target major components of the neurotransmitter release apparatus and thus participate in the entire cellular machinery by which clostridial toxin proteolytically cleaves substrate. The light chains of clostridial toxins are approximately 420-460 amino acids in length and contain an enzyme domain. The study showed that the entire length of the clostridial toxin is not necessary for enzymatic activity of the enzymatic domain. As a non-limiting example, the first eight amino acids of the BoNT/A light chain are not essential for enzymatic activity. As another non-limiting example, the first eight amino acids of the TeNT light chain are not essential for enzymatic activity. Likewise, the carboxyl end of the light chain is not essential for activity. As a non-limiting example, the last 32 amino acids of the BoNT/A light chain (residues 417-448) are not essential for enzymatic activity. As another non-limiting example, the last 31 amino acids of the TeNT light chain (residues 427-457) are not required for enzymatic activity. Thus, aspects of this embodiment may include clostridial toxin light chains comprising an enzyme domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, at least 425 amino acids, and at least 450 amino acids. Other aspects of this embodiment may include clostridial toxin light chains comprising an enzyme domain having a length of, for example, no more than 350 amino acids, no more than 375 amino acids, no more than 400 amino acids, no more than 425 amino acids, and no more than 450 amino acids.

The non-cytotoxic protease component of the present invention preferably comprises an L chain of serotype BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G or BoNT/X (or a fragment or variant thereof) .

The polypeptides of the present invention, especially their protease component, may be pegylated, which may help improve stability, eg, the duration of action of the protease component. PEGylation is particularly preferred when the protease includes BoNT/A protease B or C ₁ . Pegylation preferably involves attaching a PEG to the N-terminus of the protease moiety. As an example, the N-terminus of the protease may be extended by one or more amino acid residues (eg cysteine), which may be the same or may be different. One or more of these amino acid residues may have their own attached (eg, covalently linked) PEG molecule. An example of this technology is described in WO2007/104567, which is incorporated by reference in its entirety.

A translocation domain is a molecule that allows for the translocation of a protease into a target cell such that functional expression of protease activity occurs in the cytosol of the target cell. The presence of any molecule (eg, protein or peptide) of the desired translocation function of the present invention can be confirmed by any of a number of conventional methods of analysis.

For example, Shone C. (1987) described an in vitro assay method using liposomes loaded with a test molecule. The presence of the desired translocation function is confirmed by the release from liposomes of K ⁺ and/or labeled NAD, which can be easily monitored [see. Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

The following example is provided by Blaustein R. (1987), which describes a simple in vitro assay using planar phospholipid bilayer membranes. The membranes are loaded with the test molecule and the desired translocation function is confirmed by increasing the conductivity across said membranes [cf. Blaustein (1987) F.E.B.S. Letts; vol. 226, no. 1: pp. 115-120].

Additional methodology for providing evaluation of membrane fusion and thus identification of translocation domains suitable for use in the present invention is provided in Methods in Enzymology Vol 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The present invention also encompasses translocation domain variants, preferably as long as the domain variants still exhibit the desired translocation activity. By way of example, a variant may share at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% or at least 98% amino acid sequence homology with the translocation reference domain. The term "fragment" when used in relation to a translocation domain means a peptide having at least 20, preferably at least 40, more preferably at least 80, and most preferably at least 100 amino acid residues of a translocation reference domain. In the case of a clostridial translocation domain, the fragment preferably has at least 100, preferably at least 150, more preferably at least 200, and most preferably at least 250 amino acid residues of the translocation reference domain (eg, H _N -domain). With respect to the TM “fragment” component (described above), translocation “fragments” of the present invention encompass fragments of translocation domain variants based on reference sequences.

The translocation domain is preferably capable of forming ion-permeable pores in lipid membranes under low pH conditions. Preferably, it has been found that only those portions of the protein molecule capable of forming pores in the endosomal membrane can be used.

The translocation domain may be derived from a microbial protein source, in particular a bacterial or viral protein source. Thus, in one embodiment, the translocation domain is a translocation domain of an enzyme, such as a bacterial toxin or a viral protein.

It is well known that certain domains of bacterial toxin molecules are capable of forming such pores. It is also known that certain translocation domains of virally expressed membrane fusion proteins are capable of forming such pores. Such domains may be used within the scope of the present invention.

The translocation domain may be of clostridial origin, such as the H _N domain (or a functional component thereof). H _N denotes a portion or fragment of the H chain of a clostridial neurotoxin approximately equivalent to the N-terminal half of the H chain, or a domain corresponding to a fragment of an intact H chain. The H chain may be deprived of the natural binding function of the H _C component of the H chain. In some embodiments, _HC function can be abolished by deletion of the _HC amino acid sequence (either at the level of DNA synthesis or at the post-synthesis level via nuclease or protease treatment). Alternatively, in some embodiments, the _HC function may be inactivated by chemical or biological treatment. Thus, in some embodiments, the H chain is unable to bind to a binding site on a target cell to which native clostridial neurotoxin (ie, holotoxin) binds.

Examples of suitable (reference) translocation domains include:

Botulinum neurotoxin type A - amino acid residues (449-871)

Botulinum neurotoxin type B - amino acid residues (441-858)

Botulinum neurotoxin type C - amino acid residues (442-866)

Botulinum neurotoxin type D - amino acid residues (446-862)

Botulinum neurotoxin type E - amino acid residues (423-845)

Botulinum neurotoxin type F - amino acid residues (440-864)

Botulinum neurotoxin type G - amino acid residues (442-863)

Tetanus neurotoxin - amino acid residues (458-879).

The reference sequence identified above should be considered as a guide, as there may be slight variation between subserotypes. As an example, US 2007/0166332 (incorporated herein by reference) cites slightly different clostridial sequences:

Botulinum neurotoxin type A - amino acid residues (A449-K871)

Botulinum neurotoxin type B - amino acid residues (A442-S858)

Botulinum neurotoxin type C - amino acid residues (T450-N866)

Botulinum neurotoxin type D - amino acid residues (D446-N862)

Botulinum neurotoxin type E - amino acid residues (K423-K845)

Botulinum neurotoxin type F - amino acid residues (A440-K864)

Botulinum neurotoxin type G - amino acid residues (S447-S863)

Tetanus neurotoxin - amino acid residues (S458-V879).

In the context of the present invention, various H _N regions of a clostridial toxin containing a translocation domain can be used in aspects of the present invention, preferably provided that these active moieties can promote the release of a non-cytotoxic protease (e.g., the clostridial L chain) from intracellular vesicles into the cytoplasm of the target cell and thus participate in the entire cellular mechanism by which the clostridial toxin proteolytically cleaves the substrate. The H _N regions of the heavy chains of clostridial toxins are approximately 410-430 amino acids in length and contain a translocation domain. The study showed that the entire length of the Clostridium toxin heavy chain H _N region is not necessary for translocation domain translocation activity. Thus, aspects of this embodiment may include Clostridial toxin H _N regions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids. Other aspects of this embodiment may include Clostridial toxin H _N regions comprising a translocation domain having a length of, for example, no more than 350 amino acids, no more than 375 amino acids, no more than 400 amino acids, and no more than 425 amino acids.

For further details regarding the genetic basis of toxin production in Clostridium botulinum and C. tetani, see Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term "H _N " encompasses naturally occurring portions of the neurotoxin H _N and modified portions of H _N having amino acid sequences that are not naturally occurring and/or synthetic amino acid residues, preferably as long as the modified portions of H _N still exhibit the aforementioned function. translocations.

Alternatively, the translocation domain may be of non-clostridial origin. Examples of sources of non-clostridial (reference) translocation domains include, but are not limited to, the diphtheria toxin translocation domain [O'Keefe et al., Proc. Natl. Acad. sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51], Pseudomonas type A exotoxin translocation domain [Prior et al. Biochemistry (1992) 31, 3555-3559], anthrax toxin translocation domains [Blanke et al. Proc. Natl. Acad. sci. USA (1996) 93, 8437-8442], various fusogenic or hydrophobic peptides with translocation function [Plank et al. J Biol. Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata et al. (1992) Biochem., 31, pp. 1986-1992]. The translocation domain may correspond to a translocation domain present in a naturally occurring protein, or may include amino acid variation, preferably as long as the variation does not impair the translocation capability of the translocation domain.

Specific examples of viral (reference) translocation domains suitable for use in the present invention include certain translocation domains expressed by viruses of membrane fusion proteins. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation function (ie, membrane fusion and vesiculation) of a number of fusogenic and amphiphilic peptides derived from the N-terminal region of influenza virus hemagglutinin. Other virally expressed membrane fusion proteins known to have desirable translocation activity are the Semliki Forest Virus (SFV) fusogenic peptide translocation domain, the vesicular stomatitis virus (VSV) G glycoprotein translocation domain, the SER virus F protein translocation domain, and the translocation of the envelope glycoprotein of foaming viruses. Viral encoded Aspike proteins are particularly useful in the context of the present invention, eg SFV E1 protein and VSV G protein.

The use of the (reference) translocation domains shown in the table (below) includes the use of their sequence variants. The variant may contain one or more conservative nucleic acid substitutions and/or deletions or insertions of nucleic acids, preferably provided that the variant has the desired translocation function. The variant may also contain one or more amino acid substitutions and/or amino acid deletions or insertions, preferably provided that the variant has the desired translocation function.

Source of translocation domain Amino acid residues Links diphtheria toxin 194-380 Silverman et al., 1994, J. Biol. Chem. 269, 22524-22532
London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain II of pseudomonas exotoxin 405-613 Prior et al., 1992, Biochemistry 31, 3555-3559
Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virus hemagglutinin GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 101), and
Its variants Plank et al., 1994, J. Biol. Chem. 269, 12918-12924
Wagner et al., 1992, PNAS 89, 7934-7938
Murata et al., 1992, Biochemistry 31, 1986-1992 Fusogenic protein of Semliki forest virus Translocation domain Kielian et al., 1996, J Cell Biol. 134(4), 863-872 Glycoprotein G of vesicular stomatitis virus 118-139 Yao et al., 2003, Virology 310(2), 319-332 SER virus F protein Translocation domain Seth et al., 2003, J Virol 77(11) 6520-6527 Foaming virus envelope glycoprotein glycoprotein Translocation domain Picard-Maureau et al., 2003, J Virol. 77(8), 4722-4730

Examples of clostridial neurotoxin H _C domain reference sequences include:

BoNT/A - N872-L1296

BoNT/B - E859-E1291

BoNT/C1 - N867-E1291

BoNT/D - S863-E1276

BoNT/E - R846-K1252

BoNT/F - K865-E1274

BoNT/G - N864-E1297

TeNT - I880-D1315.

For the newly identified BoNT/X, the _HC domain has been described as corresponding to its amino acids 893-1306, with domain boundaries potentially varying by about 25 amino acids (eg, 868-1306 or 918-1306).

The polypeptides of the present invention may also contain a translocation promoting domain. The specified domain facilitates the delivery of non-cytotoxic protease into the cytosol of the target cell and is described, for example, in WO 08/008803 and WO 08/008805, each of which is incorporated herein by reference.

By way of example, suitable translocation promoting domains include an enveloped virus fusogenic peptide domain, e.g., suitable fusogenic peptide domains include an influenza virus fusogenic peptide domain (e.g., a 23 amino acid influenza A virus fusogenic peptide domain), an alphavirus fusogenic peptide domain (e.g., a fusogenic peptide domain). vesiculovirus fusogenic peptide domain (e.g., 21 amino acid vesicular stomatitis virus fusogenic peptide domain), respirovirus fusogenic peptide domain (e.g., 25 amino acid Sendai virus fusogenic peptide domain), morbilivirus fusogenic peptide domain (e.g. , 25 amino acid canine distemper virus fusogenic peptide domain), avulavirus fusogenic peptide domain (e.g., 25 amino acid Newcastle disease virus fusogenic peptide domain), henipavirus fusogenic peptide domain (e.g., 25 amino acid Hendra virus fusogenic peptide domain), fusogenic peptide domain a metapneumovirus (eg, a 25 amino acid human metapneumovirus fusogenic peptide domain) or a spumavirus fusogenic peptide domain, such as a simian foaming virus fusogenic peptide domain; or their fragments or variants.

As a further example, a translocation promoting domain may include the H _CN domain of a clostridial toxin, or a fragment or variant thereof. In more detail, the translocation promoting domain H _CN of the clostridial toxin may be at least 200 amino acids long, at least 225 amino acids long, at least 250 amino acids long, at least 275 amino acids long. Therefore, the translocation promoting domain H _CN of the clostridial toxin is preferably no more than 200 amino acids, no more than 225 amino acids, no more than 250 amino acids, or no more than 275 amino acids. Specific (reference) examples include:

Botulinum neurotoxin type A - amino acid residues (872-1110)

Botulinum neurotoxin type B - amino acid residues (859-1097)

Botulinum neurotoxin type C - amino acid residues (867-1111)

Botulinum neurotoxin type D - amino acid residues (863-1098)

Botulinum neurotoxin type E - amino acid residues (846-1085)

Botulinum neurotoxin type F - amino acid residues (865-1105)

Botulinum neurotoxin type G - amino acid residues (864-1105)

Tetanus neurotoxin - amino acid residues (880-1127).

The above sequence positions may vary somewhat depending on the serotype/subtype, and the following examples of suitable (reference) clostridial toxin H _CN domains include:

Botulinum neurotoxin type A - amino acid residues (874-1110)

Botulinum neurotoxin type B - amino acid residues (861-1097)

Botulinum neurotoxin type C - amino acid residues (869-1111)

Botulinum neurotoxin type D - amino acid residues (865-1098)

Botulinum neurotoxin type E - amino acid residues (848-1085)

Botulinum neurotoxin type F - amino acid residues (867-1105)

Botulinum neurotoxin type G - amino acid residues (866-1105)

Tetanus neurotoxin - amino acid residues (882-1127).

Any of the facilitating domains described above can be combined with any of the previously described translocation domain peptides that are suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain can be combined with a non-clostridial translocation domain peptide or with a clostridial domain translocation peptide. Alternatively, the translocation promoting domain H _CN of a clostridial toxin can be combined with a peptide of a non-clostridial translocation domain. Alternatively, the clostridial toxin H _CN promoting domain can be combined with a clostridial translocation domain peptide, examples of which include:

Botulinum neurotoxin type A - amino acid residues (449-1110)

Botulinum neurotoxin type B - amino acid residues (442-1097)

Botulinum neurotoxin type C - amino acid residues (450-1111)

Botulinum neurotoxin type D - amino acid residues (446-1098)

Botulinum neurotoxin type E - amino acid residues (423-1085)

Botulinum neurotoxin type F - amino acid residues (440-1105)

Botulinum neurotoxin type G - amino acid residues (447-1105)

Tetanus toxin - amino acid residues (458-1127).

It is contemplated that embodiments relating to various methods are equally applicable to other methods, polypeptides, eg, labelable or labeled polypeptides, nucleic acids, and vice versa.

SEQUENCE HOMOLOGY

Any of various alignment methods can be used to determine percent identity, including, but not limited to, global methods, local methods, and hybrid methods such as, for example, segment approach methods. Protocols for determining percent identity are standard techniques and are within the ability of one skilled in the art. Global methods align sequences first to the end of the molecule and determine the best alignment by summing the scores of individual residue pairs and by applying gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, for example, Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22 ) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conservative motifs common to all of the input sequences. Non-limiting methods include, for example, Match-box, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, for example, C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262 (5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize alignment scores using a deletion insertion penalty of 10, a deletion continuation penalty of 1, and a "blosum 62" scoring matrix, Henikoff and Henikoff (ibid.), as shown below (amino acids are indicated by the standard one-letter code).

The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions between the sequences. Thus, % identity can be calculated as the number of identical nucleotides/amino acids per total number of nucleotides/amino acids multiplied by 100. The calculation of % sequence identity also takes into account the number of gaps and the length of each gap that needs to be made to optimize the alignment of two or more sequences. Comparison of sequences and determination of percent identity between two or more sequences can be performed using special mathematical algorithms such as BLAST, which are known to the person skilled in the art.

ALIGNMENT PERFORMANCE FOR IDENTIFICATION OF SEQUENCES

The percent identity is then calculated as follows:

Total number of identical matches

__________________________________________ × 100

[length of the longest sequence plus the number of gaps introduced into the longer sequence to align the two sequences]

Essentially homologous polypeptides are characterized by the presence of one or more amino acid substitutions, deletions or insertions. These changes are preferably minor, ie. are conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding and activity of the polypeptide; small deletions, typically one to about 30 amino acids; and small N- or C-terminal extensions such as an N-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

CONSERVATIVE AMINO ACID SUBSTITUTIONS

Basic: arginine, lysine, histidine

Acidic: glutamic acid, aspartic acid

Polar: glutamine, asparagine

Hydrophobic: leucine, isoleucine, valine

Aromatic: phenylalanine, tryptophan, tyrosine

Small: glycine, alanine, serine, threonine, methionine.

In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can be substituted for the amino acid residues of the polypeptides of the present invention. The amino acid residues of the polypeptide can be replaced by a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and non-natural amino acids. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be used where nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for amino acid synthesis and tRNA aminoacylation are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system containing E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. sci. USA 90:10145-9, 1993). In the second method, translation is carried out in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In a third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). ). A non-naturally occurring amino acid is included in the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring forms by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Proteins Sci. 2:395-403, 1993).

Amino acid residues of the polypeptides of the present invention may be substituted with non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and non-natural amino acids.

Essential amino acids in the polypeptides of the present invention can be identified using techniques known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). The sites of biological interaction can also be determined by physical analysis of the structure, when determined by such methods as nuclear magnetic resonance, crystallography, electrogography or photoaffinity labeling, in conjunction with the mutation of the putative amino acids of the contact sites. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The type of essential amino acids can also be determined by analyzing homologues with related components (eg, translocation components or protease components) of the polypeptides of the present invention.

A variety of amino acid substitutions can be made and tested using known mutagenesis and screening methods such as those described by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86 :2152-6, 1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selecting a functional polypeptide, and then sequencing the mutated polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., US Patent No. 5223409; Huse, WIPO publication WO 92/06204) and mutagenesis, targeting an area (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

A variety of amino acid substitutions can be made and tested using known mutagenesis and screening methods such as those described by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86 :2152-6, 1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selecting a functional polypeptide, and then sequencing the mutated polypeptides to determine the spectrum of valid substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., US Patent No. 5223409; Huse, WIPO publication WO 92/06204) and directed to mutagenesis region (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Unless otherwise defined, all technical and scientific terms used in the present description have the same meaning, which is usually meant by a person skilled in the field to which the present invention relates. In Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) in this area a general vocabulary of many of the terms used in the present description.

The present invention is not limited to the exemplary methods and materials described herein, and any methods and materials similar or equivalent to those described herein may be used to practice or test embodiments of the invention. Numeric ranges include numbers that define the range. Unless otherwise indicated, any nucleic acid sequences are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in N to C orientation, respectively.

The headings given in the present description are not limitations of the various aspects and embodiments of the present invention.

Amino acids are referred to herein using the amino acid name, the three-letter abbreviation, or the one-letter abbreviation. The term "protein" as used herein includes proteins, polypeptides and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. In the present description and claims, conventional one-letter and three-letter codes for amino acid residues can be used. 3-letter amino acid code as determined in accordance with the Joint Commission on Biochemical Nomenclature (JCBN) IUPACIUB. It is also understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

Throughout the present description, other definitions may be given. Before describing the exemplary embodiments in more detail, it should be understood that the present invention is not limited to the specific embodiments described, and as such may vary. It is also understood that the terminology used in the present specification is for the purpose of describing specific embodiments only and is not intended to be limiting as the scope of the present invention is defined by the appended claims.

When a range of values is provided, it is understood that each value within the range up to a tenth of one is also specifically provided, unless the context otherwise requires, between the upper and lower limits of that range. The invention encompasses every smaller range between any specified value or its included value in the specified range and any other specified or included value in this specified range. The upper and lower limits of these smaller ranges can be independently included or excluded from the range, and the invention covers each range where either, neither, or both of these limits are included in the smaller ranges, subject to any particular excluded limit in said range. When the specified range includes one or both of the limits, the invention also includes any or both of these included limits.

It should be noted that, as used within the scope of the invention and in the appended claims, the singular form includes the plural unless the context clearly indicates otherwise. Thus, for example, a reference to "polypeptide" includes a plurality of such candidate agents, and a reference to "polypeptide" includes a reference to one or more polypeptides and their equivalents known to those skilled in the art, and so on.

The publications described in this specification are provided for disclosure only prior to the filing date of this application. Nothing in this specification should be construed as an admission that such publications constitute prior art for the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are described below, by way of example only, with the aid of the accompanying drawings and examples.

Figure 1 is a diagram of a strategy for double labeling polypeptides with ligands. The protein contains a SrtA recognition site at the C-terminus followed by a Strep tag. At the N-terminus, the protein contains a site of glycine residues protected by a TEV cleavage site. Also received a peptide containing a plot of glycine residues associated with the preferred choice of fluorophore, and the second peptide containing the site of recognition of SrtA and 6 His-tag (HT). Two different SrtA enzymes allow site-specific labeling of fluorophores of different colors at the N- and C-termini.

Figure 2 shows the SNAP-25 cleavage analysis for unlabeled singly labeled and doubly labeled polypeptides. A. Cleavage of SNAP-25 in cortical neurons by 3, 10, 30, 100, 300 and 1000 nM unlabeled EGF Ligand polypeptide, TxRed labeled EGF polypeptide, SNAP594 labeled EGF Ligand polypeptide, SrtA singly labeled EGF polypeptide ligand and doubly labeled with SrtA polypeptide with EGF ligand. Ligand-free (ligand-free) polypeptide was used as a control for all concentrations. Polypeptide exposure was performed for 24 hours. B. SNAP-25 cleavage in cortical neurons with 3, 10, 30, 100, 300, and 1000 nM unlabeled nociceptin ligand polypeptide and doubly labeled with SrtA nociceptin polypeptide. Ligand-free (ligand-free) polypeptide was used as a control for all concentrations. The impact of polypeptides was carried out for 24 hours.

Figure 3 shows intravital cofocal imaging of a doubly labeled polypeptide with an EGF ligand. A. Confocal in vivo imaging photograph showing A549 cells treated with an EGF ligand polypeptide labeled with HF555 at the N-terminus and HF488 at the C-terminus. The images (right) are photographs of the boxed area shown in the large image (left) taken at various intervals starting at 0.5 minutes after protein addition. The formation of agglomerates characteristic of this polypeptide can be observed after 3 minutes. B. In vivo confocal imaging photograph showing A549 cells treated with an EGF ligand polypeptide tagged with HF555 at the N-terminus and HF488 at the C-terminus. The images (right) are photographs of the boxed area shown in the large image (left) taken at various intervals starting at 30 minutes after protein addition. The disappearance of agglomerates can be observed after 45 minutes.

4 is a diagram of a doubly labeled full-length proteolytically inactivated BoNT/A1 mutant, referred to as BoNT/A(0). The donor and acceptor sites of sortase and the protocol are the same as in Fig.1.

Figure 5 shows the SDS-PAGE analysis of doubly labeled proteolytically inactivated BoNT/A (BoNT/A(0)), visualized using fluorescent staining (left) and Coomassie staining (right). Lanes 1 and 4 represent the protein molecular weight marker, lanes 2 and 5 represent unreduced doubly labeled BoNT/A(0) and lanes 3 and 6 represent reduced doubly labeled (L-chain below and H-chain above) BoNT/A (0).

6 shows single molecule time-of-flight TIRF microscopy images of singly labeled BoNT/A(0) taken at 5 second intervals. The white arrow shows the movement of a single molecule over time in seconds.

SEQUENCE LIST

When the initial Met amino acid residue or the corresponding initial codon is listed in any of the following SEQ ID NOs, the specified residue/codon is optional. In case of any difference between the sequences described in the description and the sequences in the ST.25 sequence listing, the sequences in the description take precedence.

SEQ ID NO: 1 - Nucleotide sequence of an EGF ligand polypeptide (EGFTM) with SrtA double-labeled sites.

SEQ ID NO: 2 - Polypeptide sequence of an EGF ligand polypeptide (EGF TM ) with SrtA doubly labeled regions.

SEQ ID NO: 3 - Nucleotide sequence of a nociceptin ligand polypeptide (nociceptin TM) with SrtA double-labeled sites

SEQ ID NO: 4 - Polypeptide sequence of a nociceptin ligand (nociceptin TM) polypeptide with SrtA doubly labeled regions

SEQ ID NO: 5 - Nucleotide sequence of polypeptide with EGF ligand (EGF TM)

SEQ ID NO: 6 - Polypeptide sequence of polypeptide with EGF ligand (EGF TM)

SEQ ID NO: 7 - Nucleotide sequence of the nociceptin ligand polypeptide (nociceptin TM)

SEQ ID NO: 8 - Polypeptide sequence of a nociceptin ligand polypeptide (nociceptin TM)

SEQ ID NO: 9 - Nucleotide sequence of GFP-tagged polypeptide with EGF ligand

SEQ ID NO: 10 - Polypeptide sequence of GFP-tagged polypeptide with EGF ligand

SEQ ID NO: 11 - Nucleotide sequence of SNAP-tagged polypeptide with EGF ligand

SEQ ID NO: 12 - Polypeptide sequence of SNAP-tagged polypeptide with EGF ligand

SEQ ID NO: 13 - Sortase A Nucleotide Sequence (LPESG Targeting)

SEQ ID NO: 14 - Sortase A polypeptide sequence (LPESG targeting)

SEQ ID NO: 15 - Sortase A Nucleotide Sequence (LAETG Targeting)

SEQ ID NO: 16 - Sortase A polypeptide sequence (LAETG targeting)

SEQ ID NO: 17 - BoNT/A - UniProt P10845

SEQ ID NO: 18 - BoNT/B - UniProt P10844

SEQ ID NO: 19 - BoNT/C - UniProt P18640

SEQ ID NO: 20 - BoNT/D - UniProt P19321

SEQ ID NO: 21 - BoNT/E - UniProt Q00496

SEQ ID NO: 22 - BoNT/F - UniProt A7GBG3

SEQ ID NO: 23 - BoNT/G - UniProt Q60393

SEQ ID NO: 24 - BoNT/X polypeptide sequence

SEQ ID NO: 25 - TeNT - UniProt P04958

SEQ ID NO: 26 - Polypeptide sequence of labeled EGF TM polypeptide

SEQ ID NO: 27 - C. ternatea 1 butelase polypeptide sequence (plus signal peptide)

SEQ ID NO: 28 - C. ternatea butelase 1 polypeptide sequence (minus signal peptide)

SEQ ID NO: 29 - Peptide with conjugated detectable label and sortase donor site

SEQ ID NO: 30 - Peptide with conjugated detectable label and acceptor site for sortase

SEQ ID NO: 31 - Staphylococcus aureus sortase A polypeptide sequence

SEQ ID NO: 32 - Staphylococcus aureus sortase B polypeptide sequence

SEQ ID NO: 33 - Streptococcus pneumoniae sortase A polypeptide sequence

SEQ ID NO: 34 - Streptococcus pneumoniae sortase B polypeptide sequence

SEQ ID NO: 35 - Streptococcus pneumoniae sortase C polypeptide sequence

SEQ ID NO: 36 - Streptococcus pneumoniae sortase D polypeptide sequence

SEQ ID NO: 37 - Streptococcus pyogenes sortase A polypeptide sequence

SEQ ID NO: 38 - Polypeptide sequence of proteolytically inactive BoNT/A(0) mutant

SEQ ID NO: 39 - Nucleotide sequence of full-length proteolytically inactive BoNT/A(0) mutant with SrtA double-labeled sites

SEQ ID NO: 40 - Polypeptide sequence of full length proteolytically inactive BoNT/A(0) mutant with SrtA double tagged sites

SEQ ID NO: 41 - PATG Prochloron didemni polypeptide sequence

SEQ ID NO: 42 - PCY1 Saponaria vaccaria polypeptide sequence

SEQ ID NO: 43 - POPB Galerina marginata polypeptide sequence

SEQ ID NO: 44 - Oldenlandia affinis OaAEP1b butelase homologue polypeptide sequence (plus signal peptide)

SEQ ID NO: 45 - Oldenlandia affinis OaAEP1b butelase homologue polypeptide sequence (minus signal peptide)

SEQ ID NO: 1 - Nucleotide sequence of EGF ligand polypeptide with SrtA double-labeled sites

TGGCGAATGGGACGCGCCCTGTAGCGGCCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAACAAAATATTAACG CTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGG TCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAAGTTTTATTGTTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCGTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTT TTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTC GCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGTCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCAT ACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCCAACAGTCCCCC GGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGAATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAA ATAATTTTGTTTAACTTTAAAGGAGATACATATgggatccatgGAGAACCTGTATTTCAGGGCGGCGGTGGCAGCGGCGCAGCGGCGGCAGCcctttcgttaacaaacagttcaactataaagacccagttaacggtgttgacattgcttacatcaaaatcccgaacgctggccagatgcagccggtaaaggcattcaaaatccacaacaaa atctgggttacccggaacgtgatacctttactaacccggaagaaggtgacctgaacccgccaccggaagcgaaacaggtgccggtatcttactatgactccacctacctgtctaccgataacgaaaaggacaactacctgaaaggtgttactaaactgttcgagcgtatttactccaccgacctgggccgtatgctgctgactagcatcg ttcgcggtatcccgttctggggcggttctaccatcgataccgaactgaaagtaatcgacactaactgcatcaacgtttattcagccggacggttcctatcgttccgaagaactgaacctggtgatcatcggcccgtctgctgatatcatccagttcgagtgtaagagctttggtcacgaagttctgaacctcacccgtaacggctacggttccactcagta catccgtttctctccggacttcaccttcggttttgaagaatccctggaagtagacacgaacccactgctgggcgctggtaaattcgcaactgatcctgcggttaccctggctcacgaactgattcatgcaggccaccgcctgtacggtatcgccatcaatccgaaccgtgtcttcaaagttaacaccaacgcgtattacgagatgtccggtct ggaagttagcttcgaagaactgcgtacttttggcggtcacgacgctaaattcatcgactctctgcaagaaaacgagttccgtctgtactactataacaagttcaaagatatcgcatccaccctgaacaaagcgaaatccatcgtgggtaccactgcttctctccagtacatgaagaacgtttttaaagaaaaatacctgctcagcgaagacacc tccggcaaattctctgtagacaagttgaaattcgataaactttacaaaatgctgactgaaatttacaccgaagacaacttcgttaagttctttaaagttctgaaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaacatcgtgccgaaagttaactacactatctacgatggtttcaacctgcgtaacaccaacctgg ctgctaattttaacggccagaacacggaaatcaacaacatgaacttcacaaaactgaaaaacttcactggtctgttcgagttttacaagctgctgtgcgtcgacggcatcatcattacctccaaaactaaatctctgatagaaggtagaaacaaagcgctgaacctgcagtgtatcaaggttaacaactgggatttattcttcagcccga gtgaagacaacttcaccaacgacctgaacaaaggtgaagaaatcacctcagatactaacatcgaagcagccgaagaaaacatctcgctggacctgatccagcagtactacctgacctttaatttcgacaacgagccggaaaacatttctatcgaaaacctgagctctgatatcatcggccagctggaactgatgccgaacatcgaacgtttcccaa acggtaaaaagtacgagctggacaaatataccatgttccactacctgcgcgcgcaggaatttgaacacggcaaatcccgtatcgcactgactaactccgttaacgaagctctgctcaacccgtcccgtgtatacaccttcttctctagcgactacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttgggtt gggttgaacagcttgtttatgattttaccgacgagacgtccgaagtatctactaccgacaaaattgcggatatcactatcatcatcccgtacatcggtccggctgaacattggcaacatgctgtacaaagacgacttcgttggcgcactgatcttctccggtgcggtgatcctgctggagttcatcccggaaatcgccatcccgg tactgggcacctttgctctggtttcttacattgcaaacaaggttctgactgtacaaaccatcgacaacgcgctgagcaaacgtaacgaaaaatgggatgaagtttacaaatatatcgtgaccaactggctggctaaggttaatactcagatcgacctcatccgcaaaaaaatgaaagaagcactggaaaaccaggcggaagctaccaa ggcaatcattaactaccagtacaaccagtacaccgaggaagaaaaaaacaacatcaacttcaacatcgacgatctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaacaagttcctgaaccagtgctctgtaagctatctgatgaactccatgatcccgtacggtgttaaacgtctggaggacttcgatgcg tctctgaaagacgccctgctgaaatacatttacgacaaccgtggcactctgatcggtcaggttgatcgtctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagctcagtaaatatgtcgataaccaacgccttttgtccactctagaaggcggTGGCGGTAGCGGTGGCGGTGGCAGCGGCGTGGCGGTAGCGCACT AGacAACAGCGACCCTAAATGCCCACTgAGTCATGAAGGATACTGCCTTAATGATGGTGTTTGTATGTACATAGGAACATTGGACCGTTATGCTTGCAATTGTGTAGTGGGCTATGTCGGGGAAAGGTGTCAATATCGATCTCAAGCTGGCAGAGTTAAGAgggctagaagcaGGCGGCAGCGGCGGCGGCAGCGGCCTGCCCGAAAGCGGTGGCGGATCTGCTTGG TCTCACCCGCAGTTCGAAAAAGGTGGTGGTTCTGGTGGTGGTTCTGGTGGTTCTGCTTGGTCTCACCCGCAGTTCGAAAAAtaatgaAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTT GAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

SEQ ID NO: 2 - Polypeptide sequence of EGF ligand polypeptide with SrtA double-labeled sites

MENLYFQGGGGSGGSGGSPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYG STQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVF KINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININK FLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELRGLEAGGSGGGSGLPESGGGSAWSHPQFEKGGGSGGGSG GSAWSHPQFEK

SEQ ID NO: 3 - Nucleotide sequence of the nociceptin ligand polypeptide with SrtA double-labeled sites

TGGCGAATGGGACGCGCCCTGTAGCGGCCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAACAAAATATTAACG CTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGG TCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAAGTTTTATTGTTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCGTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTT TTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTC GCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGTCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCAT ACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCCAACAGTCCCCC GGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGAATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAA ATAATTTTGTTTAACTTTAAAGGAGATACATatgGAGAACCTGTATTTCAGGGCGGCGGTGGCAGCGGCGGCAGCGGCGCAGCGGCAGCATGcctTTTGTGAACAAACAGTTCAACTATAAGGATCCGGTTAATGGTGTGGATATCGCCTATATCAAAATTCCGAATGCAGGTCAGATGCAGCCGGTTAAAGCCTTTAAAATCCATAACAAAATTTGGGTGATTCCGGA ACGTGATACCTTTACCAATCCGGAAGAAGGTGATCTGAATCCGCCTCCGGAAGCAAAACAGGTTCCGGTTAGCTATTATGATAGCACCTATCTGAGCACCGATAACGAGAAAGATAACTATCTGAAAGGTGTGACCAAACTGTTTGAACGCATTTATAGTACCGATCTGGGTCGTATGCTGCTGACCAGCATTGTTCGTGGTATTCCGTTTTGGGGTGGTAGCACCATTGATA CCGAACTGAAAGTTATTGACACCAACTGCATTAATGTGATTCAGCCGGATGGTAGCTATCGTAGCGAAGAACTGAATCTGGTTATTATTGGTCCGAGCGCAGATATCATTCAGTTTGAATGTAAATCCTTTGGCCACGAAGTTCTGAATCTGACCCGTAATGGTTATGGTAGTACCCAGTATATTCGTTTCAGTCCGGATTTTACCTTTGGCTTTTGAAGAAAGCCTGGA AGTTGATACAAATCCGCTGTTAGGTGCAGGTAAATTTGCAACCGATCCGGCAGTTACCCTGGCACATGAACTGATTCATGCCGGTCATCGTCTGTATGGTATTGCAATTAATCCGAACCGTGTGTTCAAAGTGAATACCAACGCATATTATGAAATGAGCGGTCTGGAAGTGTCATTTGAAGAACTGCGTACCTTTGGTGGTCATGATGCCAAATTTATCGATAGCCTGCAAGAAA ATGAATTTCGCCTGTACTACTATAACAAATTCAAGGATATTGCGAGCACCCTGAATAAAGCCAAAAGCATTGTTGGCACCACCGCAAGCCTGCAGTATATGAAAAATGTGTTTAAAGAAAAAATATCTGCTGAGCGAAGATACCAGCGGTAAATTTAGCGTTGACAAACTGAAATTCGATAAACTGTACAAGATGCTGACCGAGATTTATACCGAAGATAACTTCGTGAAGTTTTTCAAAGTGCTGA ACCGCAAAACCTACCTGAACTTTGATAAAGCCGTGTTCAAAATCAACATCGTGCCGAAAGTGAACTATACCATCTATGATGGTTTTAACCTGCGCAATACCAATCTGGCAGCAAACTTTAATGGTCAGAACACCGAAATCAACAACATGAACTTTACCAAACTGAAGAACTTCACCGGTCTGTTCGAATTTTACAAACTGCTGTGTGTGTGGATGGCATTATTACCAGCAAAACCAAATCCGATGATGA CGATAAATTCGGTGGTTTTACCGGTGCACGTAAAAGCGCACGTAAACGTAAAAATCAGGCACTGGCAGGCGGTGGTGGTAGCGGTGGCGGTGGTTCAGGTGGTGGTGGCTCAGCACTGGTTCTGCAGTGTATTAAAGTTAATAACTGGGACCTGTTTTTAAGCCCGAGCGAGGATAATTTCACCAACGATCTGAACAAAGGCGAAGAAATTACCAGCGATACCAATATT GAAGCAGCCGAAGAAAACATTAGCCTGGATCTGATTCAGCAGTATTATCTGACCTTCAACTTCGATAATGAGCCGGAAAATATCAGCATTGAAAACCTGAGCAGCGATATTTGGCCAGCTGGAACTGATGCCGAATATTGAACGTTTTCCGAACGGCAAAAATACGAGCTGGATAAATACACCATGTTCCATTATCTGCGTGCCCAAGAATTTGAACATGGTAAAAGCCGTATTGCACTGA CCAATAGCGTTAATGAAGCACTGCTGAACCCGAGCCGTGTTTATACCTTTTTTAGCAGCGATTACGTGTGAAAAAGGTTAACAAAGCAACCGAAGCAGCCATGTTTTTAGGTTGGGTTGAACAGCTGGTTTATGATTTCACCGATGAAACCAGCGAAGTTAGCACCACCGATAAAATTGCAGATATTACCATCATCATCCCGTATATCGGTCCGGCACTGAATATTGGCAATATGCTGT ATAAAGACGATTTTGTGGGTGCCCTGATCTTTAGCGGTGCAGTTATTCTGCTGGAATTTATTCCGGAAATTGCCATTCCGGTTCTGGGCACCTTTGCACTGGTGAGCTATATTGCAAATAAAGTTCTGACCGTGCAGACCCATCGATAATGCACTGAGCAAACGTAACGAAAAATGGGATGAAGTGTACAAGTATATCGTGACCAATTGGCTGGCAAAAGTTAACACCCAGATTGA CCTGATTCGCAAGAAGATGAAAGAAGCACTGGAAAACCAGGCAGAAGCAACCAAAGCCATTATTAACTATCAGTACAACCAGTACACCGAAGAAGAGAGAATAACATCAACTTCAACATCGATGATCTGAGCAGCAAGCTGAATGAAAGCATCAACAAAGCCATGATCAACATTAACAAATTTCTGAATCAGTGCAGCGTGAGCTATCTGATGAATAGCATGATTCCGTATGGTGTGAA ACGTCTGGAAGATTTTGATGCAAGCCTGAAAGATGCCCTGCTGAAATATATCTATGATAATCGTGGCACCCTGATTGGTCAGGTTGATCGTCTGAAAGATAAAGTGAACAACACCCTGAGTACCGATATTTCCTTTTCAGCTGAGCAAATATGTGGATAATCAGCGTCTGCTGAGTACCCTGGATGGCGGCAGCGGCGGCGGCAGCGGCCTGCCCGAAAGCGGTGGCGGATCTGCT TGGTCTCACCCGCAGTTCGAAAAAGGTGGTGGTTCTGGTGGTGGTTCTGGTGGTTCTGCTTGGTCTCACCCGCAGTTCGAAAAAtaatgaAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGCCTCTAAACGGG TCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

SEQ ID NO: 4 - Polypeptide sequence of the nociceptin ligand polypeptide with SrtA double-labeled sites

MENLYFQGGGGSGGSGGSGSMPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNG YGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDK AVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKFGGFTGARKSARKRKNQALAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFP NGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEAT KAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLDGGSGGGSGLPESGGGSAWSHPQFEKGGG

SEQ ID NO: 5 - Nucleotide sequence of EGF ligand polypeptide

TGGCGAATGGGACGCGCCCTGTAGCGGCCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAACAAAATATTAACG CTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGG TCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAAGTTTTATTGTTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCGTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTT TTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTC GCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGTCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCAT ACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCCAACAGTCCCCC GGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGAATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAA ATAATTTTGTTTAACTTTAAAGGAGATACATATGggatccatggagttcgttaacaaacagttcaactataaagacccagttaacggtgttgacattgcttacatcaaaatcccgaacgctggccagatgcagccggtaaaggcattcaaaatccacaacaaaatctgggttatcccggaacgtgataccttttactaacccggaagaaggtgacct gaacccgccaccggaagcgaaacaggtgccggtatcttactatgactccacctacctgtctaccgataacgaaaaggacaactacctgaaaggtgttactaaactgttcgagcgtatttactccaccgacctgggccgtatgctgctgactagcatcgttcgcggtatcccgttctggggcggttctaccatcgataccgaactgaaagtaat cgacactaactgcatcaacgtttattcagccggacggttcctatcgttccgaagaactgaacctggtgatcatcggcccgtctgctgatatcatccagttcgagtgtaagagctttggtcacgaagttctgaacctcacccgtaacggctacggttccactcagtacatccgtttctctccggacttcaccttcggttttgaagaatccctggaagtagacacgaac ccactgctgggcgctggtaaattcgcaactgatcctgcggttaccctggctcacgaactgattcatgcaggccaccgcctgtacggtatcgccatcaatccgaaccgtgtcttcaaagttaacaccaacgcgtattacgagatgtccggtctggaagttagcttcgaagaactgcgtacttttggcggtcacgacgctaaat tcatcgactctctgcaagaaaacgagttccgtctgtactactataacaagttcaaagatatcgcatccaccctgaacaaagcgaaatccatcgtgggtaccactgcttctctccagtacatgaagaacgtttttaaagaaaaatacctgctcagcgaagacacctccggcaaattctctgtagacaagttgaaattcgataaactttacaaaatg ctgactgaaatttacaccgaagacaacttcgttaagttctttaaagttctgaaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaacatcgtgccgaaagttaactacactatctacgatggtttcaacctgcgtaacaccaacctggctgctaattttaacggccagaacacggaaatcaacaacatgaacttcacaaaactga aaaacttcactggtctgttcgagttttacaagctgctgtgcgtcgacggcatcattacctccaaaactaaatctctgatagaaggtagaaacaaagcgctgaacctgcagtgtatcaaggttaacaactgggatttattcttcagcccgagtgaagacaacttcaccaacgacctgaacaaaggtgaagaaatcacctcagatact aacatcgaagcagccgaagaaaacatctcgctggacctgatccagcagtactacctgacctttaatttcgacaacgagccggaaaacatttctatcgaaaacctgagctctgatatcatcggccagctggaactgatgccgaacatcgaacgtttcccaaacggtaaaaagtacgagctggacaaatataccatgttccactacctgcgg cgcaggaatttgaacacggcaaatcccgtatcgcactgactaactccgttaacgaagctgctcaacccgtcccgtgtatacaccttcttctctagcgactacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttgggttgggttgaacagcttgtttatgattttaccgacgagacgtccgaagtatctactact accgaca aa caaaccatcgacaacgcgctgagcaaacgtaacgaaaaatgggatgaagtttacaaattatcgtgaccaactggctggctaaggttaatactcagatcgacctcatccgcaaaaaaatgaaagaagcactggaaaaccaggcggaagctaccaaggcaatcattaactaccagtacaaccagtacaccgaggaagaaaaaaacaacatca acttcaacatcgacgatctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaacaagttcctgaaccagtgctctgtaagctatctgatgaactccatgatcccgtacggtgttaaacgtctggaggacttcgatgcgtctctgaaagacgccctgctgaaatacatttacgacaaccgtggcactctgatc ggtcaggttgatcgtctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagctcagtaaatatgtcgataaccaacgccttttgtccactctagaaggcggTGGCGGTAGCGGTGGCGGTGGCAGCGGCGGTGGCGGTAGCGCACTAGacAACAGCGACCCTAAATGCCCACTgAGTCATGAAGGATACTGCCTTAATGATGGTGTT TGTATGTACATAGGAACATTGGACCGTTATGCTTGCAATTGTGTAGTGGGCTATGTCGGGGAAAGGTGTCAATATCGAGATCTCAAGCTGGCAGAGTTAAGAgggctagaagcaCACCATCATCACcaccatcaccatcaccattaatgaAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCT GCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

SEQ ID NO: 6 - Polypeptide sequence of an EGF ligand polypeptide

MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEV DTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLR NTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATE AAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYG VKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELRGLEAHHHHHHHHHH

SEQ ID NO: 7 - Nucleotide sequence of the nociceptin ligand polypeptide

TGGCGAATGGGACGCGCCCTGTAGCGGCCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAACAAAATATTAACG CTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGG TCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAAGTTTTATTGTTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCGTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTT TTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTC GCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGTCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCAT ACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCCAACAGTCCCCC GGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGAATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAA ATAATTTTGTTTAACTTTAAAGGAGATACATATGGGCAGCATGGAATTTGTGAACAAACAGTTCAACTATAAGGATCCGGTTAATGGTGTGGATATCGCCTATATCAAAATTCCGAATGCAGGTCAGATGCAGCCGTTAAAGCCTTTAAAATCCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTTACCAATCCGGAAGAAGGTGATCTGAATCCGCCTCCGGAAGCA AAACAGGTTCCGGTTAGCTATTATGATAGCACCTATCTGAGCACCGATAACGAGAAAGATAACTATCTGAAAGGTGTGACCAAACTGTTTGAACGCATTTATAGTACCGATCTGGGTCGTATGCTGCTGACCAGCATTGTTCGTGGTATTCCGTTTTGGGGTGGTAGCACCATTGATACCGAACTGAAAGTTATTGACACCAACTGCATTAATGTGATTCAGCCGGATGGTAG CTATCGTAGCGAAGAACTGAATCTGGTTATTATTGGTCCGAGCGCAGATATCATTCAGTTTGAATGTAAATCCTTTGGCCACGAAGTTCTGAATCTGACCCGTAATGGTTATGGTAGTACCCAGTATATTCGTTTCAGTCCGGATTTTACCTTTGGCTTTGAAGAAAGCCTGGAAGTTGATACAAATCCGCTGTTAGGTGCAGGTAAATTTGCAACCGATCCGGCAG TTACCCTGGCACATGAACTGATTCATGCCGGTCATCGTCTGTATGGTATTGCAATTAATCCGAACCGTGTGTTCAAAGTGAATACCAACGCATATTATGAAATGAGCGGTCTGGAAGTGTCATTTGAAGAACTGCGTACCTTTGGTGGTCATGATGCCAAATTTATCGATAGCCTGCAAGAAAATGAATTTCGCCTGTACTACTATAACAAATTCAAGGATATTGCGAGCACCCTGAATAAA GCCAAAAGCATTGTTGGCACCACCGCAAGCCTGCAGTATATGAAAAATGTGTTTAAAGAAAATATCTGCTGAGCGAAGATACCAGCGGTAAATTTAGCGTTGACAAACTGAAATTCGATAAACTGTACAAGATGCTGACCGAGATTTATACCGAAGATAACTTCGTGAAGTTTTTCAAAGTGCTGAACCGCAAAACCTACCTGAACTTTGATAAAGCCGTGTTCAAAATCAACATCGTGCCGAA AGTGAACTATACCATCTATGATGGTTTTAACCTGCGCAATACCAATCTGGCAGCAAACTTTAATGGTCAGAACACCGAAATCAACAACATGAACTTTACCAAACTGAAGAACTTCACCGGTCTGTTCGAATTTTACAAACTGCTGTGTGTGGATGGCATTATTACCAGCAAAACCAAATCCGATGATGACGATAAATTCGGTGGTTTTACCGGTGCACGTAAAAGCGCACGTAAACGTAAAAATC AGGCACTGGCAGGCGGTGGTGGTAGCGGTGGCGGTGGTTCAGGTGGTGGTGGCTCAGCACTGGTTCTGCAGTGTATTAAAGTTAATAACTGGGACCTGTTTTTTAGCCCGAGCGAGGATAATTTCACCAACGATCTGAACAAAGGCGAAGAAATTACCAGCGATACCAATATTGAAGCAGCCGAAGAAAACATTAGCCTGGATCTGATTCAGCAGTATTATCTGACCTT CAACTTCGATAATGAGCCGGAAAATATCAGCATTGAAAACCTGAGCAGCGATATTATTGGCCAGCTGGAACTGATGCCGAATATTGAACGTTTTCCGAACGGCAAAAAATACGAGCTGGATAAATACACCATGTTCCATTATCTGCGTGCCCAAGAATTTGAACATGGTAAAAGCCGTATTGCACTGACCAATAGCGTTAATGAAGCACTGCTGAACCCGAGCCGTGTTTATACCTTTTAGCA GCGATTACGTGAAAAAGGTTAACAAAGCAACCGAAGCAGCCATGTTTTTAGGTTGGGTTGAACAGCTGGTTTATGATTTCACCGATGAAACCAGCGAAGTTAGCACCACCGATAAAATTGCAGATATTACCATCATCATCCCGTATATCGGTCCGGCACTGAATATTGGCAATATGCTGTATAAAGACGATTTTGTGGGTGCCCTGAATCTTTAGCGGTGCAGTTATTCTGCTG GAATTTATTCCGGAAATTGCCATTCCGGTTCTGGCACCTTTGCACTGGTGAGCTATATTGCAAATAAAGTTCTGACCGTGCAGACCCATCGATAATGCACTGAGCAAACGTAACGAAAAATGGGATGAAGTGTACAAGTATATCGTGACCAATTGGCTGGCAAAAGTTAACACCCAGATTGACCTGATTCGCAAGAAGATGAAAGAAGCACTGGAAAACCAGGCAGAAGCAACCAAA GCCATTATTAACTATCAGTACAACCAGTACACCGAAGAAGAGAAGAATAACATCAACTTCAACATCGATGATCTGAGCAGCAAGCTGAATGAAAGCATCAACAAAGCCATGATCAACATTAACAAATTTCTGAATCAGTGCAGCGTGAGCTATCTGATGAATAGCATGATTCCGTATGGTGTGAAACGTCTGGAAGATTTTGATGCAAGCCTGAAAGATGCCCTGCTGAAATATATCTATGATA ATCGTGGCACCCTGATTGGTCAGGTTGATCGTCTGAAAGATAAAGTGAACAACACCCTGAGTACCGATATTCCCTTTTCAGCTGAGCAAATATGTGGATAATCAGCGTCTGCTGAGTACCCTGGATCATCATCACCATCACCACTAAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACC GCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

SEQ ID NO: 8 - Polypeptide sequence of the nociceptin ligand polypeptide

MGSMEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEE SLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGF NLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKFGGFTGARKSARKRKNQALAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQ EFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNN INFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLDHHHHHH

SEQ ID NO: 9 - Nucleotide sequence of GFP-tagged polypeptide with EGF ligand

TGGCGAATGGGACGCGCCCTGTAGCGGCCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAACAAAATATTAACG CTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGG TCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAAGTTTTATTGTTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCGTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTT TTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTC GCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGTCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCAT ACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCCAACAGTCCCCC GGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGAATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAA ATAATTTTGTTTAACTTTAAAGGAGATACATATgATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACC CTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTG GAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAG TTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGGGCGGCAGCGGCGGCGCAGCGGCGGCggatccatggagttcgttaacaaacagttcaactataaagacccagttaacggtgttgacattgcttacatcaaaatcccgaacgctggccagatgcagccggtaaaggcattcaaaatccacaacaaaatctgggttatccc ggaacgtgatacctttactaacccggaagaaggtgacctgaacccgccaccggaagcgaaacaggtgccggtatcttactatgactccacctacctgtctaccgataacgaaaaggacaactacctgaaaggtgttactaaactgttcgagcgtatttactccaccgacctgggccgtatgctgctgactagcatcgttcgcggtatc ccgttctggggcggttctaccatcgataccgaactgaaagtaatcgacactaactgcatcaacgttattcagccggacggttcctatcgttccgaagaactgaacctggtgatcatcggcccgtctgctgattcatccagttcgagtgtaagagctttggtcacgaagttctgaacctcacccgtaacggctacggttccactcagtacatccgtttctctcc ggacttcaccttcggttttgaagaatccctggaagtagacacgaacccactgctgggcgctggtaaattcgcaactgatcctgcggttaccctggctcacgaactgattcatgcaggccaccgcctgtacggtatcgccatcaatccgaaccgtgtcttcaaagttaacaccaacgcgtattacgagatgtccggtctggaagttagcttc gaagaactgcgtacttttggcggtcacgacgctaaattcatcgactctctgcaagaaaacgagttccgtctgtactactataacaagttcaaagatatcgcatccaccctgaacaaagcgaaatccatcgtgggtaccactgcttctctccagtacatgaagaacgtttttaaagaaaaatacctgctcagcgaagacacctccggcaaattct ctgtagacaagttgaaattcgataaactttacaaaatgctgactgaaatttacaccgaagacaacttcgttaagttctttaaagttctgaaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaacatcgtgccgaaagttaactacactatctacgatggtttcaacctgcgtaacaccaacctggctgctaattttaac ggccagaacacggaaatcaacaacatgaacttcacaaaactgaaaaacttcactggtctgttcgagttttacaagctgctgtgcgtcgacggcatcattacctccaaaactaaatctctgatagaaggtagaaacaaagcgctgaacctgcagtgtatcaaggttaacaactgggatttattcttcagcccgagtgaagacaacttca ccaacgacctgaacaaaggtgaagaaatcacctcagatactaacatcgaagcagccgaagaaaacatctcgctggacctgatccagcagtactacctgacctttaatttcgacaacgagccggaaaacatttctatcgaaaacctgagctctgatcatcggccagctggaactgatgccgaacatcgaacgtttcccaaacggtaaaaagt acgagctggacaaatataccatgttccactacctgcgcgcgcaggaatttgaacacggcaaatcccgtatcgcactgactaactccgttaacgaagctctgctcaacccgtcccgtgtatacaccttcttctctctagcgactacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttgggttgggttgaacagctt gtttatgattttaccgacgagacgtccgaagtatctactaccgacaaaattgcggatatcactatcatcatcccgtacatcggtccggctctgaacattggcaacatgctgtacaaagacgacttcgttggcgcactgatcttctccggtgcggtgatcctgctggagttcatcccggaaatcgccatcccggtactgggcacctttt gctctggtttcttacattgcaaacaaggttctgactgtacaaaccatcgacaacgcgctgagcaaacgtaacgaaaaatgggatgaagtttacaaatatatcgtgaccaactggctggctaaggttaatactcagatcgacctcatccgcaaaaaaatgaaagaagcactggaaaaccaggcggaagctaccaaggcaatcattaactacca gtacaaccagtacaccgaggaagaaaaaaacaacatcaacttcaacatcgacgatctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaacaagttcctgaaccagtgctctgtaagctatctgatgaactccatgatcccgtacggtgttaaacgtctggaggacttcgatgcgtctctgaaagacg ccctgctgaaatacatttacgacaaccgtggcactctgatcggtcaggttgatcgtctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagctcagtaaatatgtcgataaccaacgccttttgtccactctagaaggcggTGGCGGTAGCGGTGGCGGTGGCAGCGGCGGTGGCGGTAGCGCACTAGacAACAGCGACCCT AAATGCCCACTaAGTCATGAAGGATACTGCCTTAATGATGGTGTTTGTATGTACATAGGAACATTGGACCGTTATGCTTGCAATTGTGTAGTGGGCTATGTCGGGGAAAGGTGTCAATATCGAGATCTCAAGCTGGCAGAGTTAAGAgggctagaagcaCACCATCATCACcaccatcaccatcaccattaatgaAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGA TCCGGCCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

SEQ ID NO: 10 - Polypeptide sequence of GFP-tagged polypeptide with EGF ligand

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPV LLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGGSGGGSGGGSMEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTTFNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRS EELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYK MLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYEL DKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELRGLEAH HHHHHHHH

SEQ ID NO: 11 - Nucleotide sequence of SNAP-tagged polypeptide with EGF ligand

TGGCGAATGGGACGCGCCCTGTAGCGGCCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAACAAAATATTAACG CTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGG TCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAAGTTTTATTGTTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCGTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTT TTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTC GCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGTCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCAT ACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCCAACAGTCCCCC GGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGAATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAA ATAATTTTGTTTAACTTTAAAGGAGATACATATgATGGACAAAGACTGCGAAATGAAGCGCACCACTGGATAGCCCTCTGGGCAAGCTGGAACTGTCTGGGTGCGAACAGGGCCTGCACCGTATCATCTTCCTGGGCAAAGGAACATCTGCCGCCGACGCCGTGGAAGTGCCTGCCCCAGCCGCCGTGCTGGGCGGACCAGAGCCACTGATGCAGGCCACCGCCTGG CTCAACGCCTACTTTCACCAGCCTGAGGCCATCGAGGAGTTCCCTGTGCCAGCCCTGCACCACCCAGTGTTCCAGCAGGAGAGCTTTACCCGCCAGGTGCTGTGGAAACTGCTGAAAGTGGTGAAGTTCGGAGAGTCATCAGCTACAGCCACCTGGCCGCCCTGGCCGGCAATCCCGCCGCCACCGCCCGTGAAAACCGCCCTGAGCGGAAATCCCGTGCCCATTCTGATCCCC TGCCACCGGGTGGTGCAGGGCGACCTGGACGTGGGGGCTACGAGGGCGGGCTCGCCGTGAAAGAGTGGCTGCTGGCCCACGAGGGCACAGACTGGGCAAGCCTGGGCTGGGTGGCGGCAGCGGCGGCGGCAGCGGCGGCggatccatggagttcgttaacaaacagttcaactataaagacccagttaacggtgttgacattgcttacatca aaatcccgaacgctggccagatgcagccggtaaaggcattcaaaatccacaacaaaatctgggttatcccggaacgtgatacctttactaacccggaagaaggtgacctgaacccgccaccggaagcgaaacaggtgccggtatcttactatgactccacctacctgtctaccgataacgaaaaggacaactacctgaaaggtgttacta aactgttcgagcgtatttactccaccgacctgggccgtatgctgctgactagcatcgttcgcggtatcccgttctggggcggttaccatcgataccgaactgaaagtaatcgacactaactgcatcaacgttattcagccggacggttcctatcgttccgaagaactgaacctggtgatcatcggcccgtctgctgatatcatccagttcgag tgtaagagctttggtcacgaagttctgaacctcacccgtaacggctacggttccactcagtacatccgttctctccggacttcaccttcggttttgaagaatccctggaagtagacacgaacccactgctgggcgctggtaaattcgcaactgatcctgcggttaccctggctcacgaactgattcatgcaggccaccgcctgtacggtatcgccatcaat ccgaaccgtgtcttcaaagttaacaccaacgcgtattacgagatgtccggtctggaagttagcttcgaagaactgcgtacttttggcggtcacgacgctaaattcatcgactctctgcaagaaaacgagttccgtctgtactactactataacaagttcaaagatatcgcatccaccctgaacaaagcgaaatccatcgtgggtacc actgcttctctccagtacatgaagaacgtttttaaagaaaaatacctgctcagcgaagacacctccggcaaattctctgtagacaagttgaaattcgataaactttacaaaatgctgactgaaatttacaccgaagacaacttcgttaagttctttaaagttctgaaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaacat cgtgccgaaagttaactacactatctacgatggtttcaacctgcgtaacaccaacctggctgctaattttaacggccagaacacggaaatcaacaacatgaacttcacaaaactgaaaaacttcactggtctgttcgagttttacaagctgctgtgcgtcgacggcatcattacctccaaaactaaatctctgatagaaggtagaaacaa agcgctgaacctgcagtgtatcaaggttaacaactgggatttattcttcagcccgagtgaagacaacttcaccaacgacctgaacaaaggtgaagaaatcacctcagatactaacatcgaagcagccgaagaaaacatctcgctggacctgatccagcagtactacctgacctttaatttcgacaacgagccggaaaacatttctatcgaaa acctgagctctgatatcatcggccagctggaactgatgccgaacatcgaacgtttcccaaacggtaaaaagtacgagctggacaaatataccatgttccactacctgcgcgcgcaggaatttgaacacggcaaatcccgtatcgcactgactaactccgttaacgaagctctgctcaacccgtcccgtgtatacaccttcttctctag cgactacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttgggttgggttgaacagcttgtttatgattttaccgacgagacgtccgaagtatctactaccgacaaaattgcggatatcactatcatcatcccgtacatcggtccggctctgaacattggcaacagctgctgtacaaagacgacttcgttggcgcactga tcttctccggtgcggtgatcctgctggagttcatcccggaaatcgccatcccggtactgggcacctttgctctggtttcttacattgcaaacaaggttctgactgtacaaaccatcgacaacgcgctgagcaaacgtaacgaaaaatgggatgaagtttacaaattatcgtgaccaactggctggctaaggttaatactcagatc gacctcatccgcaaaaaaatgaaagaagcactggaaaaccaggcggaagctaccaaggcaatcattaactaccagtacaaccagtacaccgaggaagaaaaaaaacaacatcaacttcaacatcgacgatctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaacaagttcctgaaccagtgctctgtaagctatct gatgaactccatgatcccgtacggtgttaaacgtctggaggacttcgatgcgtctctgaaagacgccctgctgaaatacatttacgacaaccgtggcactctgatcggtcaggttgatcgtctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagctcagtaaatatgtcgataaccaacgccttttgtcc actctagaaggcggTGGCGGTAGCGGTGGCGGTGGCAGCGGCGGTGGCGGTAGCGCACTAGacAACAGCGACCCTAAATGCCCACTaAGTCATGAAGGATACTGCCTTAATGATGGTGTTTGTATGTACATAGGAACATTGGACCGTTATGCTTGCAATTGTGTAGTGGGCTATGTCGGGGAAAGGTGTCAATATCGAGATCTCAAGCTGGCAGTTAAGAgggct agaagcaCACCATCATCACcaccatcaccatcaccattaatgaAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTGCTGAAAGGAGGAACTATATCCGGAT

SEQ ID NO: 12 - Polypeptide sequence of SNAP-tagged polypeptide with EGF ligand

MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAVKEWLLAHEGHRLGKPGLGGGSGGGS GGGSMEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEE SLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGF NLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKV NKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIP YGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELRGLEAHHHHHHHHHH

SEQ ID NO: 13 - Sortase A Nucleotide Sequence (LPESG Targeting)

TGGCGAATGGGACGCGCCCTGTAGCGGCCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAACAAAATATTAACG CTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGG TCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAAGTTTTATTGTTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCGTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTT TTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTC GCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGTCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCAT ACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCCAACAGTCCCCC GGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGAATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAA ATAATTTTGTTTAACTTTAAAGGAGATATCATATGCAGGCAAAACCGCAGATTCCGAAAGATAAAAGCAAAGTGGCAGGCTATATTGAAATTCCGGATGCCGATATTAAAGAACCGGTTTATCCGGGTCCTGCAACACGTGAACAGCTGGATCGTGGTGTTTGTTTTGTTGAAGAAAATGAGAGCCTGGATGATCAGAACATTAGCATTACCGGTCATACCGCAATTGATCGTCC GAATTATCAGTTTACCAATCTGCGTGCAGCCAAACCGGGTAGCATGGTTTATCTGAAAGTTGGTAATGAAACCCGCATCTACAAAATGACCAGCATTCGTAATGTTAAACCGACCGCAGTTGGTGTTCTGGATGAACAAAAAGGTAAAGATAAACAGCTGACCCTGGTTACCTGTGTGATGATTATAACTTTGAAACCGGTGTTTGGGAAACGCGCAAAATCTTTGTTGC AACCGAAGTTAAACATCACCATCACCACCATCATCACCATTAAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

SEQ ID NO: 14 - Sortase A polypeptide sequence (LPESG targeting)

MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLDRGVCFVEENESLDDQNISITGHTAIDRPNYQFTNLRAAKPGSMVYLKVGNETRIYKMTSIRNVKPTAVGVLDEQKGKDKQLTLVTCDDYNFETGVWETKIFVATEVKHHHHHHHHH

SEQ ID NO: 15 - Sortase A Nucleotide Sequence (LAETG Targeting)

TGGCGAATGGGACGCGCCCTGTAGCGGCCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTT GATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAACAAAATATTAACG CTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGG TCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAAGTTTTATTGTTCATGACCAAAATCCC TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCGTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTT TTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTC GCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGTCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCAT ACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCCAACAGTCCCCC GGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGAATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAA ATAATTTTGTTTAACTTTAAAGGAGATACATATGCAGGCAAAACCGCAGATTCCGAAAGATAAAAGCAAAGTGGCAGGCTATATTGAAATTCCGGATGCCGATATTAAAGAACCGGTTTATCCGGGTCCTGCAACACGTGAACAGCTGAATCGTGGTGTTTGTTTTCACGATGAAAATGAGAGCCTGGATGATCAGAATATTAGCATTGCAGGCCATACCTTTATTGATCGTCCGAAT TATCAGTTCACCAATCTGAAAGCAGCAAAACCGGGTAGCATGGTTTATTTCAAAGTTGGTAATGAAACCCGCATTCTACAAAATGACCAGCATTCGTAAAGTTCATCCGAATGCAGTTGGTGTTCTGGATGAACAAGAAGGCAAAGATAAACAGCTGACCCTGGTTACCTGTGATGATTATAACGAAGAAACCGGTGTTTGGGAAAGCCGTAAAATCTTTGTTGCAACCGAAG TGAAACATCATCACCACCATCACCATCATCATCACTAAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

SEQ ID NO: 16 - Sortase A polypeptide sequence (LAETG targeting)

MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVCFHDENESLDDQNISIAGHTFIDRPNYQFTNLKAAKPGSMVYFKVGNETRIYKMTSIRKVHPNAVGVLDEQEGKDKQLTLVTCDDYNEETGVWESRKIFVATEVKHHHHHHHHH

SEQ ID NO: 17 - BoNT/A - UniProt P10845

MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTTFTNPEEGDLN

PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI

GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN

EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT

NRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELN

EKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR

GSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA

GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAK

LVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO: 18 - BoNT/B - UniProt P10844

MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN

KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG

DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH

FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY

GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV

DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN

IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA

YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN

YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY

LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND

FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLI

PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY

KALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSV

SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL

SIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFK

LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS

GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYING

KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSY

SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLY

IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD

SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS

KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

SEQ ID NO: 19 - BoNT/C - UniProt P18640

MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK

PPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN

NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF

AAQEGFGALSIIISSPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG

IAIPNDQTISSVSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI

AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTE

FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA

LRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLRK

DINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQN

VDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLM

WANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILL

EAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQF

NNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNIN

KFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSF

QNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQ

LNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIID

SVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNM

KIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKEL

DGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNNN

DFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYA

IGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYR

HNYLVPTVKQGNYASLLESTSTHWGFVPVSE

SEQ ID NO: 20 - BoNT/D - UniProt P19321

MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSK

PPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDS

STPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSN

PSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG

INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDI

AKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSE

VVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPA

LQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDE

TNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYL

NSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANE

VVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFP

EFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHIN

YQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIR

ECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTM

PFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTI

YTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNS

GWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYIN

GELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQI

LRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSV

SDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNY

GIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLS

TSSFWKFISRDPGWVE

SEQ ID NO: 21 - BoNT/E - UniProt Q00496

MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS

LKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTP

DNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGS

IAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPL

ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYK

DVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKL

SNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKG

IRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESA

PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSS

IDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADIS

IVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNK

NKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIE

SKYNSYTLEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKIN

KLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDKILISYF

NKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNI

SQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEII

WTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL

GNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYL

LYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDN

LVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNF

KNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

SEQ ID NO: 22 - BoNT/F - UniProt A7GBG3

MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFD

PPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGN

EHTPINEFHPVTRTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVY

DPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGAR

GVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATR

LSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKF

KVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKG

LVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFVASESSYNENDINTPKEIDDTTNLN

NNYRNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFF

YLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEEFINTINKPVHAALFISWINQVIR

DFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELL

IPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRK

EQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERF

ITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNN

SIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIY

STNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDC

IRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGN

SRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETL

YSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSN

TRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKL

IRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTS

SNGCFWSFISKEHGWQEN

SEQ ID NO: 23 - BoNT/G - UniProt Q60393

MPVNIKXFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFN

ASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLG

NASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSIMIMNGH

SPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLY

GIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIA

NRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNL

AGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAY

EEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYN

TQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGD

SLFEYLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWVK

GVIDDFTSESTQKSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAFEIGGAAILMEFI

PELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTI

KERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFI

NQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKDS

IPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSSGYGATMNVGSDVIFND

IGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTII

SCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEEYSIKDNISDYINKWFSITITNDRLG

NANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVS

SLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAAI

NYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQ

LFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYD

NYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE

SEQ ID NO: 24 - BoNT/X polypeptide sequence

MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNT

NDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSIP

LPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEG

TLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGIS

NRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISE

RLNTTVVENDLLKYIKNKIPVQGRRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVR

KHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFI

KICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISN

KDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELY

EPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPF

KNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNI

GNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRD

QKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAK

IKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDK

FIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNL

GAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNF

SISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI

AFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLL

DQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWS

SFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMG

ISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETS

KPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED

SEQ ID NO: 25 - TeNT - UniProt P04958

MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFN

PPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGN

SYSLLDKFDTNNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDN

KNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLH

GLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYK

AIANKLSQVTSCNDPNIDIDSYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTE

IELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNT

NAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLTFIAE

KNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAP

EYKSNAASTIEIHNIDDNTTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVI

SKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYEGN

FIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYK

LVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKN

KLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIG

ITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDIS

GFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPK

VSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLP

DKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNN

NQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDV

QLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYV

SYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDD

KNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

SEQ ID NO: 26 - Polypeptide sequence of labeled EGF TM polypeptide

*HHHHHLAETGGSGGSGGSEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAV FKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNP SRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININ KFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELRGLEAGGSGGGSGLPESGK†

* = HiLyte555; †=HiLyte488

SEQ ID NO: 27 - C. ternatea 1 butelase 1 polypeptide sequence (plus signal peptide)

MKNPLAILFLIATVVAVVSGIRDDFFLRLPSQASKFFQADDNVEGTRWAVLVAGSKGYVNY

RHQADVCHAYQILKKGGLKDENIIVFMYDDIAYNESNPHPGVIINHPYGSDVYKGVPKDY

VGEDINPPNFYAVLLANKSALTGTGTGSGKVLDSGPNDHVFIYYTDHGGAGVLGMPSKPYIA

ASDLNDVLKKKHASGTYKSIVFYVESCESGSMFDGLLPEDHNIYVMGASDTGESSWVTYC

PLQHPSPPPEYDVCVGDLFSVAWLEDCDVHNLQTETFQQQYEVVKNKTIVALIEDGTHVV

QYGDVGLSKQTLFVYMGTDPANDNNTFTDKNSLGTPRKAVSQRDADLIHYWEKYRRAPEG

SSRKAEAKKQLREVMAHRMHIDNSVKHIGKLLFGIEKGHKMLNNVRPAGLPVVDDWDCFK

TLIRTFETHCGSLSEYGMKHMRSFANLCNAGIRKEQMAEASAQACVSIPDNPWSSLHAGF

SV

SEQ ID NO: 28 - C. ternatea butelase 1 polypeptide sequence (minus signal peptide)

IRDDFLRLPSQASKFFQADDNVEGTRWAVLVAGSKGYVNYRHQADVCHAYQILKKGGLKD

ENIIVFMYDDIAYNESNPHPGVIINHPYGSDVYKGVPKDYVGEDINPPNFYAVLLANKSA

LTGTGSGKVLDSGPNDHVFIYYTDHGGAGVLGMPSKPYIAASDLNDVLKKKHASGTYKSI

VFYVESCESGSMFDGLLPEDHNIYVMGASDTGESSWVTYCPLQHPSPPPEYDVCVGDLFS

VAWLEDCDVHNLQTETFQQQYEVVKNKTIVALIEDGTHVVQYGDVGLSKQTLFVYMGTDP

ANDNNTFTDKNSLGTPRKAVSQRDADLIHYWEKYRRAPEGSSRKAEAKKQLREVMAHRMH

IDNSVKHIGKLLFGIEKGHKMLNNVRPAGLPVVDDWDCFKTLIRTFETHCGSLSEYGMKH

MRSFANLCNAGIRKEQMAEASAQACVSIPDNPWSSLHAGFSV

SEQ ID NO: 29 - Peptide with conjugated detectable label and sortase donor site

GGGGK†

†=HiLyte488

SEQ ID NO: 30 - Peptide with conjugated detectable label and acceptor site for sortase

*HHHHHHLAETGGG

*=HiLyte555

SEQ ID NO: 31 - Staphylococcus aureus sortase A polypeptide sequence

MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDKKQQ

AKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGH

TFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLT

LITCDDYNEKTGVWEKRKIFVATEVK

SEQ ID NO: 32 - Staphylococcus aureus sortase B polypeptide sequence

MRMKRFLTIVQILLVVIIIIFGYKIVQTYIEDKQERANYEKLQQKFQMLMSKHQEHVRPQ

FESLEKINKDIVGWIKLSGTSLNYPVLQGKTNHDYLNLDFEREHRRKGSIFMDFRNELKN

LNHNTILYGHHVGDNTMFDVLEDYLKQSFYEKHKIIEFDNKYGKYQLQVFSAYKTTTKDN

YIRTDFENDQDYQQFLDETKRKSVINSDVNVTVKDRIMTLSTCEDAYSETTKRIVVVAKI

IKVS

SEQ ID NO: 33 - Streptococcus pneumoniae sortase A polypeptide sequence

MEKLYIHLKNLRKVAVVMLLVFTTFYLLLMFLNQSDNQEIAKNIEKFNDSVIVAKTDNTK

ADIKEIEKNIEKVRKIEGGNVERVNQLTSENEKVKENIDLNIEEEEIIENSYKSLETTDNF

EKLGIIEIPKIDLNLSIFKGKPFVNTKNRQDTMLYGAVTNKKNQKMGRENYVLASHIISN

SNLLFTSINQLEKGDVITLKDSEYSYQYTVYNNFIVSKDETWILNDIKDYSILTLYTCYD

DSTKLPENRVVIRAVLTDIN

SEQ ID NO: 34 - Streptococcus pneumoniae sortase B polypeptide sequence

MAKTKKQKRNNLLLGVVFFIGXAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDE

RMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPAIDVDLPVYAG

TAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKE

VMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTTCTPYMINTHRLLVRGHRIPYVAEVEEE

FIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKRQSERALKALKEATKEVKVEDE

where X is Met or Ile.

SEQ ID NO: 35 - Streptococcus pneumoniae sortase C polypeptide sequence

MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQM

DKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQ

EIPMYVGTSEDILQKGAGLLEGASLPVGGKNTHTVITAHRGLPTAELFSQLDKMKKGDIF

YLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPY

TAPIAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD

SEQ ID NO: 36 - Streptococcus pneumoniae sortase D polypeptide sequence

MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSL

AYNQRLASQNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGM

GLAHVDGTPLPMDGTGIRSVIAGHRAEPSHVFFRHLDQLKVGDALYYDNGQEIVEYQMMD

TEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVA

FTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK

SEQ ID NO: 37 - Streptococcus pyogenes sortase A polypeptide sequence

MVKKQKRRKIKSMSWARKLLIAVLLILGLALLFNKPIRNTLIARNSNKYQVTKVSKKQIK

KNKEAKSTFDFQAVEPVSTESVLQAQMAAQQLPVIGGIAIPELGINLPIFKGLGNTELIY

GAGTMKEEQVMGGENNYSLASHHIFGITGSSQMLFSPLERAQNGMSIYLTDKEKIYEYII

KDVFTVAPERVDVIDDTAGLKEVTLVTCTDIEATERIIVKGELKTEYDFDKAPADVLKAF

NHSYNQVST

SEQ ID NO: 38 - Polypeptide sequence of the proteolytically inactive BoNT/A(0) mutant

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEV DTNPLLGAGKFATDPAVTLAHQLIYAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFN LRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATE AAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYG VKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQ MINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVE KILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO: 39 - Nucleotide sequence of full-length proteolytically inactive BoNT/A(0) mutant with SrtA double-labeled sites

ATGGAGAACCTGTATTTCAGGGCGGCGGTGGCAGCGGCGGCAGCGGCGCAGCCCGTTTGTGAACAAGCAGTTCAACTATAAAGATCCGGTTAATGGTGTGGATATCGCCTATATCAAAATTCCGAATGCAGGTCAGATGCAGCCGGTTAAAGCCTTTAAAATCCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTTACCAATCCGGAAGAAGGTGATCTGA ATCCGCCTCCGGAAGCAAAACAGGTTCCGGTTAGCTATTATGATAGCACCTATCTGAGCACCGATAACGAGAAAGATAACTATCTGAAAGGTGTGACCAAACTGTTTGAACGCATTTATAGTACCGATCTGGGTCGTATGCTGCTGACCAGCATTGTTCGTGGTATTCCGTTTTGGGGTGGTAGCACCATTGATACCGAACTGAAAGTTATTGACACCAACTGCATTAATGTGAT TCAGCGGATGGTAGCTATCGTAGCGAAGAACTGAATCTGGTTATTATTGGTCCGAGCGCAGATATCATTCAGTTTGAATGTAAAAGCTTTGGCCACGAAGTTCTGAATCTGACCCGTAATGGTTATGGTAGTACCCAGTATATTCGTTTCAGTCCGGATTTTACCTTTGGCTTTTGAAGAAAGCCTGGAAGTTGATACAAATCCGCTGTTAGGTGCAGGTAAAT TTGCAACCGATCCGGCAGTTACCCTGGCACACCAGCTGATTTATGCCGGTCATCGTCTGTATGGTATTGCCATTAATCCGAATCGTGTGTTCAAAGTGAATACCAACGCCTATTATGAAATGAGCGGTCTGGAAGTGAGTTTTGAAGAACTGCGTACCTTTGGTGGTCATGATGCCAAATTTATCGATAGCCTGCAAGAAAATGAATTTCGCCTGTACTATAACAAATTCAAGGATATT GCGAGCACCCTGAATAAAGCCAAAAGCATTGTTGGCACCACCGCAAGCCTGCAGTATATGAAAAATGTGTTTAAAGAAAATATCTGCTGAGCGAAGATACCAGCGGTAAATTTAGCGTTGACAAACTGAAATTCGATAAACTGTACAAGATGCTGACCGAGATTTATACCGAAGATAACTTCGTGAAGTTTTTCAAAGTGCTGAACCGCAAAACCTACCTGAACTTTGATAAAGCCGTGTTCAAA ATCAACATCGTGCCGAAAGTGAACTATACCATCTATGATGGTTTTAACCTGCGCAATACCAATCTGGCAGCAAACTTTAATGGTCAGAACACCGAAATCAACAACATGAACTTTACCAAACTGAAGAACTTCACCGGTCTGTTCGAATTTTACAAACTGCTGTGTGTTCGTGGCATTATTACCAGCAAAACCAAAAGTCTGGATAAAGGCTACAATAAAGCCCTGAATGATCTGTGCATTAAGG TGAATAATTGGGACCTGTTTTTTAGCCCGAGCGAGGATAATTTCACCAACGATCTGAACAAAGGCGAAGAAATTACCAGCGATACCAATATTGAAGCAGCCGAAGAAAACATTAGCCTGGATCTGATTCAGCAGTATTATCTGACCTTCAACTTCGATAATGAGCCGGAAAATATCAGCATTGAAAACCTGAGCAGCGATATTATTGGCCAGCTGGAACTGATGCCGAATATTGAACGTTTTCCGAAC GGCAAAAAATACGAGCTGGATAAATACACCATGTTCCATTATCTGCGTGCCCAAGAATTTGAACATGGTAAAAGCCGTATTGCACTGACCAATAGCGTTAATGAAGCACTGCTGAACCCGAGCCGTGTTTATACCTTTTTTAGCAGCGATTACGTGAAAAAGGTTAACAAAGCAACCGAAGCAGCCATGTTTTAGGTTGGGTTGAACAGCTGGTTTATGATTTCACCGATG AAACCAGCGAAGTTAGCACCACCGATAAAATTGCAGATATTACCATCATCATCCCGTATATCGGTCCGGCACTGAATATTGGCAATATGCTGTATAAAGACGATTTTGTGGGTGCCCTGATTTTTTAGCGGTGCAGTTATTCTGCTGGAATTTATTCCGGAAATTGCCATTCCGGTTCTGGGCACCTTTGCACTGGTGAGCTATATTGCAAATAAAGTTCTGACCGTGCAGACCATCGATA ATGCACTGAGCAAACGTAACGAAAAATGGGATGAAGTGTACAAGTATATCGTGACCAATTGGCTGGCAAAAGTTAACACCCAGATTGACCTGATTCGCAAGAAGATGAAAGAAGCACTGGAAAATCAGGCAGAAGCAACCAAAGCCATTATCAACTATCAGTATAACCAGTACACCGAAGAAGAGAAAAATAACATCAACTTCAACATCGACGATCTGTCCAGCAAACTGAACGAAAGCAT CAACAAAGCCATGATTAACATTAACAAATTTCTGAACCAGTGCAGCGTGAGCTATCTGATGAATAGCATGATTCCGTATGGTGTGTGAAACGTCTGGAAGATTTTGATGCAAGCCTGAAAGATGCCCTGCTGAAATATATCTATGATAATCGTGGCACCCTGATTGGTCAGGTTGATCGTCTGAAAGATAAAGTGAACAACACCCTGAGTACCGATATTCCCTTTTCAGCTGAGCAAATATGTG GATAATCAGCGTCTGCTGTCAACCTTTACCGAATACATTAAGAACATCATCAACACAGCATTCTGAACCTGCGTTATGAAAGCAATCATCTGATTGATCTGAGCCGTTATGCCAGCAAAATCAATATAGGCAGCAAGGTTAACTTCGACCCGATTGACAAAAATCAGATACAGCTGTTTAATCTGGAAAGCAGCAAAATTGAGGTGATCCTGAAAAACGCCATTGTGTATAATAGCATGTACG AGAATTTCTCGACCAGCTTTTGGATTCGTATCCCGAAATACTTTAATAGCATCAGCCTGAACAACGAGTACACCATTATTAACTGCATGGAAAACAATAGCGGCTGGAAAGTTAGCCCTGAATTATGGCGAAATTATCTGGACCCTGCAGGATACCCAAGAAATCAAAACAGCGTGTGGTTTTCAAATACAGCCAGATGATTAATATCAGCGACTATATCAACCGCTGGATTTTTGTGACCATTA CCAATAATCGCCTGAATAACAGCAAGATCTATATTAACGGTCGTCTGATTGACCAGAAACCGATTAGTAATCTGGGTAATATTCATGCGAGCAACAACATCATGTTTAAACTGGATGGTTGTCGTGATACCCATCGTTATATTTGGATCAAGTACTTCAACCTGTTCGATAAAAGTTGAACGAAAAAGAAATTAAAGACCTGTATGATAACCAGAAGCAACAGCGGTATTCTGAAGGATT TTTGGGGAGATTATCTGCAGTATGACAAACCGTATTATATGCTGAATCTGTACGACCCGAATAAATACGTGGATGTGAATAATGTTGGCATCCGTGGTTATATGTACCTGAAAGGTCCGCGTGGTAGCGTTATGACCACAAACATTTATCTGAATAGCAGCCTGTATCGCGGAACCAAATTCATCATTAAAAAGTATGCCAGCGGCAACAAGGATAATATTGTGCGTAATAATGATCGCG TGTACATTAACGTTGTGGTGAAGAATAAAGAATATCGCCTGGCAACCAATGCAAGCCAGGCAGGCGTTGAAAAAATTCTGAGTGCCCTGGAAATTCCGGATGTTGGTAATCTGAGCCAGGTTGTTGTGATGAAAAGCAAAAATGATCAGGCATCACCAACAAGTGCAAAATGAATCTGCAGGACAATAACGGCAACGATATTGGTTTTATTGGCTTCCACCAGTTCAACA ATATTGCGAAACTGGTTGCAAGCAATTGGTATAATCGTCAGATTGAACGTAGCAGTCGTACCCTGGGTTGTAGCTGGGAATTTATCCCTGTGTGGATGATGGTTGGGGTGAACGTCCGCTGGGCGGCAGCGGCGGCGGCAGCGGCCTGCCCGAAACGGTGGCGGATCTGCTTGGTCTCACCCGCAGTTCGAAAAAGGTGGTGGTTCTGGTGGTGG TTCTGGTGGTTCTGCTTGGTCTCACCCGCAGTTCGAAAAATAATGA

SEQ ID NO: 40 - Polypeptide sequence of full length proteolytically inactive BoNT/A(0) mutant with SrtA double tagged sites

MENLYFQGGGGSGGSGGSPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYG STQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHQLIYAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAV FKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININK FLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTL QDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPPRGSVMTTNIYLNSSLYRGTKDNIVFIIKKYASGNKDNIVRNNDRVYIN VVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLGGSGGGSGLPESGGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

SEQ ID NO: 41 - PATG Prochloron didemni polypeptide sequence

MFSIMITIDYPFTVSLNRDIQVTSTEDYYTLQVTESDPSAWLTFATTPAMDMAFDHLKAGTTTESLVQTLAELGGPAAREQFALTLQQLDERGWLSYAVLPLAEAIPMVESAELNLPGNPHWMETGVTLSRFAYQHPYEGTMVLESPLSKFRVKLLDWRASALLAQLAQPQTLGTIAPPPYLGPETAYQFLNLLWATGF LASDHEPVSLQLWDFHNLLFHSRSRLGRHDYPGTDLNVDNWSDFPVVKPPMSDRIVPLPRPNLEALMSNDATLTEAIETRKSVREYDDDNPITIEQLGELLYRAARVTKLLSPEERFGKLWQQNKPVFEEAGVDEGEFSHRPYPGGGAMYELEIYPVVRLCQGLSQGVYHYDPLNHQLEQIVESKDDIFAVSGSPLASKLGPHV LLVITARFGRLFRLYRSVAYALVLKHVGVLQQNLYLVATNMGLAPCAGGAGDSDAFAQVTGIDYVEESAVGEFILGSLASEVESDVVEGEDEIESAGVSASEVESSATKQKVALHPHDLDERIPGLADLHNQTLGDPQITIVIIDGDPDYTLSCFEGAEVSKVFPYWHEPAEPITPEDYAAFQSIRDQGLKGKEKEEALEAVIPD TKDRIVLNDHACHVTSTIVGQEHSPVFGIAPNCRVINMPQDAVIRGNYDDVMSPLNLARAIDLALELGANIIHCAFCRPTQTSEGEEILVQAIKKCQDNNVLIVSPTGNNSNESWCLPAVLPGTLAVGAAKVDGTPCHFSNWGGNNTKEGILAPGEEILGAQPCTEEPVRLTGTSMAAPVMTGISALLMSLQVQQGKPVDAEAVRTALLKTA IPCDPEVVEEPERCLRGFVNIPGAMKVLFGQPSVTVSFAGGQATRTEHPGYATVAPASIPEPMAERATPAVQAATATEMVIAPSTEPANPATVEASTAFSGNVYALGTIGYDFGDEARRDTFKERMADPYDARQMVDYLDRNPDEARSLIWTLNLEGDVIYALDPKGPFATNVYEIFLQMLAGQLEPETSADFIERLSVPARRTTRTVELFS GEVMPVVNVRDPRGMYGWNVNALVDAALATVEYEEADEDSLRQGLTAFLNRVYHDLHNLGQTSRDRALNFTVTNTFQAASTFAQAIASGRQLDTIEVNKSPYCRLNSDCWDVLLTFYDPEHGRRSRRVFRFTLDVVYVLPVTVGSIKSWSLPGKGTVSK

SEQ ID NO: 42 - PCY1 Saponaria vaccaria polypeptide sequence

MATSGFSKPLHYPPVRRDETVVDDYFGVKVADPYRWLEDPNSEETKEFVDNQEKLANSVLEECELIDKFKQKIIDFVNFPRCGVPFRRANKYFHFYNSGLQAQNVFQMQDDLDGKPEVLYDPNLREGGRSGLSLYSVSEDAKYFAFGIHSGLTEWVTIKILKTEDRSYLPDTLEWVKFSPAIWTHDNKGFFYCPY PPLKEGEDHMTRSAVNQEARYHFLGTDQSEDILLWRDLENPAHHLKCQITDDGKYFLLYILDGCDDANKVYCLDLTKLPNGLESFRGREDSAPFMKLIDSFDASYTAIANDGSVFTFQTNKDAPRKKLVRVDLNNPSVWTDLVPESKKDLLESAHAVNENQLILRYLSDVKHVLEIRDLESGALQHRLPIDIGSVDGITARRRDSV VFFKFTSILTPGIVYQCDLKNDPTQLKIFRESVVPDFDRSEFEVKQVFVPSKDGTKIPIFIAARKGISLDGSHPCEMHGYGGFGINMMPTFSASRIVFLKHLGGVFCLANIRGGGEYGEEWHKAGFRDKKQNVFDDFISAAEYLISSGYTKARRVAIEGGSNGGLLVAACINQRPDLFGCAEANCGVMDMLRFHKFTLGYL WTGDYGCSDKEEEFKWLIKYSPIHNVRRPWEQPGNEETQYPATMILTADHDDRVVPLHSFKLLATMQHVLCTSLEDSPQKNPIIARIQRKAAHYGRATMTQIAEVADRYGFMAKALEAPWID

SEQ ID NO: 43 - POPB Galerina marginata polypeptide sequence

MSSVTWAPGNYPSTRRSDHVDTYQSASKGEVPVPDPYQWLEESTDEVDKWTTAQADLAQSYLDQNADIQKLAEKFRASRNYAKFSAPTLLDDGHWYWFYNRGLQSQSVLYRSKEPALPDFSKGDDNVGDVFFDPNVLAADGSAGMVLCKFSPDGKFFAYAVSHLGGDYSTIYVRSTSSPLSQASVAQGVDGR LSDEVKWFKFSTIIWTKDSKGFLYQRYPARERHEGTRSDRNAMMCYHKVGTTQEEDIIVYQDNEHPEWIYGADTSEDGKYLYQFKDTSKKNLLWVAELDEDGVKSGIHWRKVVNEYAADYNIITNHGSLVYIKTNLNAPQYKVITIDLSKDEPEIRDFIPEEKDAKLAQVNCANEEYFVAIYKRNVKDE IYLYSKAGVQLTRLAPDFVGAASIANRQKQTHFFLTLSGFNTPGTIARYDFTAPETQRFSILRTTKVNELDPDDFESTQVWYESKDGTKIPMFIVRHKSTKFDGTAAAIQYGYGGFATSADPFFSPIILTFLQTYGAIFAVPSIRGGGEFGEEWHKGGRRETKVNTFDDFIAAAQFLVKNKYAAPGKVAINGASNGGLLVMGSIVRAP EGTFGAAVPEGGVADLLKFHKFTGGQAWISEYGNPSIPEEFDYIYPLSPVHNVRTDKVMPATLITVNIGDGRVVPMHSFKFIATLQHNVPQNPHPLLIKIDKSWLGHGMGKPTDKNVKDAADKWGFIARALGLELKTVE

SEQ ID NO: 44 - Oldenlandia affinis OaAEP1b butelase homologue polypeptide sequence (plus signal peptide)

MVRYLAGAVLLLVVLSVAAAAVSGARDGDYLHLPSEVSRFFRPQETNDDHGEDSVGTRWAVLIAGSKGYANYRHQAGVCHAYQILKRGGLKDENIVVFMYDDIAYNESNPRPGVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKS AITGGSGKVVDSGPNDHIFIYYTDHGAAGVIGMPSKPYLYADELNDAL KKKHASGTYKSLVFYLEACESGSMFEGILPEDLNIYALTSTNTTESSWCYYCPAQENPPPPEYNVCLGDLFSVAWLEDSDVQNSWYETLNQQYHHVDKRISHASHATQYGNLKLGEEGLFVYMGSNPANDNYTSLDGNALTPSSIVVNQRDADLLHLWEKFRKAPEGSARKEVAQTQIFKAMSHRVHIDSSIKLIGKLLF GIEKCTEILNAVRPAGQPLVDDWACLRSLVGTFETHCGSLSEYGMRHTRTIA NICNAGISEEQMAEAASQACASIP

SEQ ID NO: 45 - Oldenlandia affinis OaAEP1b butelase homologue polypeptide sequence (minus signal peptide)

ARDGDYLHLPSEVSRFFRPQETNDDHGEDSVGTRWAVLIAGSKGYANYRHQAGVCHAYQILKRGGLKDENIVVFMYDDIAYNESNPRPGVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKSAITGGSGKVVDSGPNDHIFIYYTDHGAAGVIGMPSKPYLYADELNDALKKKHASGTYKSLVFYLEACESGS MFEGILPEDLNIYALTSTNTTESSWCYYCPAQENPPPPEYNVCLGDLFSVAWLEDSDVQNSWYETLNQQYHHVDKRISHASHATQYGNLKLGEEGLFVYMGSNPANDNYTSLDGNALTPSSIVVNQRDADLLHLWEKFRKAPEGSARKEVAQTQIFKAMSHRVHIDSSIKLIGKLLFGIEKCTEILNAVRPAGQPLVDDWA CLRSLVGTFETHCGSLSEYGMRHTRTIANICNAGISEEQMAEAASQACASIP

EXAMPLES

EXAMPLE 1

Construction of Texas Red, eGFP, SNAP, and SrtA singly and doubly labeled EGF ligand polypeptide

Several labeling strategies have been tried. The goal was to obtain a labeled version of the polypeptide that did not affect its structural characteristics and its ability to transport into cells and cleave SNARE proteins efficiently and similarly to the unlabeled version.

Four different strategies for labeling the polypeptide with EGF ligand have been tried (Fonfria, E., S. Donald and V.A. Cadd (2016). "Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments." J Recept Signal Transduct Res 36(1): 79-88). After cloning, if necessary, the polypeptide was expressed by recombinant methods and purified using standard techniques as previously published (Masuyer, G., M. Beard, V.A. Cadd, J.A. Chaddock and K.R. Acharya (2011). "Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B." J Struct Biol 174(1): 52-57, Somm, E., N. Bonnet, A. Martinez, P. M. Marks, V. A. Cadd, M. Elliott, A. Toulotte, S. L. Ferrari, R. Rizzoli , P.S. Huppi, E. Harper, S. Melmed, R. Jones and M.L. Aubert (2012) "A botulinum toxin-derived targeted secretion inhibitor downregulates the GH/IGF1 axis." J Clin Invest 122(9): 3295-3306 ). Briefly, the polypeptide was expressed by recombinant methods in competent E. coli bacteria. The expressed polypeptide was purified using an affinity column, followed by anion exchange chromatography, enzymatic activation to give a double-stranded complex, and finally a polishing step using hydrophobic interaction.

1. The unmodified EGF Ligand peptide, purified as described above, was labeled using the Texas Red-X Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Successful labeling of the protein was confirmed by confocal microscopy and intravital imaging. The nucleotide and polypeptide sequences for the labeled polypeptide are shown as SEQ ID NOs: 5 and 6, respectively.

2. The EGF ligand polypeptide was labeled at the N-terminus with enhanced green fluorescent protein (eGFP) by standard cloning techniques. Nucleotide and polypeptide sequences are shown as SEQ ID NOs: 9 and 10, respectively. Protein expression and purification were performed as described above. After expression, an unsuccessful attempt was made to purify the eGFP labeled EGF ligand polypeptide.

3. The EGF ligand polypeptide was labeled at the N-terminus with a SNAP tag substrate (New England Biolabs) using standard cloning techniques. Nucleotide and polypeptide sequences are shown as SEQ ID NOs: 11 and 12, respectively. Expression and purification of this protein was successful. Labeling of the SNAP-tagged polypeptide with EGF ligand was performed using SNAP-Surface 594 fluorescent substrate (New England Biolabs) according to the manufacturer's protocol. Successful labeling of the protein was confirmed by confocal microscopy and intravital imaging.

4. Attempts have also been made to obtain polypeptides containing non-natural amino acids for site-specific labeling. However, these attempts were unsuccessful due to difficult expression and/or purification.

5. An EGF ligand polypeptide (ie, a polypeptide having EGFTM) was labeled with two different sortase A (SrtA) recognition sites, one at the N-terminus and one at the C-terminus. The use of SrtA allowed the conjugation of two fluorophores of different colors on the same protein. The polypeptide was designed as illustrated in Fig.1. Two mutant versions of SrtA were selected (Dorr, B.M., H.O. Ham, C. An, E.L. Chaikof and D.R. Liu (2014). "Reprogrammming the specificity of sortase enzymes". Proc Natl Acad Sci U S A 111(37): 13343-13348) (SEQ ID NOS: 14 and 16). They have been shown to have 100% specificity for their respective recognition sites. The EGF ligand polypeptide was cloned with a LPESG recognition site for the first SrtA at the C-terminus followed by a dual StrepTag recognition site (IBA-lifesciences), allowing initial affinity-mediated protein purification. Nucleotide and polypeptide sequences are shown as SEQ ID NOs: 1 and 2, respectively. Separately, a peptide containing a region of glycine residues conjugated to a selected fluorophore (Eurogentec) was prepared. The sequence of this peptide was: GGGGK(HF488) (SEQ ID NO: 29). During the SrtA-mediated reaction, glycine in the LPESG region was cleaved off by SrtA (SEQ ID NO: 14) and the region of glycine residues present on the fluorescent peptide was recognized by SrtA and used to mediate conjugation between polypeptide and peptide. This provided a singly fluorescently labeled polypeptide with an EGF ligand. It should be noted that the labeled polypeptide no longer had a Strep tag and a reverse affinity-mediated purification step was used to select the labeled portion of the polypeptide. For a doubly labeled polypeptide with an EGF ligand, a cloning 3 glycine residue region at the N-terminus of the polypeptide after the start codon and tobacco etch virus (TEV) recognition region for cleavage. The TEV site was added to help protect the glycine residue sites from circularization during the initial SrtA C-terminal reaction as detailed above. Separately, a peptide containing the LAETG recognition site conjugated to a selected fluorophore (Eurogentec) was prepared. The sequence of this peptide was: HiLyte Fluor™ 555-HHHHHHLAETGGG (SEQ ID NO: 30). In addition, a 6 His tag (6HT) was placed in front of the LAETG region for ease of purification of the protein after the SrtA reaction (SEQ ID NO: 16). The SrtA reaction was carried out similarly to the C-terminal region and the final doubly labeled EGF ligand protein was purified using a His affinity purification step. Successful single and double labeling of the protein was confirmed by SDS-PAGE gel electrophoresis, confocal microscopy and in vivo microscopy.

Sortase A (SrtA) proteins having a C-terminal His tag were expressed in competent E. coli bacteria and purified using an affinity capture column.

Sortase conjugation of the polypeptide and fluorescent peptides was performed overnight at 4° C. using an equivalent ratio of polypeptide and SrtA and fluorescent peptide of 1 to 2 to 20, respectively.

In the present example, the EGF ligand polypeptide was conjugated to the HiLyte 555 fluorophore at the C-terminal portion of the translocation-ligand domain and the HiLyte 488 fluorophore at the N-terminal portion of the light chain. Expression of a polypeptide containing SrtA recognition sites and two SrtA variants was successful. Advantageously, by providing a polypeptide that can be labeled with two different colored fluorophores, one can visualize the transport mechanisms of both the light chain (containing a non-cytotoxic protease) and the translocation domain-ligand portions of the protein.

EXAMPLE 2

Construction of SrtA doubly labeled nociceptin ligand polypeptide

A polypeptide having a nociceptin ligand with TM (nociceptin ligand polypeptide) was prepared for double fluorescent labeling using the strategy used for the EGF ligand polypeptide. The design, purification and fluorescent peptides used to double label this polypeptide were exactly the same as for the EGF ligand polypeptide. Successful double labeling of the polypeptide was confirmed by SDS-PAGE gel electrophoresis, confocal microscopy and in vivo imaging. The nucleotide and polypeptide sequences for the polypeptide containing the sortase regions are shown as SEQ ID NOs: 3 and 4, respectively.

Confirmation of labeled proteins using SNAP25 cleavage assay

To determine that labeling polypeptides with ligands does not affect their ability to bind to their respective receptors, transport into cells, and translocation, SNAP25 cleavage assays were performed to determine the relative potency of labeled polypeptides compared to unlabeled versions. A similar efficacy profile may indicate that the labeled polypeptide is transported similarly to the unlabeled version. SNAP25 cleavage assay was performed as previously described (Fonfria, E., S. Donald and V.A. Cadd (2016). "Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments." J Recept Signal Transduct Res 36(1): 79-88). Briefly, cortical neurons were treated with 3-1000 nM each of labeled and unlabeled protein for 24 hours. After treatment, cells were collected in NuPAGE lysis buffer (Thermo Fischer Scientific) supplemented with 0.1 M dithiothreitol and 250 units/ml benzonase (Sigma). Lysates were separated by SDS-PAGE and subjected to Western blotting using primary antibodies against SNAP-25 (Sigma). These antibodies allow recognition of both the cleaved and uncleaved portion of SNAP25. Relative potency was determined by the ratio of cleaved SNAP25 to uncleaved SNAP25 (FIG. 2). Figure 2A shows the dose response efficacy of an EGF ligand polypeptide. Compared to the unlabeled polypeptide, the Texas Red and SNAP594 labeled versions showed a marked decrease in potency with values similar to the control polypeptide without ligand. In contrast, SrtA singly and doubly labeled polypeptides demonstrated similar efficacy to the unlabeled version, showing that this labeling strategy does not affect protein architecture and its cellular transport mechanisms. Similarly, double labeling of the polypeptide with nociceptin ligand did not affect its efficacy in cortical neurons (FIG. 2B) compared to the unlabeled control polypeptide.

In general, simple and clear labeling methods were initially tried, such as non-site-specific labeling using Texas red dye and a SNAP tag, the site-specific version originally tested. However, although these labeling strategies were successful, they were shown to affect the efficiency of the polypeptides compared to the unlabeled counterpart, indicating that the addition of several fluorescent molecules in the case of Texas Red or SNAP tag affected the transport properties of the labeled polypeptide. An attempt to produce an eGFP-tagged polypeptide with an EGF ligand was unsuccessful due to the lack of expression of the tagged protein. In contrast, the SNAP25 cleavage assay confirmed that the two fluorophores on the EGF Ligand and Nociceptin Ligand polypeptides did not affect their efficacy, indicating that the mechanisms of action of the labeled polypeptides are similar to their unlabeled counterparts. This was unexpected due to the negative impact that labeling with SNAP and Texas Red had on performance.

EXAMPLE 3

Visualization of doubly labeled polypeptide with EGF ligand in immortalized cell lines

The SrtA-mediated double labeling method was chosen as the optimal strategy for labeling the polypeptides of the invention. To visualize the labeled polypeptide in mammalian cells, in vivo confocal 3D microscopy was performed. Human lung adenocarcinoma (A549) cells were treated with 50 nM doubly labeled EGF ligand polypeptide and visualized continuously over time using a Zeiss 880 confocal microscope equipped with AiryScan (Zeiss). For these experiments, the EGF ligand polypeptide was labeled at the N-terminus with the HiLyte 555 fluorophore (AnaSpec) and at the C-terminus with the HiLyte 488 fluorophore (AnaSpec). Figure 3 shows photographs of double stained agglomerates formed from the EGF ligand polypeptide during internalization into A549 cells. From figa it can be seen that the agglomerates appeared 3 minutes after adding the polypeptide to the cells, and their size and number increased over time. On figv shows the disappearance of the fluorescent agglomerate over time with complete disappearance after 65 minutes after adding the polypeptide.

In vivo imaging using a doubly labeled EGF ligand polypeptide fully confirmed this labeling method and the ability to monitor intra vivo internalization and transport of labeled polypeptides.

Once it has been demonstrated that sortase labeling is advantageous and does not affect performance, it can then be used for other clostridial neurotoxins, including BoNT serotypes (and derivatives).

EXAMPLE 4

Construction of a BoNT/A polypeptide with SrtA mediated double labeling

The full-length proteolytically inactive BoNT/A(0) mutant (SEQ ID NO: 38) was modified to allow sortase double fluorescent labeling (see FIG. 4). The doubly labeled polypeptide sequence is shown as SEQ ID NO: 40, while the nucleotide sequence encoding said polypeptide is given as SEQ ID NO: 39. Construction, purification, and fluorescent peptides used to double label SEQ ID NO: 40, were the same as for the EGF ligand polypeptide according to Example 1. Successful double labeling of the polypeptide was confirmed by SDS-PAGE (FIG. 5). In more detail, using Coomassie staining, both bands corresponding to the L-chain and H-chain domains of the polypeptide can be visualized, while exposing the gel to UV radiation demonstrated (via fluorescence) successful labeling of both the L-chain and the H-chain .

EXAMPLE 5

Visualization of singly labeled BoNT/A(0) polypeptide in primary cortical neurons

To visualize the labeled BoNT/A(0) polypeptide in primary neuronal cells, in vivo TIRF microscopy of single molecules was performed in neurons treated with it. Primary cortical neurons were treated with 1 nM singly labeled BoNT/A(0) polypeptide and imaged continuously over time using specialized single molecule TIRF microscopy. For these experiments, the BoNT/A(0) polypeptide was labeled at the N-terminus with either HiLyte 555 fluorophore or HiLyte 488 fluorophore (AnaSpec). Figure 6 shows images over time of a singly stained BoNT/A(0) molecule transported to primary cortical neurons. From Figure 6, it can be seen that a single BoNT/A(0) molecule (white arrow) moves rapidly within the selected neuronal region. In vivo TIRF imaging of single molecules of the BoNT/A(0) singly labeled polypeptide clearly demonstrates that single BoNT/A(0) molecules transported into neurons can be visualized using specialized high resolution microscopy techniques.

After demonstrating that single BoNT/A(0) labeling can be visualized at the single molecule level in primary neurons, this method can be further applied to other serotypes and clostridial neurotoxin derivatives, including serotypes with non-cytotoxic protease activity.

All publications mentioned in the above description are included in the present description by reference. Various modifications and variations of the described methods and systems of the present invention will become apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods of carrying out the invention, which are obvious in the field of biochemistry and biotechnology or related fields, are intended to be included in the scope of the following claims.

Claims (74)

1. A method for producing a labeled polypeptide, including: a. obtaining a polypeptide containing: i. an acceptor site for sortase or a donor site for sortase; ii. a non-cytotoxic protease containing the L-chain of clostridial neurotoxin, or a proteolytically inactive mutant thereof; iii. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And iv. translocation domain; b. incubation of the polypeptide with: sortase; And a labeled substrate containing a sortase donor site or an acceptor site for sortase, respectively, and a conjugated detectable label; where sortase catalyzes: conjugation between an amino acid of a sortase acceptor site in the polypeptide and an amino acid of a sortase donor site in the labeled substrate; or conjugation between an amino acid of a sortase acceptor site in the labeled substrate and an amino acid of a sortase donor site in the polypeptide; thereby effecting labeling of the polypeptide; And c. obtaining a labeled polypeptide, where the labeled polypeptide has no reduced efficiency compared to the equivalent unlabeled polypeptide and/or where the labeled polypeptide shows similar cell binding, translocation and cleavage of the SNARE protein with the equivalent unlabeled polypeptide. 2. The method according to claim 1, where: a. the sortase acceptor or donor site is C-terminal of the TM or where the sortase acceptor or donor site is N-terminal of the non-cytotoxic protease or a proteolytically inactive mutant thereof; and/or b. i. the acceptor site for sortase contains: L(A/P/S)X(T/S/A/C)(G/A); NPQTN; YPRTG; IPQTG; VPDTG; or LPXTGS, where X is any amino acid; and/or ii. where the donor site for sortase contains G _n or A _n where n is 1-10; or c. i. the acceptor site for sortase contains: L(A/P/S)X(T/S/A/C)G, where X is any amino acid; NPQTN; YPRTG; IPQTG; VPDTG; or LPXTGS, where X is any amino acid; and/or ii. where the donor site for sortase contains G _n where n is 1-10; and/or d. sortase is sortase A (SrtA). 3. The method according to claim 1, where: a. the sortase acceptor or donor site is C-terminal of the TM or where the sortase acceptor or donor site is N-terminal of the non-cytotoxic protease or a proteolytically inactive mutant thereof; and/or b. i. L(A/P/S)X(T/S/A/C)G, where X is any amino acid; NPQTN; YPRTG; IPQTG; VPDTG; or LPXTGS, where X is any amino acid; and/or ii. where the donor site for sortase consists of G_nor A_n, where n equals 1-10; or c. i. the acceptor site for sortase consists of: L(A/P/S)X(T/S/A/C)G, where X is any amino acid; NPQTN; YPRTG; IPQTG; VPDTG; or LPXTGS, where X is any amino acid; and/or ii. where the donor site for sortase consists of G _n where n is 1-10; and/or d. sortase is sortase A (SrtA). 4. The method according to claim 1, where the polypeptide contains at least two acceptor sites for sortase; at least two sortase donor sites; or at least one sortase acceptor site and at least one sortase donor site. 5. The method according to claim 4, where at least two sections are different, preferably where at least two sections have different amino acid sequences, Where: the first sortase acceptor or donor site is C-terminal of the TM and the second sortase acceptor or donor site is N-terminal of the non-cytotoxic protease or a proteolytically inactive mutant thereof; or the first sortase acceptor or donor site is N-terminal of the non-cytotoxic protease or a proteolytically inactive mutant thereof; and the second sortase acceptor or donor site is C-terminal of the TM. 6. Labeled polypeptide for its visualization, containing: i. a detectable label conjugated to the polypeptide; ii. amino acid sequence that contains: a. L(A/P/S)X(T/S/A/C)G _n where X is any amino acid and n is 1-10; b. L(A/P/S)X(T/S/A/C)A _n , where X is any amino acid and n is 1-10; c. NPQTN; d. YPRTG; e. IPQTG; f. VPDTG; or g. LPXTGS, where X is any amino acid; iii. a non-cytotoxic protease containing the L-chain of clostridial neurotoxin, or a proteolytically inactive mutant thereof; iv. a targeting portion (TM) that is capable of binding to a binding site on a target cell; And v. translocation domain, where the labeled polypeptide has no reduced potency compared to an equivalent unlabeled polypeptide and/or where the labeled polypeptide exhibits similar cell binding, translocation and cleavage of the SNARE protein to an equivalent unlabeled polypeptide. 7. Labeled polypeptide according to claim 6, where: a. an amino acid sequence that contains: L(A/P/S)X(T/S/A/C)G _n ; L(A/P/S)X(T/S/A/C)A _n ; NPQTN; YPRTG; IPQTG; VPDTG; or LPXTGS, located on the C-terminal side of the TM; or b. where the amino acid sequence that contains: L(A/P/S)X(T/S/A/C)G _n ; L(A/P/S)X(T/S/A/C)A _n ; NPQTN; YPRTG; IPQTG; VPDTG; or LPXTGS, is N-terminal to a non-cytotoxic protease or a proteolytically inactive mutant thereof. 8. A labeled polypeptide according to claim 6, containing an additional detectable label conjugated to the polypeptide and an additional amino acid sequence that contains: a. L(A/P/S)X(T/S/A/C)G _n where X is any amino acid and n is 1-10; b. L(A/P/S)X(T/S/A/C)A _n , where X is any amino acid and n is 1-10; c. NPQTN; d. YPRTG; e. IPQTG; f. VPDTG; or g. LPXTGS where X is any amino acid. 9. Labeled polypeptide according to claim 8, where a. the amino acid sequence of claim 6 is different from the additional amino acid sequence; and/or b. the amino acid sequence of claim 6 is C-terminal of TM and the additional amino acid sequence is N-terminal of the non-cytotoxic protease or proteolytically inactive mutant thereof; or the amino acid sequence of claim 6 is N-terminal from the non-cytotoxic protease or proteolytically inactive mutant thereof, and the additional amino acid sequence is from the C-terminal side of TM. 10. The method according to any one of paragraphs. 1-5 or labeled polypeptide according to any one of paragraphs. 6-9, where the translocation domain includes a clostridial neurotoxin translocation domain; and/or where TM is an _HC -peptide of a clostridial neurotoxin. 11. The method according to any one of paragraphs. 1-5 or labeled polypeptide according to any one of paragraphs. 6-9, where the polypeptide is a clostridial neurotoxin; and/or where the polypeptide is a botulinum neurotoxin (BoNT); and/or where the polypeptide is selected from: BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X or TeNT. 12. The method according to any one of paragraphs. 1-5 or labeled polypeptide according to any one of paragraphs. 6-9, where the polypeptide does not contain a functional H domain_WITHclostridial neurotoxin. 13. A method for determining at least the internalization and/or intracellular transport of a labeled polypeptide, comprising: a. bringing the target cell into contact with the labeled polypeptide according to claim 6; And b. detecting a detectable label. 14. The method according to any one of paragraphs. 1-5 or 13 or a labeled polypeptide according to any one of paragraphs. 6-9 wherein the detectable label is a fluorescent dye, preferably wherein the fluorescent dye is selected from: HiLyte, AlexaFluor, Atto, Quantum Dots and Janelia Fluor. 15. The method according to any one of paragraphs. 1-5 or 13 or a labeled polypeptide according to any one of paragraphs. 6-9, wherein the labeled polypeptide contains two or more detectable labels, preferably wherein the two or more detectable labels are different colored fluorophores.

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