Method of intracellular peptide ligation

Abstract

Disclosed herein are methods and kits for producing ligated peptides. The methods comprise culturing a cell comprising a first and second peptide sequences and an AEP catalytic domain construct, such that the AEP catalytic domain construct hydrolyses an Asn-containing recognition sequence of the first peptide sequence and ligates the resulting Asn-containing sequence to the N-terminus of the second peptide sequence such that the first peptide sequence is ligated to the second peptide sequence. The methods and kits find application, for example, in the identification of transcription factor antagonists.

Description

[0001] Method of intracellular peptide ligation This application claims priority from GB2315373.7 filed 6 October 2023, the contents and elements of which are herein incorporated by reference for all purposes. Field of the Invention The present disclosure relates to methods of intracellular peptide ligation and particularly, although not exclusively, to a method of peptide cyclisation, and ligated peptides obtained according to said method. The present disclosure further relates to methods of preparing and screening peptide libraries, use of an AEP catalytic domain construct for use in a method of intracellular peptide ligation, and kits for use in any of the methods described. Background Asparaginyl endopeptidase (AEP) are proteases present in the vacuole of plant cells (Hara-Nishimura et al., 1993). AEPs react selectively at an internal asparagine (Asn) or Aspartate (Asp) residue of a peptide backbone. Pro-AEP is synthesised as an inactive precursor (zymogen) that adopts a dimeric state at neutral pH which prevents enzymatic activity that would result in unwanted degradation of different substrates. A monomeric state is adopted at pH 4.0 and the protonation of salt bridges surrounding the active site of the AEP enables the auto-proteolytic cleavage of the pro-domain to generate the active enzyme (Tang et al., 2021). In addition to AEP’s proteolytic function leading to degradation or activation of different substrates, some AEPs also show non-proteolytic ligase activity which facilitates post- translational modification to produce backbone cyclised peptides (Tang et al., 2021). The catalytically active core domain of AEP hydrolyses the C-terminal amide bond of an internal Asn or Asp residue present in a short recognition sequence P1-P1’-P2’ and mediates a nucleophilic attack on the amide carbonyl of the internal Asn to form a thioester acyl enzyme (Harris et al., 2015). For most AEPs, a water molecule acts as nucleophile, thereby completing the peptide bond hydrolysis (Dall et al., 2013). In addition to AEP’s proteolytic function leading to degradation or activation of different substrates, some AEPs also show non-proteolytic ligase activity. For ligase-type AEPs, the N-terminal amine of an incoming peptide adopts the role of the nucleophile to resolve the thioester intermediate. The nucleophilic amine can be either from the same peptide, resulting in cyclisation of the peptide, or from a different peptide, resulting in an intermolecular ligation (Harris et al., 2015; Nguyen et al., 2014; Nguyen et al., 2016). Legumain is the only known mammalian AEP that is highly expressed in different tissues such as kidney, liver, spleen, placenta, and testis. Similar to other non-mammalian AEPs, legumain shows both proteolytic activity and non-proteolytic ligase activity (Solberg et al., 2022). Legumain activity is tightly regulated, both by its environment through pH dependent activation, and by endogenous inhibitors to prevent potentially damaging proteolysis inside the cell. Indeed, increased legumain expression and / or activity has been linked to age-related degenerative diseases, neurodegeneration, and cancer (Solberg et al., 2022; Song et al., 2022). Cyclisation is a powerful strategy to enhance stability of proteins and peptides (Hayes et al., 2021). Cyclic peptides in plants demonstrate superior stability compared with their linear counterparts (Colgrave et al., 2004). In peptide drug discovery, cyclisation is often employed to improve drug-like properties as constraining the peptide into the bioactive conformation can enhance target binding affinity (Sohrabi et al., 2020; Lu et al., 2018). Moreover, head-to-tail (H2T) cyclisation removes cleavable N- and C-termini which improves peptide stability against exopeptidases (Qian et al., 2017) and can assist in conferring cell penetrance owing to the removal of the amnio- and carboxy-terminal charges. Protein cyclisation can also improve thermal stability (Si et al., 2016; Scott et al., 1999) a highly desirable attribute for industrially relevant enzymes (Rigoldi et al., 2018). However, the production of cyclic peptides is challenging and inefficient, requiring high temperatures (typically around 50-60 ºC). In addition, it often employs toxic and unsustainable reagents for chemical synthesis, such as amide coupling reagents and organic solvents. AEPs such as butelase 1 and Oldenlandia affinis asparaginyl endopeptidase 1 (OaAEP1) have been employed to facilitate peptide ligation and H2T cyclisation reactions in vitro. OaAEP1 facilitates H2T cyclisation of peptides during the biosynthesis of cyclotides via an Asn-containing recognition sequence in the plant species Oldenlandia affinis (Harris et al., 2015). Mutagenesis studies of OaAEP1 demonstrated that substitutions of the gatekeeper residue (Cys247) to larger residues such as Ile, Leu and Met abolished ligase activity, whereas replacements with smaller Ala and Gly residues at the same position resulted in an increase in peptide cyclisation activity (Yang et al., 2017). Peptide ligation by the engineered OaAEP1-C247A variant proceeds with impressive catalytic efficiency and offers in site- specific protein modification to study post-translational modification in cell biology research (Harmand et al., 2021), production of protein-drug conjugation (Rehm et al., 2019) and protein semi-synthesis (Mikula et al., 2018; Mikula et al., 2017). WO 2018 / 056899 describes the use OaAEPI Cys247Ala in methods of extracellular peptide ligation. Recombinant OaAEP1-C247A is commonly prepared as an inactive zymogen, that prevents unwanted substrate proteolysis. The zymogen can only be activated in acidic conditions (pH 3.6 – 4.0) after purification (Yang et al., 2017; Harmand et al., 2017; Mikula et al.2018; Mikula et al., 2017; Rehm et al., 2019). To simplify recombinant preparation and improve usability of OaAEP1-C247A, studies have been performed to identify truncated yet functional constructs which bypasses this acid activation step (Tang et al., 2020). While these biocatalytic approaches offer a much safer and more sustainable alternative, they require additional purification during recombinant preparation which is time and labour intensive. Existing approaches to direct intracellular peptide ligation include the use of transpeptidase (Strijbis et al., 2012) and protein tags such as intein (Shah et al., 2012; Tavassoli et al., 2007) and SpyTag / SpyCatcher. However, long recognition sequences are needed for sortase A-mediated ligation (LPXTG) (Strijbis et al., 2012; Antos et al., 2017) and the SpyTag / Catcher system (Si et al., 2016). As recognition sequences remain in the modified protein, these methods introduce a traceable scar which can alter the native protein fold. Ligation by intein splicing proceeds with a small ligation scar (C / S), however splice rates are slow (k = 3.7 × 10−2 s−1 for Npu intein), with solubility issues reported in some intein-fused constructs (Luk et al., 2015). Consequently, there remains a need for improved methods for generating ligated peptides intracellularly. The present invention has been devised in light of the above considerations. Summary of the Invention The present inventors have showed, for the first time, that it is possible to carry out AEP catalysis in a live recombinant organism outside of plant species without disrupting intracellular biochemical processes and causing cytotoxicity. This absence of any cytotoxic effects upon intracellular expression of the AEP enzyme was surprising in view of the tight control mechanisms that exist for activation of naturally occurring AEPs. An enzyme construct containing only the catalytic core domain of an AEP was shown to enzymatically cleave and ligate a peptide of interest in live recombinant E. coli cells. The recombinant intracellular expression of this catalytic core domain bypasses the zymogen stage so that cells can be used for intracellular screening assays without the need for further activating steps, which normally take place in vitro under acidic conditions, not compatible with culturing living cells. The ability to introduce post-translational modifications within a host organism during recombinant expression offers a significant advantage over in vitro approaches as it negates the need to purify individual protein components thus enabling more efficient peptide ligation. Moreover, it offers a sustainable and cost-effective method to produce high-value modified peptides and proteins and novel applications such as in situ modification during intracellular screening for peptide drug discovery. Accordingly, in a first aspect, the present invention provides method of intracellular peptide ligation, the method comprising the steps of: In a first aspect, there is provided a method of intracellular peptide ligation, the method comprising the steps of: a) providing a cell comprising: i. a first peptide sequence comprising P1-P1’- P2’-Xn-COOH / CONH2, wherein P1 is Asn; P1’ is any amino acid except Pro; P2’ is an amino acid selected from the group Phe, Ile, Leu, Met, and Trp; X is any amino acid and n is an integer of 0 or 1 or more, ii. a second peptide sequence comprising H2N-P1”-P2” wherein P1” and P2” are any amino acid; and iii. an asparaginyl endopeptidase (AEP) catalytic domain construct, b) culturing the cell comprising the first and second peptide sequences and the AEP catalytic domain construct, such that the AEP catalytic domain construct hydrolyses the bond between P1 and P1’ of the first peptide sequence and ligates the resulting Asn-containing sequence to the N- terminus of the second peptide sequence such that the first peptide sequence is ligated to the second peptide sequence. In some embodiments, the AEP catalytic domain construct comprises P1-P1’- P2’-Xn-COOH / CONH2 at its C terminus, wherein P1 is Asn; P1’ is any amino acid except Pro; P2’ is an amino acid selected from the group Phe, Ile, Leu, Met, and Trp; X is any amino acid and n is an integer of 0 or 1 or more, and H2N-P1”-P2” at its N terminus, wherein P1” and P2” are any amino acid. In some embodiments, the first peptide sequence and the second peptide sequence are part of the same peptide. In some embodiments, the first peptide sequence, the second peptide sequence, and the AEP catalytic domain construct are part of the same peptide. In some embodiments, P1’ is any naturally occurring amino acid except Pro; and / or P2’ is a naturally occurring amino acid selected from the group Phe, Ile, Leu, Met, and Trp; and / or X is any naturally occurring amino acid; and / or P1” is any naturally occurring amino acid, and / or P2” is a naturally occurring amino acid, optionally a naturally occurring amino acid selected from Val, Leu, or Ile. In some embodiments, P1’ is Cys or Gly; and P2’ is Leu; P1” is Gly; and P2” is Leu. In some embodiments, the AEP catalytic domain construct comprises the catalytic domain of Oldenlandia affinis, oaAEP1, or a mutant thereof. For example, the oaAEP1 mutant may comprise a C247A mutation (“C247 mutant”). In some embodiments, the AEP catalytic domain construct is as set out in SEQ ID NO: 5 (“C247A mutant”) or a variant thereof. The variant may have at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5. In preferred embodiments, the variant has at least 95% sequence identity to SEQ ID NO: 5. In some embodiments the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions. In preferred embodiments, the variant has no more than 10 substitutions. In some embodiments, the variant comprises or consists of a sequence according to SEQ ID NO: 6. In some embodiments, the variant has one or more functional properties that are at least the same compared to the C247A mutant (SEQ ID NO: 5). The one or more functional properties may comprise activity, stability, kinetic properties and / or solubility. In some embodiments, the variant has at least the same activity compared with the C247A mutant. optionally wherein the activity is determined by measuring the cyclisation of a substrate peptide, optionally wherein the substrate peptide is GLPVSTKPVATRNGL (SEQ ID NO: 9) or GLPYSTKPYATRNGL (SEQ ID NO: 20). Measuring the cyclisation of substrate peptide may be carried out by liquid chromatography-mass spectrometry (LC–MS). In some embodiments, the variant has at least the same stability compared with the C247A mutant. In preferred embodiments, the stability is determined by a thermal shift assay. In some embodiments, the variant has at least the same solubility compared with the C247A mutant. The solubility may be determined by measuring the absorbance of the variant at 280 nm, or by using a biuretic methods (e.g. BCA assay, Lowry assay), colorimetric dye based method (e.g. Bradford (Coomassie) assay), or fluorescent dye method. In some embodiments, the variant has at least the same kinetic properties compared with the C247A mutant. The kinetic properties may be determined by fluorescence energy transfer (FRET), surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC). In some embodiments, the AEP catalytic domain construct is cyclic. In some embodiments, the first peptide fragment and / or second peptide fragment comprises a labelling moiety. In some embodiments, the labelling moiety is an affinity tag, e.g. a His tag, and / or a detectable label, e.g. a GFP tag, and / or degradation tag, e.g. a SSRA tag. In some embodiments, the cell is a bacterial cell, optionally an Escherichia coli cell. In some embodiments, the method is performed at a temperature lower than 50 ºC, lower than 40 ºC, or lower than 35 ºC. In some embodiments, method is performed at a temperature of between about 30 ºC and about 37 ºC. In some embodiments, the method is performed at a temperature of about 30 ºC. In some embodiments, the method is carried out in the absence of amide coupling reagents, and / or in the absence of organic solvents. In some embodiments, the method further comprises isolating the ligated peptide from the cell. In some embodiments, providing in the cell the first and second peptide sequence comprises delivering one or more nucleic acid(s) encoding the first and second peptide sequences to the cell, such that the cell expresses the first and second peptide sequences. In some embodiments, providing in the cell the AEP catalytic domain construct comprises delivering one or more nucleic acid(s) encoding the AEP catalytic domain construct, such that the cell expresses the AEP catalytic domain construct. In a second aspect, there is provided a ligated peptide and / or cells obtainable according to any of the preceding claims. In a third aspect, there is provided a method for screening peptide libraries for transcription factor antagonists, the method comprising; a) providing a cell, wherein the cell comprises a peptide ligated according to the method of the first aspect of the invention, and first and second candidate binding partners, and b) assaying whether the ligated peptide is able to inhibit association between the first and second candidate binding partner. In some embodiments, assaying for whether the ligated peptide is able to inhibit association between the first and second candidate binding partners comprises determining whether the ligated peptide is able to modulate expression and / or activity of a reporter protein. In some embodiments, association of the first and second candidate binding partners forms a DNA- binding complex that binds one or more binding site(s) in a nucleic acid encoding the reporter protein, wherein binding of the DNA-binding complex to the binding site inhibits expression of the reporter protein. In some embodiments, some or all of the binding sites are located in the transcribed sequence of the nucleic acid encoding the reporter protein. In some embodiments, the first and second candidate binding partners are transcription factors, for example a bZIP transcription factor, e.g. c-Jun, CREB, and / or BZLF1. In a fourth aspect, there is provided an AEP catalytic domain construct for use in a method of intracellular peptide ligation. In some embodiments, the AEP catalytic domain comprises the catalytic domain of Oldenlandia affinis, oaAEP1, or a mutant thereof. In some embodiments, the oaAEP1 mutant comprises a C247A mutation (“C247 mutant”). In some embodiments, the AEP catalytic domain construct is as set out in SEQ ID NO: 5 (“C247A mutant”) or a variant thereof. In some embodiments, the variant has at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5, optionally wherein the variant has at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions, optionally wherein the variant has no more than 10 substitutions. In some embodiments, the variant comprises or consists of a sequence as set out in SEQ ID NO: 6. In some embodiments, the variant has one or more functional properties that are at least the same, or improved, compared with the C247A mutant of SEQ ID NO: 5. The one or more functional properties may comprise kinetic properties and / or solubility. In some embodiments, the AEP catalytic domain construct is cyclic. In a fifth aspect, there is provided a kit for use in a method according to the first and / or second aspect of the invention, wherein the kit comprises an AEP catalytic domain construct. The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. Summary of the Figures Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which: Figure 1 shows a schematic of the recombinant gene expression system for linear (red box) and cyclic mDHFR (green box). Figure 2 shows the plasmid map and amino acid sequence of the mDHFR substrate. The protein (amino acid sequence in orange) was appended with a short linker, His6 tag (in blue) for purification and AEP recognition sites at the N and C termini (black, underlined). NGL sequence near the N-terminus enables OaAEP1 to remove the upstream amino acids, revealing the cognate N-terminal recognition sequence (GL) for OaAEP1-mediated cyclisation. Figure 3 shows the plasmid map and amino acid sequences of recombinant OaAEP1-C247A employed for the production of cyclic mDHFR. Residues 24-328 of OaAEP1 (black, underlined) was employed with a C247A mutation. The enzyme was appended with a His6 tag (in blue) to enable purification and a ubiquitin fusion (in orange) to enhance solubility. Figure 4 shows SDS PAGE analysis of the protein contents in the host cell upon IPTG-induced gene expression (left), purified cyclic and linear mDHFR (right). Figure 5 shows the deconvoluted mass spectra of purified (A) cyclic and (B) linear mDHDR, demonstrating that upon cyclisation, 590Da are lost. Figure 6 shows circular dichroism (CD) analysis of cyclic and linear mDHFR (10 µM). (A) CD spectra (20 °C) of mDHFR before and after thermal denaturation. (B) Thermal denaturation profile by monitoring the MRE at 209 nm indicate an increase in Tm of 3.7 °C for the H2T cyclic protein. Fraction of unfolded protein was calculated using equation (1). Tm were estimated by converting the data to MRE and fitting to the Boltzmann sigmoidal equation using OriginPro graphing and analysis software. Tm is reported here as mean average of triplicate experiments with standard error of the mean. Figure 7 shows a schematic of the mDHFR catalysed reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as cofactor. Figure 8 shows an activity assay for cyclic mDHFR following the change in absorbance at 340nm by NADPH during a mDHFR catalysed reaction. UV / Vis analysis monitoring the change in absorbance at 340 nm for turnover of NADPH at 20 °C (closed circles) showing comparable activity for both linear and cyclic versions. Following incubation at 45 °C for 15 mins, the experiment was repeated at 20 °C and mDHFR activity again monitored (open circles). Specific activity was calculated from the linear initial rate (first 2.5 min) using equation (2). Data reported are averages with standard error of the mean from triplicate experiments. Figure 9 shows UV / Vis analysis monitoring the change in absorbance at 340 nm for turnover of NADPH at 20 °C. Linear (red) and cyclic (green) mDHFR were incubated at 20 °C (filled circle), 45 °C (hollow circle), or 65 °C (triangle) before use in the assay (performed at 20 °C). A negative control of DHF and NADPH with no enzyme was employed as a blank (black filled circle). Figure 10 shows Electrospray Mass Spectrometry analysis of recombinantly produced Kalata B1 according to SEQ ID NO: 8 extracted from E. coli using 50% Acetonitrile with 1% Formic acid. The 2889.1 indicates the presence of cyclised Kalata B1, which has an expected mass of 2890.14 Da. Figure 11 shows the intracellular activity of the OaAEP1 variant according to SEQ ID NO: 7. (A) SDS - page results of mDHFR co-expressed with OaAEP1 variants identified using a library screen as well as the OaAEP1-C247A (“WT”) enzyme. Hit 4 corresponds to the OaAEP1 mutant according to SEQ ID NO: 7 and is able to cyclise mDHFR whereas the enzyme variants corresponding to “Hit1” and “Hit 3” are not. (B) shows the mass-spec results of mDHFR co-expressed with the OaAEP1 variant corresponding to “Hit 4”. The expected and observed masses were as follows: Cyclic mDHFR: Expected mass = 22990.44, Observed mass = 22991.01; Linear mDHFR: expected mass = 23580.15, observed mass = 23581.00; Cyclic MRN-mDHFR = expected mass: 23391.93, observed mass: 23391.90. Figure 12 shows the in vitro activity of the OaAEP1 variant according to SEQ ID NO: 7. (A) Shows that the OaAEP1 variant (Frac 16) can be purified for in vitro applications using size exclusion chromatography. The band around 36.5Da indicates the presence of the enzyme. (B) shows the mass- spec results of the substrate sequence according to SEQ ID NO: 9 incubated with a control (top) and OaAEP1 variant (bottom). Figure 13 shows the results of an SDS PAGE analysis of the protein contents in E. coli upon IPTG- induced expression of OaAEP1 (SEQ ID NO: 21) after overnight incubation at 18º and 25ºC. Expected mass for the linear peptide is 31.5 kDa. Expected mass for the cyclised protein is 31.0 kDa. Detailed Description of the Invention Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference. Described herein are methods of intracellular peptide ligation, comprising the steps of: a) providing a cell comprising: i. a first peptide sequence comprising P1-P1’- P2’-Xn-COOH / CONH2, wherein P1 is Asn; P1’ is any amino acid except Pro; and P2’ is an amino acid selected from the group Phe, Ile, Leu, Met, and Trp; and X is any amino acid and n is an integer of 0 or 1 or more; ii. a second peptide sequence comprising H2N-P1”-P2” wherein P1” and P2” are any amino acid; and iii. an asparaginyl endopeptidase (AEP) catalytic domain construct, b) culturing the cell comprising the first and second peptide sequences and the AEP catalytic domain construct, such that the AEP catalytic domain construct hydrolyses the bond between Asn and P1’ of the first peptide sequence and ligates the resulting Asn-containing sequence to the N-terminus of the second peptide sequence such that the first peptide sequence is ligated to the second peptide sequence. The method as described herein is performed intracellularly, that is within a live cell. The term “intracellular” is intended to encompass experiments that take place involving cells and may be in cultured cells or may be in cells or tissues that have been taken from an organism. The methods described herein are not practiced on the human or animal body. The cell used in the methods described herein may be a prokaryotic cell or a eukaryotic cell. Typically, cells are isolated cells. The cell used in the methods described herein may be a bacterial cell, such as an Escherichia coli cell, for example BL21 (DE3), XL-1, RV308, Shuffle T7, or DH5alpha cells. Methods where the cell is a bacterial cell may involve culturing the bacterial cell in suitable media. Such techniques are well known to those of skill in the art. Alternatively, the cell is a eukaryotic cell such as a yeast cell (e.g such as Saccharomyces cerevisiae or Pichia pastoris), a plant cell, insect cell or a mammalian cell. In some embodiments, the cell is a mammalian cell, for example a human cell. Mammalian cells, especially human cells, may be somatic cells. Screening methods where the cell is a eukaryotic cell may involve culture or fermentation of the eukaryotic cell. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Culture, fermentation and separation techniques are well known to those of skill in the art. As used herein, “peptide ligation” is intended to mean the formation of a new peptide bond between two peptide sequences. In some embodiments, the peptide bond is formed between two ends of the same peptide, i.e. between a first and second peptide sequence of the same peptide, resulting in intramolecular cyclisation of the peptide. A peptide comprising the first and second peptide sequences which can be added to the cell may also be referred to herein as a ‘peptide to be cyclised’ and following cyclisation referred to as a ‘cyclised peptide’. In some embodiments, the peptide may be Kalata B1. Kalata B1 (UNIPROT ID: P56254) is a cyclotide derivable from the African plant Oldenlandia Affinis. The propeptide of Kalata B1 exists as a precursor protein, which may be cut by an asparaginyl endopeptidase (AEP) catalytic domain construct according to the invention, followed by ligation of the N- and C-termini of the processed peptide, resulting in a 29 amino acid long cyclic product (Yayci et al., 2024). In some embodiments, the first peptide, second peptide, and AEP catalytic domain construct are part of the same peptide, resulting in auto-cyclisation of the AEP catalytic domain construct. In alternative embodiments, the peptide bond is formed between two peptide sequences that are part of different peptides, thus resulting in intermolecular ligation of the two peptides. Techniques for assessing peptide ligation and / or cyclisation, such as Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-page), High-Performance Liquid Chromatography (HPLC), and / or mass spectrometry (MS) are well known to those of skill in the art. Peptide ligation is mediated by an asparaginyl endopeptidase (AEP) catalytic domain construct. AEP, as used herein refers to endopeptidase identified from the vacuole of plant cells that selectively hydrolyses substrates at an Asn-containing recognition sequence (P1-P1’-P2’). AEPs described herein have ligase activity. Examples of AEP variants with ligase activity include those derived from varies plant species including Oldenlandia Affinis (OaAEP1b, OaAEP3, OaAEP4, OaAEP5), Clitoria Ternatea (Butelase 1); Helianthus annuus (HaAEP1), Viola yedonesis (VyPAL2) and Momordica cochinchinensis (MCoAEP2). The AEP catalytic domain construct used in methods of the invention may comprise the recognition sequence so that it is able to auto-ligate. That is, the AEP catalytic domain may comprise P1-P1’- P2’-Xn- COOH / CONH2 at its C terminus and H2N-P1”-P2” at its N terminus, such that it self-catalyses and ligates resulting in a cyclic AEP catalytic domain construct. Cyclisation of the AEP catalytic domain construct may result in enhanced stability compared with a linear construct. Thus, in some embodiments, the AEP catalytic domain construct comprises P1-P1’- P2’-Xn-COOH / CONH2 at its C terminus, wherein P1 is Asn; P1’ is any amino acid except Pro; P2’ is an amino acid selected from the group Phe, Ile, Leu, Met, and Trp; X is any amino acid and n is an integer of 0 or 1 or more, and H2N-P1”-P2” at its N terminus wherein P1” and P2” are any amino acid. Structurally, AEPs include a catalytic core domain which adopts a protein fold with six stranded β-sheets surrounded by five α-helices. In the proenzyme, the prodomain prevents substrate access to the catalytic domain. Activation of the catalytic domain takes place upon a change from neutral to acidic pH, which enables the cleavage of the pro-domain to generate the active enzyme (Tange et al., 2021). The methods described herein use an AEP catalytic domain construct, also referred to as the catalytic core domain construct, which is intended to cover the whole or part (also referred to as “truncated form”) of the AEP catalytic core domain, but always excluding the prodomain. The omission of the prodomain means that acidic conditions, i.e. low pH, are not required for the enzyme activation, which normally involves multiple purification steps. In some embodiments the AEP catalytic domain construct comprises the catalytic domain of Oldenlandia affinis, OaAEP1. The AEP catalytic domain construct may comprise a mutant OaAEP1 catalytic domain. In some embodiments, the OaAEP1 mutant catalytic domain comprises a C247A mutation. In some embodiments, the AEP catalytic domain is as set out in SEQ ID NO: 5 (C247A mutant), or a variant thereof. In some embodiments, the variant has one or more functional properties that are at least the same, i.e., not substantially worse, or improved compared to the C247A mutant. A variant that is not substantially worse than the C247A mutant has a functional property that is at least 90% of the functional property of the C247A mutant when measured using a suitable assay. Functional properties as described herein include, but are not limited to, properties such as enzymatic activity, stability, solubility, and / or kinetic properties. Methods for testing such properties are known to those skilled in the art. Methods for testing enzymatic activity of the AEP catalytic domain construct include a peptide cyclisation assay as described in Tang et al., 2020. Briefly, activity of the variant may be tested by measuring the cyclisation of a substrate peptide, such as ‘GLPVSTKPVATRNGL’ (SEQ ID NO: 9), using liquid chromatography-mass spectrometry (LC–MS), where increased concentrations of cyclised substrate peptide compared with linear substrate indicate increased enzymatic activity. Accordingly, in some embodiments, the variant has at least the same activity compared with the C247A mutant as determined by measuring the cyclisation of a substrate peptide. The substrate peptide may be GLPVSTKPVATRNGL (SEQ ID NO: 9) or GLPYSTKPYATRNGL (SEQ ID NO: 20). Testing the cyclisation of substrate peptide may be carried out by liquid chromatography-mass spectrometry (LC–MS). Assays for determining stability include thermal shift assays, which measure changes in thermal denaturation temperature of the enzyme. The person skilled in the art will be familiar with different methods for measuring thermal shifts, including differential scanning fluorimetry (DSF) (Huynh et al., 2015). Accordingly, in some embodiments, the variant has at least the same stability compared with the C247A mutant as determined by a thermal shift assay. Methods for determining enzyme solubility are well-known and include measuring the absorbance at 280 nm (UV range), biuretic methods (e.g. BCA assay, Lowry assay), colorimetric dye based methods (e.g. Bradford (Coomassie) assay), fluorescent dye methods. Accordingly, in some embodiments the variant has at least the same solubility compared with the C247A mutant as determined by measuring the absorbance of the variant at 280 nm (where higher absorbance indicates improved solubility), or by using a biuretic methods (e.g. BCA assay, Lowry assay), colorimetric dye based method (e.g. Bradford (Coomassie) assay), or fluorescent dye method. Assays for measuring kinetic properties are well known and include real time methods such as fluorescence energy transfer (FRET) (for example, as described in Ma et al, 2023), biolayer interferometry (BLI), surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC). Kinetic properties as used herein refers to, for example, KM and Kcat. In some embodiments, the variant may comprise one or more modifications, such as insertions, deletions, or substitutions. In some embodiments, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modifications (e.g. substitutions). In some embodiments, the variant comprises between 1 and 10, between 2 and 10, between 3 and 10, between 4 and 10, between 5 and 10, between 6 and 10, between 7 and 10, between 8 and 10, or between 9 and 10 substitutions. In some embodiments, the variant comprises between 1 and 9, 2 and 9, 3 and 9, 4 and 9, 5 and 9, 6 and 9, 7 and 9, 8 and 9, 1 and 7, 2 and 7, 3 and 7, 4 and 7, 5 and 7, 6 and 7, 1 and 5, 2 and 5, 3 and 5, 4 and 5, 1 and 4, 2 and 4, 3 and 4, 1 and 3, 1 and 2 substitutions. In some embodiments, the variant has not more than 15 substitutions. In some embodiments, the variant has not more than 10 substitutions. In some embodiments, the variant has not more than 9 substitutions. In some embodiments the variant has at least 60% sequence identity to the amino acid sequence of the C247 mutant (SEQ ID NO: 5), more preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the variant comprises or consists of a sequence according to SEQ ID NO: 6. In some embodiments, the AEP catalytic domain construct is cyclic. Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in a comparative sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences. Where the aligned sequences are of different length, sequence identity of the shorter comparison sequence may be determined over the entire length of the longer given sequence or, where the comparison sequence is longer than the given sequence, sequence identity of the comparison sequence may be determined over the entire length of the shorter given sequence. Sequence identity may be defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). Methods for producing an AEP catalytic domain construct are well known in the art and include DNA recombinant technology. For example, the AEP catalytic domain may be cloned and expressed in in E. coli as an ubiquitin fusion protein followed by isolation ion exchange and size exclusion as described in Harris et al., 2015, and Tang et al., 2020. The terms “peptide” and “peptide sequence” are used interchangeably herein and refer to chains of amino acids linked by peptide bonds. The first peptide sequence as described herein comprises P1-P1’- P2’-Xn-COOH / CONH2. P1, P1’, P2’ as referred to herein refer to amino acid residues surrounding the cleavage sites for substrates of AEP according to the numbering conventions adopted by Schechter and Berger for substrates of proteases (Schechter et al., 1967). In brief, from the cleavage site, that is between Asn (or P1 as per the numbering convention of Schechter and Berger) and P1’, to the N-terminus, amino acid residues are numbered in ascending order starting from P1 (i.e. P2, P3, P4,… Pn). Amino acids P2 to Pn may be any amino acid. It is to be understood that the first peptide sequence may comprise any number of amino acids between the cleavage site, starting with P2, and its N-terminus. In other words, the first peptide sequence may be any peptide sequence, provided it comprises P1-P1’- P2’-Xn-COOH / CONH2. Accordingly, in some embodiments, the first peptide sequence comprises the sequence Pz-P1-P1’- P2’-Xn-COOH / CONH2, wherein P is any amino acid, more preferably any naturally occurring amino acid, and z is an integer of 1 or more (e.g.2 or more, 3 or more, 4 or more, 5 or more, …etc). On the other side of the cleavage site amino acids are denoted with a single prime and numbered in ascending order towards the C-terminus starting with P1. For peptide ligation, the amino acid positions on the incoming peptide, also referred to herein as the second peptide sequence, are labelled with a double prime and numbered in ascending order from the N-terminus starting with P1”. Accordingly, the second peptide sequence as described herein comprises H2N-P1”-P2”. It is to be understood that the second peptide sequence may comprise any number of amino acids between H2N-P1”-P2” and its C-terminus. In other words, the second peptide sequence may be any peptide sequence provided it comprises H2N-P1”-P2”. In embodiments wherein the first peptide sequence and the second peptide sequence are part of the same peptide to be cyclised, this peptide to be cyclised may comprise or consist of the sequence H2N-P1”-P2”-Pz-P1-P1’- P2’-Xn- COOH / CONH2, or Asn-P1”-P2”-Pz-P1-P1’- P2’-Xn-COOH / CONH2. The first and second peptide are expressed recombinantly in the cell, that is expressed within a cell which does not naturally express the peptides. Typically, the recombinant peptides are produced by recombinant DNA technology. “Amino acid” as used herein refers to natural and / or unnatural, including D- and L- amino acids, synthetic amino acids, amino acid analogues and derivatives. “Naturally occurring amino acids” refers to the 20 common proteogenic amino acids, namely Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. “Unnatural amino acid” refers to amino acids that are not naturally encoded. These are non-proteinogenic amino acids that occur naturally post-translationally without incorporation in proteins or peptides (also referred to as unusual amino acids) or are created synthetically. Examples of unusual acids include citrulline (Cit), hydroxyproline (Hyp), beta-alanine, ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, pyroglutamic acid (Pyr), Α-Aminobutyric Acid (Abu), 2,4-Diaminobutyric Acid (DAB). The method described provide a first peptide sequence comprising P1-P1’- P2’-Xn-COOH / CONH2, wherein P1 is Asn; P1’1 is any amino acid except Pro; and P2’ is an amino acid selected from the group Phe, Ile, Leu, Met, and Trp; and Xn is any amino acid and n is an integer of 0 or 1 or more, for example 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. In some embodiments, n is less than 100, or less than 50. In some embodiments, n is 0 (i.e. not present, for example such that P2’ is the C-terminal amino acid in the first peptide sequence). In some embodiments, P1’ is any naturally occurring amino acid except Pro; and P2’ is a naturally occurring amino acid selected from the group Phe, Ile, Leu, Met, and Trp; and X is any naturally occurring amino acid. For example, P1’ may be Cys or Gly; and P2’ may be Leu, and Xn may be X0 (i.e. not present). The method described further provides a second peptide sequence comprising H2N-P1”-P2” wherein P1” and P2” are any amino acid. In some embodiments, P1” is any naturally occurring amino acid, and P2” is a naturally occurring amino acid, optionally a naturally occurring amino acid selected from Val, Leu, or Ile. For example, P1” may be Gly and P2” may be Leu. In some embodiments, providing the second peptide sequence comprises providing to the cell a peptide comprising Asn-P1”-P2”, where P1”-P2” are as defined above. In such embodiments, the AEP catalytic domain construct cleaves the bond between Asn and P1” on the second peptide, resulting in the second peptide sequence comprising H2N-P1”-P2”. In embodiments, any of the peptides to be ligated may be a helical peptide. Helical peptide as used herein means that the peptide forms an alpha helix. The alpha helix typically comprises a heptad repeat consensus sequence abcdefg, where a is typically a hydrophobic amino acid, b and c are typically polar amino acid residues, d is typically a leucine or isoleucine and e, f, and g are typically polar amino acid residues. Hydrophobic amino acids may be Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, and Trp. Polar amino acid residues may be Ser, Thr, Cys, Asn, Gln, and Tyr. In some embodiments, the conformationally constrained peptide is a helix-constrained peptide. Helix-constrained as used herein is intended to mean a peptide modified to introduce a covalent cross-linker between two derivatisable amino acid resides located at anchoring positions within the peptide in order to produce a stabilised alpha helix. In some embodiments, the second peptide may be a helix-constrained peptide. In some embodiments, the ligated peptide, such as a cyclised peptide, may be modified to introduce covalent cross-linkers. Methods of inducing cross-links between amino acids are well known and include methods that introduce cross-links between the peptide backbone, e.g. between the carbonyl group and amino group as in natural alpha-helices, as well as between side-chains of the peptides. Cross linkers include disulfide bonds (e.g. as described in Leduc et al. (2003)), hydrogen bond surrogates (e.g. as described in Wang et al. (2005)), ring-closing metathesis (e.g. as described in Walensky et al. (2004)), cysteine alkylation using α-haloacetamide derivatives (e.g. as described in Woolley (2005)) or biaryl halides (e.g. as described in Muppidi et al. (2011)), lactam rings (e.g. as described in Fujimoto et al. (2008)), hydrazine linkage (e.g. as described in Cabezas & Satterthwait (1999)), oxime linkage (e.g. as described in Haney et al. (2011)), metal chelation (e.g. as described in Ruan et al. (1990)), and “click” chemistry (e.g. as described in Holland-Nell & Meldal (2011)). The cross-linker may be used to cross-link cysteine residues. Hence, the peptide may comprise a cysteine (C) at positions i and i+4, or i and i+7, in its amino acid sequence. As described in Jo et al. (2012), the introduction of cysteine residues at i and i+4 positions is useful because this spacing brings two thioether residues into proximity when in the alpha-helix. Suitable cross-linking agents for stabilising an alpha-helix within the peptide containing a cysteine (C) at position i and i+4 are described in Jo et al. (2012). For example, the cross-linking agent could be a cross-linker selected from the group consisting of an alkyl bromide, an alkyl iodide, a benzyl bromide, an allyl bromide, a maleimide, and an electrophilic difluorobenzene. Suitable cross-linkers are known in the art for crosslinking cysteine (see for example: Fairlie & Dantas de Araujo, 2016 and Jo et al., 2012). In some embodiments, the cross-linking agent is an m-xylene based, o-xylene based, or p-xylene based benzyl bromide, more preferably a m-xylene based benzyl bromide. In some embodiments, the cross-linker is a compound of formula 1: Formula 1 wherein n is an integer selected from 1 to 3; m is an integer selected from 0 to 2; A is selected from C2-6-alkenylene, C5-12-arylene and C5-12-heteroarylene; Y is a covalent bond, C1-6alkylene or –N(H)C(=O)CH2-; R1is selected from Cl, Br, I, or F;and each L is independently selected from –C(=O)-, -C≡C-, -N=N-, C1-6alkylene and a covalent bond. The R1groups provide reactive groups (e.g. leaving groups) for reaction with the cysteine. The A groups provide the linkers with structures suitable for conformationally constraining a peptide in a cell when cross inked via the two derivatisable amino acid residues. For example, the A group may be conformationally constrained into a geometry suitable for linking the two derivatisable amino acid residues. In some embodiments, R1is Br. In some embodiments A is selected from C5-12-arylene and C5-12-heteroarylene. In some embodiments m is 0. In some embodiments Y is methylene. In some embodiments L is a covalent bond. In some preferred embodiments, the cross-linking agent is 1,3-dibromomethylbenzene (DBMB) having the following chemical formula: DBMB can be used to react with derivatisable amino acid residues at the i and i+3 or i and i+4 in the amino acid sequence of the peptide. In some preferred embodiments, the cross-linking agent is 4,4ʹ-bisbromomethyl-biphenyl (Bpy) having the following chemical formula: Bpy can be used to react with derivatisable amino acid residues at the i and i+7 in the amino acid sequence of the ligated peptide. Cross-linking cysteine residues in peptides can be carried out using known methods, such as those described in Timmerman et al., 2005 or WO 2021 / 260074. Briefly, this method may comprise reacting the cross-linker (e.g. DBMB) with the peptide in the presence of tris(2-carboxyethyl) phosphine (TCEP) and ammonium bicarbonate, and reacted at pH 8.0 and room temperature for 4 to 5 hours in the dark. The method may comprise providing a cell (e.g. a bacterial cell, such as an E. coli cell, or a eukaryotic cell, such as a human cell) containing the ligated peptide or the peptide(s) to be ligated (i.e. containing the first and second peptide sequences), contacting the cell with the cross-linker (e.g. as part of the cell culture media) and culturing the cell in the presence of the cross-linker. The cross-linker may be present at a concentration of between 1 µM and 1 mM (e.g. between 100 µM and 1 mM, or between 100 µM and 800 µM, or between 100 µM and 500 µM), and for a period of at least 20 minutes (e.g. between 20 minutes and 10 hours). Further details of a suitable in cellulo cross-linking method are provided for example in WO 2021 / 260074. Advantageously, since the ligation reaction described herein is also carried out inside the cell, this allows for both the cross-linking and ligation reaction to take place inside the cell, reducing the need to carry out separate ligation and cross-linking reactions and further improving efficiency of the process. The crosslinker forms thioether cross-links with the at least a pair of cysteines such that the ligated peptide may comprise the structure: Y, L, R1, n, m and A are as defined for formula 1. R1arepresents a bond or CH2-CH2- linker derived from the appropriate R1 group in formula 1. In embodiments where the alkyl cross-link formed between two C residues is formed by DBMB, the ligated peptide may comprise the structure: The cross-linker may be used to cross-link lysine (K) and aspartic acid (D) in the peptide. Hence, the peptide may comprise a lysine (K) and aspartic acid (D) at i and i+4 positions in its amino acid sequence. That is, position i is a lysine (K) and position i+4 is an aspartic acid (D), or position i is an aspartic acid (D) and position i+4 is a lysine (K). Lactamisation is useful in terms of biostability since proteases universally recognise β-strands, with the constraint providing a further steric block, denying access to the backbone (Tyndall JD et al., 2005), and potentially bioavailability and membrane permeability owing to the lipophilic nature of the constraint. A lactam bridge in a peptide refers to the side chain of lysine (K) forming an amide bond with the side chain of glutamic acid (E) or aspartic acid (D), typically aspartic acid (D). Methods of carrying out K-D lactamisation are described in the examples herein and, for example, in de Araujo et al. (2014). As noted above, the cross-link may be formed between amino acids at positions i and i+3, i and i+4, or i and i+7 in the amino acid sequence of the peptide. In some embodiments, the cross-link is between cysteine (C) residues located at these positions. In other embodiments, the cross-link is between lysine (K) and aspartic acid (D) residues at these positions. Preferably, the cross-link is formed between amino acids at positions i and i+4. In some embodiments, the cross-linker is connected to the first and / or second peptide sequence as defined herein. For example, the cross-linker may be conjugated to a peptide that comprises the first and / or second peptide sequence. In embodiments, any of the peptides to be ligated may be modified, for example by conjugation to a labelling moiety. Examples of labelling moieties include, but are not limited to, affinity tags, detectable labels, degradation tags, cell penetrating molecules, and therapeutic agents. “Affinity tag” as used herein refers to tags that help with protein purification by separating the molecules to which the affinity tag binds from those that do not. Examples of affinity tags include, biotin, hexahistidine (His) tag, chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag and glutathione-S- transferase (GST). “Detectable label” as used herein refers to a tag that is able to generate a detectable signal either directly or indirectly. Examples include but are not limited to fluorophores (e.g. a fluorescent dye such as green fluorescent protein (GFP); red fluorescent protein (RFP); 5-Carboxyfluorescein (5-FAM); 6- Carboxyfluorescein (6-FAM); Anthranilyl, 2-Aminobenzoyl (Abz); 7-Methoxycoumarinyl-4-acetyl (Mca); Cy5; Cy3; 5-[(2-Aminoethyl)amino] naphthalene-1-sulfonic acid (EDANS); 5-(Dimethylamino) naphthalene-1-sulfonyl (Dansyl)) or isotopic labels, such as radiolabels (e.g. Tritium; Iodine-125; Carbon- 11, fluorine). Methods for labelling peptides using fluorophores or radioactive labels are well-known in the art. “Degradation tag” as used herein refers to a tag that acts as a degradation signal and targets peptides for proteolysis by the ubiquitin-proteasome system. The degradation signal may be present in the peptide sequence or added by covalent modification. Examples include but are not limited to SSRA tags, PROTACs, ligand-directed degraders (LDDs), and molecular glues. “Cell penetrating molecule” as used herein refers to molecular entities attached to cell-penetrating peptides (CPPs). CPPs facilitate cellular uptake of the molecular entity or “cargo”. These molecular entities may be associated with the CPP through covalent or non-covalent interactions. Examples of CPPs include a TAT peptide (GRKKRRQRRRPQ (SEQ ID NO: 13); oligo-Arginine; MPG; PEP-1; EB1, transportan; penetratin. Further examples of CPPs are described in Reissmann, 2014. Examples of molecular entities associated with the CPP include but are not limited to nanoparticles, antisense oligonucleotides, small interfering RNA, double stranded RNA, liposomes. In some embodiments, the first and / or second peptide sequence is fused to a TAT peptide. In some embodiments the TAT peptide is conjugated to detectable label, such as a FAM label. In some embodiments, the TAT peptide comprises one or more unnatural amino acid residues as described above. “Therapeutic agent” as used herein refers to a drug, protein, peptide, gene, compound or other pharmaceutically active ingredient. Peptides modified by the labelling moiety may be prepared using standard techniques known to those skilled in the art. The method described herein may be performed at a temperature lower than 50 ºC, such as lower than 49 ºC, 48 ºC, 47 ºC, 46 ºC, 45 ºC, 44 ºC, 43 ºC, 42 ºC, 41 ºC, 40 ºC, 39 ºC, 38 ºC, 37 ºC, 36 ºC, 35 ºC, 34 ºC, 33 ºC, 32 ºC, 31 ºC, 30 ºC, 29 ºC, 28 ºC, or 25 ºC. In some embodiments, the method is performed at a temperature of between about 25 ºC and about 50 ºC, such as between about 26 ºC and about 50 ºC, about 27 ºC and about 50 ºC, about 28 ºC and about 50 ºC, about 29 ºC and about 50 ºC, about 30 ºC and about 50 ºC, about 31 ºC and about 50 ºC, about 32 ºC and about 50 ºC, about 33 ºC and about 50 ºC, about 34 ºC and about 50 ºC, about 35 ºC and about 50 ºC, about 36 ºC and about 50 ºC, about 37 ºC and about 50 ºC, about 38 ºC and about 50 ºC, about 39 ºC and about 50 ºC, about 40 ºC and about 50 ºC, about 41 ºC and about 50 ºC, about 42 ºC and about 50 ºC, about 43 ºC and about 50 ºC, about 44 ºC and about 50 ºC, about 45 ºC and about 50 ºC, about 46 ºC and about 50 ºC, about 47 ºC and about 50 ºC, about 48 ºC and about 50 ºC, about 49 ºC and about 50 ºC. In some embodiments, the method is performed at a temperature of between about 25 ºC and about 40 ºC, such as between about 26 ºC and about 40 ºC, about 27 ºC and about 40 ºC, about 28 ºC and about 40 ºC, about 29 ºC and about 40 ºC, about 30 ºC and about 40 ºC, about 31 ºC and about 40 ºC, about 32 ºC and about 40 ºC, about 33 ºC and about 40 ºC, about 34 ºC and about 40 ºC, about 35 ºC and about 40 ºC, about 36 ºC and about 40 ºC, about 37 ºC and about 40 ºC, about 38 ºC and about 40 ºC, about 39 ºC and about 40 ºC, about 25 ºC and about 39 ºC, about 25 ºC and about 38 ºC, about 25 ºC and about 37 ºC, about 25 ºC and about 37 ºC, about 25 ºC and about 36 ºC, about 25 ºC and about 35 ºC, about 25 ºC and about 34 ºC, about 25 ºC and about 34 ºC, about 25 ºC and about 33 ºC, about 25 ºC and about 32 ºC, about 25 ºC and about 31 ºC, about 25 ºC and about 30 ºC, about 25 ºC and about 29 ºC, about 25 ºC and about 28 ºC, about 25 ºC and about 27 ºC, about 25 ºC and about 26 ºC, about 26 ºC and about 39 ºC, about 26 ºC and about 38 ºC, about 26 ºC and about 37 ºC, about 26 ºC and about 36 ºC, about 26 ºC and about 35 ºC, about 26 ºC and about 34 ºC, about 26 ºC and about 33 ºC, about 26 ºC and about 32 ºC, about 26 ºC and about 31 ºC, about 26 ºC and about 30 ºC, about 26 ºC and about 29 ºC, about 26 ºC and about 28 ºC, about 26 ºC and about 27 ºC, about 27 ºC and about 39 ºC, about 27 ºC and about 38 ºC, about 27 ºC and about 37 ºC, about 27 ºC and about 36 ºC, about 27 ºC and about 35 ºC, about 27 ºC and about 34 ºC, about 27 ºC and about 33 ºC, about 27 ºC and about 32 ºC, about 27 ºC and about 31 ºC, about 27 ºC and about 30 ºC, about 27 ºC and about 29 ºC, about 27 ºC and about 28 ºC, about 28 ºC and about 39 ºC, about 28 ºC and about 37 ºC, about 28 ºC and about 36 ºC, about 28 ºC and about 35 ºC, about 28 ºC and about 34 ºC, about 28 ºC and about 33 ºC, about 28 ºC and about 34 ºC, about 28 ºC and about 33 ºC, about 28 ºC and about 32 ºC, about 28 ºC and about 31 ºC, about 28 ºC and about 30 ºC, about 28 ºC and about 29 ºC, about 29 ºC and about 39 ºC, about 29 ºC and about 38 ºC, about 29 ºC and about 37 ºC, about 29 ºC and about 36 ºC, about 29 ºC and about 35 ºC, about 29 ºC and about 34 ºC. about 29 ºC and about 33 ºC, about 29 ºC and about 32 ºC, about 29 ºC and about 31 ºC, about 29 ºC and about 30 ºC, about 30 ºC and about 39 ºC, about 30 ºC and about 38 ºC, about 30 ºC and about 37 ºC, about 30 ºC and about 36 ºC, about 30 ºC and about 35 ºC, about 30 ºC and about 34 ºC, about 30 ºC and about 33 ºC, about 30 ºC and about 32 ºC, about 30 ºC and about 31 ºC, about 31 ºC and about 39 ºC, about 31 ºC and about 38 ºC, about 31 ºC and about 37 ºC, about 31 ºC and about 36 ºC, about 31 ºC and about 35 ºC, about 31 ºC and about 34 ºC, about 31 ºC and about 33 ºC, about 31 ºC and about 34 ºC, about 31 ºC and about 33 ºC, about 31 ºC and about 32 ºC, about 32 ºC and about 39 ºC, about 32 ºC and about 38 ºC, about 32 ºC and about 37 ºC, about 32 ºC and about 36 ºC, about 32 ºC and about 35 ºC, about 32 ºC and about 34 ºC, about 33 ºC and about 34 ºC, about 34 ºC and about 39 ºC, about 34 ºC and about 38 ºC, about 34 ºC and about 37 ºC, about 34 ºC and about 36 ºC, about 34 ºC and about 35 ºC, about 35 ºC and about 39 ºC, about 35 ºC and about 38 ºC, about 35 ºC and about 37 ºC, about 35 ºC and about 36 ºC, about 36 ºC and about 39 ºC, about 36 ºC and about 38 ºC, about 36 ºC and about 37 ºC, about 37 ºC and about 39 ºC, about 37 ºC and about 38 ºC, about 38 ºC and about 39 ºC. In some embodiments, the method described herein is performed at a temperature of about of between about 30 ºC and about 37 ºC. The method described herein may be performed at a temperature of about 25 ºC, about 26 ºC, about 27 ºC, about 28 ºC, about 29 ºC, about 30 ºC, about 31 ºC, about 32 ºC, about 33 ºC, about 34 ºC, about 35 ºC, about 36 ºC, about 37 ºC, about 38 ºC, about 39 ºC, about 40 ºC, about 41 ºC, about 42 ºC, about 43 ºC, about 44 ºC, about 45 ºC, about 46 ºC, about 47 ºC, about 48 ºC, about 49 ºC, about 50 ºC. In some embodiments, the method is performed at a temperature of about 30 ºC. The method described herein may be carried out in the absence of amide coupling reagents and / or in the absence of organic solvents. Amide coupling as used herein refers to the formation of amide bond by condensation of a carboxylic acid and an amine on a protein backbone. Examples include, but are not limited to, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), Hydroxybenzotriazole (HOBT), Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU) Organic solvents as used herein refers to chemical compounds having a carbon-based molecular structure. Examples include, but are not limited to, N,N-Dimethylformamide (DMF) and Dichloromethane (DCM)). In some embodiments, the ligated peptide is a small macrocyclic peptide. Small macrocyclic peptides as used herein refers to peptide structures comprising one or more rings, spanning 9 or more amino acid resides, such as 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 90 or more, or 100 or more. In some embodiments, the peptides to be ligated (“the first and second peptide”) and AEP catalytic domain are expressed intracellularly from a nucleic acid. For example, the nucleic acid encoding may be delivered to the cell, optionally as part of an expression vector, or may be incorporated into the genome of the cell. An “expression vector” as used herein is a DNA molecule used for expression of foreign genetic material in a cell. Any suitable vectors known in the art may be used. Suitable vectors include plasmids, binary vectors, viral vectors and artificial chromosomes (e.g. yeast artificial chromosomes). Alternatively, the expression cassettes described herein may be incorporated into the genome of the cell. The methods described herein may comprise delivering (or “administering” one or more nucleic acids described herein to the cell. Molecular biology techniques suitable for administering nucleic acids and producing peptides such as the recombinant peptide and AEP catalytic domain described herein in cells are well known in the art, such as those set out in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989. Techniques for expressing peptides and enzymes such that they are localised intracellularly are well known in the art. In some embodiments, the methods described herein further comprises isolating the ligated peptide from the cell. Isolated ligated peptides and cells identified by the methods described herein are therefore also disclosed herein. The ligated peptide generated according to the methods described herein may be used in a method of screening peptide libraries for transcription factor antagonists. The method comprises providing a cell, wherein the cell comprises a peptide ligated according to the methods described herein, and first and second candidate binding partners, followed by assaying whether the ligated peptide is able to inhibit association between the first and second candidate binding partners. The candidate binding partners can be any peptides that associate with one another (or are expected to do so). The first and second binding partners may have an identical amino acid sequence (e.g. they may homodimerise with each other). Alternatively, the first and second binding partners may have different amino acid sequences (e.g. they may heterodimerise with each other). In some embodiments, the candidate binding partners form a DNA-binding complex upon association. Suitable candidate binding partners that form a DNA-binding complex upon association include those of the basic leucine zipper (bZIP), basic helix-loop helix (bHLH) or bHLH leucine zipper (bHLH-Zip) transcription factor families. bHLH and bHLH-Zip transcription factors are exclusively eukaryotic proteins that bind to sequence-specific double-stranded DNA as homodimers or heterodimers to either activate or repress gene transcription. bZIP transcription factors also contain leucine repeats through which the proteins dimerize to form a coiled coil, which is required for functional activity of the protein. As well as human proteins, certain viral proteins such as BZLF1 form part of the bZIP family. Details of bZIP, bHLH and bHLH-ZIP transcription factors and their consensus sequences are provided in Vinson et al. (2002), Newman & Keating (2003) and Rodriguez-Martinez et al. (2017). Typically, the DNA-binding fragment of these transcription factors will be (or will comprise) the basic portions of the transcription factors which physically interact with DNA. Exemplary bHLH transcription factors include ATOH1, AhR, AHRR, ARNT, ASCL1, BHLH2, BHLH3, BHLH9, ARNTL, ARNTL2, CLOCK, EPAS1, FIGLA, HAND1, HAND2, HES5, HES6, HEY1, HEY2, HEYL, HES1, HIF1A, HIF3A, ID1, ID2, ID3, ID4, LYL1, MESP2, MXD4, MYCL1, MYCN, MyoD, Myogenin, MYF5, MYF6, Neurogenin1, Neurogenin2, Neurogenin3, NeuroD1, NeuoD2, NPAS1, NPAS2, NPAS3, OLIG1, OLIG2, Pho4, Scleraxis, SIM1, SIM2, TAL1, TAL2, Twist and USF1. Exemplary bHLH-ZIP transcription factors include AP-4, Max, MXD1, MXD3, MITF, MNT, MLX, MLXIPL, MXI1, Myc, SREBP1 and SREBP2. In particular embodiments, the bHLH-ZIP transcription factor used may be c-Myc or Max, or a heterodimer between c-Myc and Max (c-Myc-Max). bHLH and bHLH-ZIP transcription factors typically bind to a consensus sequence called an E-box, which can have the sequence CANNTG (‘N’ being any nucleotide) and in particular cases has the sequence CACGTG. The candidate DNA binding partner may be or may comprise any of these bHLH or bHLH-Zip transcription factors. Exemplary human bZIP transcription factor subfamilies, the nucleotide sequences of their binding sites and examples of proteins of these subfamilies are set forth in Table 1 below. The candidate binding partners may be or may comprise any of these human bZIP proteins. For example, the first and second candidate binding partners may be proteins of the Fos / Jun bZip family that form a DNA-binding complex upon association.

[0002] Human bZIP Exemplary bZIP protein Nucleotide sequence(s) of Name of binding site subfamily binding site PAP1, YAP1, YAP2, YAP3, PAP YAP4, YAP5, YAP6, YAP7, Cap1 AFT4, mATFP4, ApCREB-2, TTACGTAA PAP / CREB-2 / PAR CREB-2 hCREB2, acr1 DBP, VBP / TEF, HLF, CES2, PAR TEF C / EBPα, C / EBPβ, C / EBPδ, C / EBPε, C / EBPγ, CRP1, ATTGCGCAAT (SEQ ID NO: C / EBP CCAAT CRP2, CRP3, Ig / EBP, lap, 10) DDIT3 cFos, FRA1 (FosL1), FRA2 (FosL2), cJun, JUNB, JUND, TPA response Fos / Jun TGACTCA or TGAGTCA GCN4, BATF, BATF2, element (TRE) BATF3 CREB1, ATF1, ATF2, ATF3, cAMP response CREB ARF5, ATFa, BBF-2, TGACGTCA element (CRE) CREB3L1 Maf MafA, MafB, BACH1, TGCTGA(G / C)TCAGCA BACH2 (SEQ ID NO: 11) and Maf recognition TGCTGAG(C / C)GTCAGCA element (MARE) (SEQ ID NO: 14) Table 1: Exemplary human bZIP transcription factor subfamilies, the nucleotide sequences of their binding sites and examples of proteins of these subfamilies. In other examples, the candidate binding partners may form protein aggregates, or may be expected to do so. Protein aggregates are typically formed where multiple misfolded proteins accumulate and clump together and their presence is associated with a number of diseases, in particular neurodegenerative diseases such as Alzheimer’s Disease (AD), Parkinson’s disease (PD) and prion disease (also known as transmissible spongiform encephalopathy). In some embodiments, the presence of an aggregate of the candidate binding partners in a human patient is associated with a disease or other pathological condition, such as a neurodegenerative disease. Examples of peptides and polypeptides that are capable of forming protein aggregates include those that are capable of aggregating to form amyloids, as well as those capable of aggregating to form amorphous or native-like deposits. In some embodiments, the candidate binding partners are amyloid-β (Aβ) peptides, α-synuclein (αS) polypeptides, tau proteins, or prion proteins. In other examples, PPI between the candidate binding partners may be associated with an intracellular signalling pathway. Numerous intracellular signalling pathways are associated with the interaction (which may be covalent or non-covalent) between one or more proteins, e.g. an enzyme such as a kinase. Accordingly, one of the candidate binding partners could be an enzyme such as a kinase and the other candidate binding partner be a protein that interacts (e.g. binds to) the enzyme. For example, guanine nucleotide exchange factors (GEFs) are proteins or protein domains that associate with small GTPases to induce catalytic activity of the GEF. Exemplary methodology for designing and producing peptides that can target and modulate helical PPIs associated with intracellular signalling pathways is provided in Yoo et al.2020. In some embodiments, the candidate binding partners are expressed intracellularly from one or more nucleic acids. For example, one or more nucleic acids encoding the candidate binding partners may be an expression cassette (also termed a “candidate binding partner expression cassette”), which may be delivered to the cell, optionally as part of an expression vector, or may be incorporated into the genome of the cell. Where the first and second candidate binding partners have an identical amino acid sequence, both binding partners may be expressed from the name nucleic acid. Assaying for whether the ligated peptide is able to inhibit association between the first and second candidate binding partners may involve determining whether the ligated peptide is able to modulate activity and / or expression of a reporter protein. A reporter protein as used herein is any protein that provides a phenotypic readout. Examples of reporter proteins include cell survival proteins, cell reproduction proteins, fluorescence proteins, bioluminescence proteins, enzymes that act on a substrate to produce a colorimetric signal, protein kinases, proteases, transcription factors, and regulatory proteins such as ubiquitin. The use of suitable reporter proteins in assays for determining PPIs is described, for example, in Wehr and Rossner (2016). In these assays, activity of the reporter protein is controlled by the association of the first and second candidate protein. This can be achieved in several ways. For example, the reporter protein may be split into a first and second fragments of the reporter protein, such that the first and second fragments need to be brought into sufficient proximity (e.g. non-covalently interact) in order to reconstitute activity of the reporter protein. Reporter proteins that can be split into fragments in this way can be termed “split reporters”. Several split reporters are known in the art and include beta-lactamase, dihydrofolate reductase (DHFR), focal adhesion kinase (FAK), Gal4, GFP (split-GFP), horseradish peroxidase, infrared fluorescent protein IFP1.4, an engineered chromophore-binding domain (CBD), LacZ (beta- galactosidase), luciferase, TEV (Tobacco etch virus protease) and ubiquitin. In some embodiments, the first candidate binding partner is linked (e.g. fused) to a first fragment of the reporter protein and the second candidate binding partner is linked (e.g. fused) to the second fragment of the reporter protein, where association (e.g. dimerisation) of the first and second candidate binding partners reconstitutes reporter protein activity. This assay may be termed the protein-fragment complementation assay, or PCA and is well known in the art. In cases where association of the first and second candidate binding partners reconstitutes reporter protein activity, the additional presence in the cell of a peptide that inhibits association between the first and second candidate binding partners will decrease activity of the reporter protein. Other suitable assays may make use of a DNA-binding complex to inhibit or promote expression of the reporter protein as a way of controlling activity of the reporter protein. In these assays, the cell may further comprise a nucleic acid encoding the reporter protein. The nucleic acid encoding the reporter protein may be an expression cassette (also termed a “reporter protein expression cassette”), which may be delivered to the cell, optionally as part of an expression vector, or may be incorporated into the genome of the cell. The nucleic acid encoding the reporter protein comprises a binding site that the DNA- binding complex binds to and inhibits or promotes expression of the reporter protein. This DNA-binding based assay can be used in embodiments where the first and second candidate binding partners form the DNA-binding complex. Additionally, this DNA-binding based assay can be used in embodiments where the first and second candidate binding partners are linked to components that form a DNA-binding complex when brought into sufficient proximity (i.e. though association of the first and second candidate binding partners). The DNA-binding complex may comprise any of the proteins of a particular bZIP family set forth in Table 1 above and the binding site in the nucleic acid encoding the reporter protein may the binding site associated with that bZIP family set forth in Table 1 above. In some embodiments, one or more binding sites are located in the promoter or enhancer region of the nucleic acid encoding the reporter protein. In these embodiments, the DNA-binding complex typically has transcriptional activation or transcriptional repression activity such that upon binding to the binding site(s) it is capable of promoting or inhibiting expression of the reporter protein. In other embodiments, some or all binding sites are located in the transcribed sequence (e.g. the coding sequence) of the nucleic acid encoding the reporter protein. In these embodiments, binding of the DNA- binding complex to the binding site(s) inhibits transcription of the reporter protein. Accordingly in preferred embodiments, the cell comprises the first and second candidate binding partners and a nucleic acid encoding a reporter protein, where association of the first and second candidate binding partners form a DNA-binding complex that binds to one or more binding sites in the nucleic acid encoding the reporter protein such that binding of the DNA-binding complex to the binding site(s) inhibits expression of the reporter protein. In these embodiments an increase in expression of the reporter protein in the presence of the ligated peptide indicates that the ligated peptide is capable of inhibiting association of the first and second candidate binding partners. Monitoring the activity and / or expression of the reporter protein will depend on the reporter protein used. For example, where the reporter protein is a cell survival protein, then inhibition of expression and / or activity of the cell survival protein will result in cell death. Cell death can be determined by one of a number of techniques known to the person skilled in the art, e.g. the observing of morphological changes such as cytoplasmic blebbing, cell shrinkage, internucleosomal fragmentation and chromatin condensation. Use of a cell survival protein as a reporter protein can be advantageous as it gives a simple binary readout, i.e. the cell is either dead or alive. Methods using cell survival proteins as reporter proteins in screening for inhibitors that disrupt PPIs are known. See, for example, Park et al. (2007), which describes methods involving beta-lactamase in a fragmentation complementation strategy. If the reporter protein is a cell reproduction protein, then inhibition of expression and / or activity of the cell reproduction protein will result in the cell being unable to proliferate and therefore unable to form progeny. Cell proliferation can be determined by one of a number of techniques known to the person skilled in the art, e.g. by counting of individual cells, foci or colonies, measuring metabolic activity using dyes such as MTT and WST-1, using nucleoside analogues such as bromodeoxyuridine (BrdU) and measuring incorporation of this analogue in the cells, staining dividing cells using reagents such as succinimidyl ester of carboxyfluorescein diacetate, and detecting proliferation markers such as PCNA, poisomerase IIB or phosphohistone H3. Inhibition of cell proliferation may also result in cell death, which can be measured as described above. As a further example of a reporter protein that provides an observable phenotype, the reporter protein can be a fluorescent protein, a bioluminescent protein, or an enzyme that acts on a substrate to produce a colorimetric signal. In these cases, activity of the reporter proteins results in an observable signal when active that can therefore be monitored. A ligated peptide that is able to modulate expression and / or activity of the reporter protein may be able to modulation expression and / or activity by at least 50%, by at least 2-fold, by at least 5-fold, or by at least 10-fold when compared to reporter protein expression and / or activity in an equivalent cell that lacks the conformationally constrained peptide. For example, where association of the first and second candidate binding partners results in a decrease in expression and / or activity of the reporter protein (e.g. a cell survival protein such as DHFR), an increase by at least 50% in expression and / or activity of the reporter protein (e.g. at least 50% more living cells) in the presence of the conformationally constrained peptide may indicate the constrained peptide is capable of modulating expression and / or activity of the reporter protein. The ligated peptide may elicit a greater modulation (e.g. at least 50% greater modulation, at least 2-fold greater modulation, at least 5-fold greater modulation, or at least 10-fold greater modulation) of expression and / or activity of the reporter protein when compared to the ability an un-ligated peptide to modulate expression and / or activity of the reporter protein. This may indicate that ligated peptide increases its ability to disrupt PPIs between the first and second candidate binding partner (e.g. the conformationally constrained peptide binds its target with a higher affinity). Thus, in some embodiments, the method further comprises determining whether the ligated peptide elicits greater modulation of the expression and / or activity of the reporter protein compared to unligated peptide. This may involve measuring the reporter protein expression and / or activity before and after the cell is contacted and cultured with the ligated peptide. The methods described herein are expected to have use in preparing genetically encoded peptide libraries. Genetically encoded peptide libraries are known and have been used in screening methods for identifying inhibitors of PPIs. See, for example, Mern et al. (2010). Briefly, such libraries are formed from libraries of nucleic acids, each of which encodes and is capable of directing expression of a different recombinant peptide. Accordingly, in embodiments that involve genetically encoded peptide libraries, the resulting recombinant peptides in the libraries can be designed to always contain a first peptide sequence comprising Asn-P1’- P2’-COOH / CONH2 and a second peptide sequence comprising H2N-P1”-P2” as defined above. Such genetically encoded peptidic libraries can be used with AEP catalytic domain construct as defined herein to ligate and screen multiple different test recombinant peptides at the same time. Thus, in some embodiments the cell is part of a plurality of cells comprising a library of recombinant peptides, the library of recombinant peptides comprising a mixture of recombinant peptides that differ in one or more of their amino acid residues outside the first and second peptide sequence. Described herein is an AEP catalytic domain construct for use in a method of intracellular peptide ligation. The AEP catalytic domain construct may be as defined above. Also described herein is a kit for use in any of the methods described herein, the kit comprising a AEP catalytic domain construct. The AEP catalytic domain construct may be as defined above. The kit may further comprise a cell. The cell may be is as defined above. The kit may further comprise one or more nucleic acids encoding the first and second peptide sequence, which may be as defined above. *** The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof. While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations. Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%. Sequences SEQ Description Sequence ID NO 1 DNA sequence ATGAGGAATGGATTACACCATCACCATCACCATGCTAGCGTT encoding for mDHFR CGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGG GATTGGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGA ACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAG TGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCT GGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAA TTAATATAGTTCTCAGTAGAGAACTCAAAGAACCACCACGAG GAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACT TATTGAACAACCGGAATTGGCCAGTAAAGTAGACATGGTTTG GGCAGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGAATC AACCAGGCCACCTCAGACTCTTTGTGACAAGGATCATGCAGG AATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAA ATATAAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGT CCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGA GAAGAAAGACCCAGGCAGGAATGGATTATGA 2 Amino acid sequence MRNGLHHHHHHASVRPLNCIVAVSQNMGIGKNGDLPWPPLRN encoding for mDHFR EFKYFQRMTTTSSVEGKQNLVIMGRKTWFSIPEKNRPLKDRINIV LSRELKEPPRGAHFLAKSLDDALRLIEQPELASKVDMVWAVGG SSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPE YPGVLSEVQEEKGIKYKFEVYEKKDPGRNGL 3 DNA sequence ATGGGCATGGCGCACCACCACCACCACCACATGCAGATCTT encoding for OaAEP1- CGTTAAAACCCTGACCGGCAAGACCATTACCCTGGAAGTGG C247A AACCGAGCGACACCATCGAGAACGTGAAAGCGAAGATCCAA GACAAAGAAGGTATTCCGCCGGATCAGCAACGTCTGATTTTT GCGGGCAAGCAGCTGGAGGACGGTCGTACCCTGAGCGATTA CAACATCCAAAAAGAAAGCACCCTGCATCTGGTGCTGCGTCT GCGTGGTGGCGCGCGTGACGGTGATTATCTGCACCTGCCGA GCGAGGTGTCTCGTTTCTTTCGTCCGCAGGAGACCAACGAC GATCACGGCGAAGACAGCGTGGGTACCCGTTGGGCGGTTCT GATTGCGGGTAGCAAGGGCTACGCGAACTATCGTCATCAGG CGGGCGTGTGCCATGCGTACCAAATCCTGAAACGTGGTGGC CTGAAGGATGAGAACATCGTGGTTTTCATGTACGACGATATT GCGTATAACGAAAGCAACCCGCGTCCGGGTGTTATCATTAAC AGCCCGCACGGCAGCGATGTGTATGCGGGTGTTCCGAAAGA TTATACCGGCGAGGAAGTGAACGCGAAGAACTTCCTGGCGG CGATCCTGGGTAACAAAAGCGCGATTACCGGTGGCAGCGGC AAGGTGGTTGACAGCGGTCCGAACGATCACATCTTTATTTAC TATACCGACCACGGTGCGGCGGGCGTTATCGGTATGCCGAG CAAGCCGTACCTGTATGCGGACGAGCTGAACGATGCGCTGA AGAAAAAGCACGCGAGCGGTACCTACAAAAGCCTGGTGTTC TATCTGGAGGCGTGCGAAAGCGGCAGCATGTTTGAGGGTAT CCTGCCGGAAGATCTGAACATTTACGCGCTGACCAGCACCA ACACCACCGAAAGCAGCTGGGCCTACTATTGCCCGGCGCAG GAGAACCCGCCGCCGCCGGAATATAACGTGTGCCTGGGCGA CCTGTTCAGCGTTGCGTGGCTGGAGGACAGCGATGTGCAGA ACAGCTGGTACGAAACCCTGAACCAGCAATATCACCACGTTG ATAAGCGTATTAGCCACGCGAGCCACGCGACCCAATACGGT AACCTGAAACTGGGCGAGGAAGGTCTGTTTGTGTACATGGG CAGCAACCCGGCGAACGACTAA Amino acid sequence MGMAHHHHHHMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKE encoding for OaAEP1- GIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGAR C247A DGDYLHLPSEVSRFFRPQETNDDHGEDSVGTRWAVLIAGSKGY ANYRHQAGVCHAYQILKRGGLKDENIVVFMYDDIAYNESNPRP GVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKSAITGGS GKVVDSGPNDHIFIYYTDHGAAGVIGMPSKPYLYADELNDALKK KHASGTYKSLVFYLEACESGSMFEGILPEDLNIYALTSTNTTESS WAYYCPAQENPPPPEYNVCLGDLFSVAWLEDSDVQNSWYETL NQQYHHVDKRISHASHATQYGNLKLGEEGLFVYMGSNPAND Sequence of core ARDGDYLHLPSEVSRFFRPQETNDDHGEDSVGTRWAVLIAGSK domain of OaAEP1- GYANYRHQAGVCHAYQILKRGGLKDENIVVFMYDDIAYNESNP C247A RPGVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKSAITG GSGKVVDSGPNDHIFIYYTDHGAAGVIGMPSKPYLYADELNDAL KKKHASGTYKSLVFYLEACESGSMFEGILPEDLNIYALTSTNTTE SSWAYYCPAQENPPPPEYNVCLGDLFSVAWLEDSDVQNSWYE TLNQQYHHVDKRISHASHATQYGNLKLGEEGLFVYMGSNPAND OaAEP1 variant ARDGDYLHLPSEVSRFFRPQETNDDHGEDSVGTRWAVLIAGSK GYANYRHQAGVCHAYQILKRGGLKDENIVVFMYDDIANNESNP RPGVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKSAITG GSGKVVDSGPNDHIFIYYTDHGAAGVIGMPSKPNLNADELNDAL KKKHASGTYKSLVFYLEACESGSMFEGNLPEDLNIYALTSTNTT ESSWAYYCPAQENPPPPEYNVCLGDLFSVAWLEDSDEQNSKD ETLNQQYHHVDKRISHASHATQYGNLKLGEEGLFVYMGSNPAN D oaAEP1 variant – His ARDGDYLHLPSEVSRFFRPQETNDDHGEDSVGTRWAVLIAGSK tagged GYANYRHQAGVCHAYQILKRGGLKDENIVVFMYDDIANNESNP RPGVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKSAITG GSGKVVDSGPNDHIFIYYTDHGAAGVIGMPSKPNLNADELNDAL KKKHASGTYKSLVFYLEACESGSMFEGNLPEDLNIYALTSTNTT ESSWAYYCPAQENPPPPEYNVCLGDLFSVAWLEDSDEQNSKD ETLNQQYHHVDKRISHASHATQYGNLKLGEEGLFVYMGSNPAN DHHHHHH Kalata B1 GLPVCGETCVGGTCNTPGCTCSWPVCTRN Substrate peptide GLPVSTKPVATRNGL C / EBP nucleotide ATTGCGCAAT binding sequence Maf binding sequence TGCTGA(G / C)TCAGCA Kalata B1 precursor MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL (with N-terminal SUMO RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDII fusion) EAHREQIGGNGLPVCGETCVGGTCNTPGCTCSWPVCTRNGLP SLAA TAT peptide GRKKRRQRRRPQ Maf binding sequence TGCTGAG(C / C)GTCAGCA Primer ST14I-F GGAGATATACCATGGGCATGGCG Primer ST14I-R CGGCCGCAAGCTTTTAGTCGTTCGCC Primer ST14V-F AAGCTTGCGGCCGCATAATG Primer ST14V-R GCCCATGGTATATCTCCTTATTAAAG Cyclic substrate peptide GLPYSTKPYATRN Substrate peptide GLPYSTKPYATRNGL auto-catalytic OaAEP1 MGRNGLHHHHHHGTRWAVLIAGSKGYANYRHQAGVCHAYQIL KRGGLKDENIVVFMYDDIAYNESNPRPGVIINSPHGSDVYAGVP KDYTGEEVNAKNFLAAILGNKSAITGGSGKVVDSGPNDHIFIYYT DHGAAGVIGMPSKPYLYADELNDALKKKHASGTYKSLVFYLEAC ESGSMFEGILPEDLNIYALTSTNTTESSWAYYCPAQENPPPPEY NVCLGDLFSVAWLEDSDVQNSWYETLNQQYHHVDKRISHASH ATQYGNLKLGEEGLFVYMGSNPANGL Examples The present examples demonstrate a method of intracellular ligation, in this instance head-to-tail cyclisation (H2T), by recombinantly expressing transpeptidase OaAEP1 with a substrate protein (e.g. mDHFR) in a live organism. This method allowed for isolation of the cyclic product from the host organism without the need to purify each protein component individually (Example 2). The cyclic peptide showed excellent thermal stability without affecting the protein fold or specific enzyme activity (Example 3) and retained full activity (Example 4). The method of intracellular ligation can be applied to a range of different proteins, such as Kalata B1 (Example 5). EXAMPLE 1 - Materials and Methods General Information Custom oligonucleotides and the pACYC-duet1 plasmid vector were purchased from Merck Sigma Aldrich. All restriction enzymes were purchased from ThermoFisher Scientific. Electrocompetent E. coli, buffers for molecular cloning, nucleotide triphosphate, Q5® High-Fidelity DNA polymerase, and NEBuilder® HiFi DNA assembly master mix was purchased from New England Biolabs. DNA Sanger sequencing performed by Eurofins Genomics. Molecular Cloning Linear mDHFR – The plasmid pACYC-Duet1-mDHFR was used for preparing linear mDHFR by IPTG induction. It was constructed by amplifying the genes encoding for His6-mDHFR then subcloned between KpnI and NdeI restriction sites. The ligated product was transformed into E. coli strain DH5α. Sequences of all plasmids were confirmed by DNA Sanger sequencing. Cyclic mDHFR – The plasmid pACYC-Duet1-OaAEP1-mDHFR was used for co-expression of OaAEP1- C247A and mDHFR by IPTG induction to prepare cyclic mDHFR. It was constructed by amplifying the gene encoding His6-Ubquitin-OaAEP1-C247A by PCR using primers ST14I-F and ST14I-R into the plasmid pACYC-Duet1-mDHFR linearized by PCR using primers ST14V-F and ST14V-R (see Table 1). The PCR products were digested by DpnI (ThermoFisher) then assembled by Gibson Assembly using NEBuilder® HiFi DNA assembly master mix and transformed into E. coli strain DH5α. Sequences of plasmids were confirmed by DNA Sanger sequencing. Primer ST14I-F GGAGATATACCATGGGCATGGCG (SEQ ID NO: 15) Primer ST14I-R CGGCCGCAAGCTTTTAGTCGTTCGCC (SEQ ID NO: 16) Primer ST14V-F AAGCTTGCGGCCGCATAATG (SEQ ID NO: 17) Primer ST14V-R GCCCATGGTATATCTCCTTATTAAAG (SEQ ID NO: 18) Table 2 lists the primer sequences used in the methods described herein. Recombinant expression and purification – Recombinant expression was performed in E. coli strain SHuffle T7 express. The cells were grown at 30 °C in LB medium until the OD600 reached 0.7, at which point protein production was induced by adding IPTG (to a final concentration of 0.2, 0.5 or 1.0 mM). After culturing at 16 °C for 18 h, cells were harvested by centrifugation at 4000 g and 4 °C for 20 minutes. Cell pellets were stored at -20 °C until purification. The cell pellet from a 1 L culture was suspended in 40 mL of lysis buffer containing 25 mM Tris.HCl (pH 7.5), 500 mM NaCl, 20 mM Imidazole, 0.1 mg / mL lysozyme, DNase I and cOmpete protease inhibitor cocktail. After lysis by sonication and clearance by centrifugation (14000g, 4 °C, 15 min), the supernatant was loaded onto a 5 mL of HisTrap FF (Cytiva). The column was washed with 10 column volumes of lysis buffer, and the remaining bound protein was eluted over a linear gradient from 20 to 500 mM imidazole over 20 column volumes. Fractions containing the desired protein were combined and concentrated using an Amicon Ultra‐15 centrifugal filter unit (10 kDa MWCO) (Merck Millipore), then further purified by size exclusion chromatography (Superdex 75 pg 16 / 600, GE Healthcare) that had been pre-equilibrated in 25 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM DTT. Fractions containing desired protein were combined, concentrated using an Amicon Ultra‐15 centrifugal filter unit (10 kDa MWCO) (Merck Millipore) then stored at -80 °C. Liquid Chromatography - Mass Spectrometry (LC-MS) – LC-MS analyses were performed using an Agilent QTOF 6545 with Jetstream ESI spray source coupled to an Agilent 1260 Infinity II Quat pump HPLC with 1260 autosampler, column oven compartment and variable wavelength detector (VWD). The MS was operated in positive ionisation mode (gas temperature at 350°C, drying gas flow at 11 L min-1, nebulizer gas flow at 50 psi, sheath gas temperature at 400°C, sheath gas flow at 12 L min-1). The VCap, Nozzle, Fragmentor and Skimmer voltages were set to 4000, 1000, 200 and 65 V respectively. The chromatographic separation (2 µL sample injection) was performed on an Agilent polymeric reversed phase (PLRP-S, 2.1 x 50 mm, 3 µm, 300 Å) at 0.2 mL / min using mobile phase A (H2O with 0.1% formic acid), and B (acetonitrile with 0.1% formic acid). Gradient elution started at 5% B, changed to 50% B at 8 min, then to 90% B at 9 min, held at 90% B until 10 min, then returned to 5% B for re-equilibration in a total 13 min run. Data analyses were performed in MassHunter BioConfirm 10.0. Circular Dichroism (CD) – CD measurements were taken on an Applied Photophysics Chirascan with 200 µL samples in 1 mm path-length CD cells. Protein samples were diluted in 20 mM potassium phosphate (pH 7.4), 100 mM potassium fluoride. For full spectra, three scans between 190 and 300 nm were collected with a bandwidth of 1 nm and data sampled at a rate of 0.5 s-1. These scans were averaged, a blank was subtracted, and the values were converted to molar residue ellipticity (MRE). Thermal denaturation experiments were performed by measuring the ellipticity at 209 nm over a temperature gradient from 4 to 90°C at 1°C increments. Post-melt scans at 20°C confirmed the proteins were irreversibly denatured as they did not overlay with the pre-melt scans. To determine the melting temperature (Tm), the thermal denaturation curves were converted to MRE and fitted to the Boltzmann sigmoidal equation using OriginPro graphing and analysis software. To estimate the fraction of unfolded protein during thermal denaturation, lower and upper bounds of each data set were linearly extrapolated using the 10 data points at 4 – 13°C and 76 – 85°C, respectively. The MRE from each sample was then fitted to respective lower and upper bounds using the following equation: Fraction of unfolded protein = (MRE-Lower bound) / (Upper bound-Lower bound) (Equation (1)) The fraction of unfolded protein was plotted against temperature to present normalized thermal denaturation profiles of linear and cyclic mDHFR. DHFR activity assay – A colorimetric assay kit (Merck CS0340) was used to measure the activity of purified mDHFR enzymes. Cyclic or Linear-mDHFR (50 nM) and NADPH (60 µM) were mixed in assay buffer. For the blank reaction, a volume of assay buffer was added instead of enzyme (Figure 9). Reactions were initiated by the addition of DHF (50 µM) and the absorbance at 340 nm of samples was measured using a 10 mm path-length quartz cuvette in a Varian Cary 50 UV-Vis spectrophotometer. The specific activity was calculated using the following equation: ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ((∆〖absorbance min^(-1)〗_(sample )― ∆〖absorbance min^(-1)〗_(blank ))) / (12.3 × mg protein) (Equation (2)) To investigate thermal stability, mDHFR were incubated at 20 °C, 45 °C, or 65 °C, then cooled on ice, before use in the activity assay (Figure 8, Figure 9). EXAMPLE 2 – Poly-cistronic construct for recombinant cyclic protein production The genes encoding the enzyme OaAEP1-C247A and substrate murine dihydrofolate reductase (mDHFR) were subcloned into a pACYC-Duet co-expression plasmid for IPTG-inducible recombinant gene expression (Figures 1 and 2). Specifically, the enzyme gene construct encodes the core domain of OaAEP1 C247A (residues 24-328) fused with a N-terminal His6-Ubiquitin tag (Figure 3). This construct was previously reported to generate soluble enzyme which was isolated for in vitro applications (Tang et al., 2020). The substrate gene encodes mDHFR with the addition of OaAEP1 recognition sequences at the N and C termini as well as a His6tag to enable purification (Figures 1 and 2). EXAMPLE 3 – cyclic mDHFR preparation and characterisation mDHFR production and cyclisation were detected by SDS-PAGE analysis upon IPTG induced gene expression. In the absence of OaAEP1-C247A, over-expression results in a prominent band of approximately 25 kDa corresponding to the linear mDHFR substrate protein (Figure 4). In contrast, when OaAEP1-C247A and mDHFR was co-expressed, the band relating to the linear protein was diminished with a band becoming visible below the 25 kDa marker which was assigned to the cyclic mDHFR product (Figure 4). Downward band shift in SDS PAGE analysis can be attributed to (i) the more constrained cyclic topology of the protein and (ii) the loss of four amino acid residues during the OaAEP1-driven cyclization of mDHFR (two from the N- (RN) and two from the C-terminus (GL)). Purified mDHFR products were also analysed by mass spectrometry which detected the expected mass of the target linear and cyclic proteins, clearly demonstrating the loss of the RNGL residues and subsequent formation of a peptide bond (Figures 5A and 5B). Circular dichroism (CD) studies were next performed to demonstrate that H2T cyclisation did not significantly alter the global protein structure (Figure 6A). Both linear and cyclic mDHFR display strong negative mean residue ellipticity (MRE) at 209 nm, and positive MRE signal at 195 nm which are consistent with the β-strand dominant content of the DHFR protein fold. Thermal denaturation analysis following the loss of MRE signal at 209 nm displayed a thermal denaturation midpoint (Tm) of 39.9 °C for cyclic mDHFR, an increase of 3.7 °C over the linear protein (36.2 °C) (Figure 6B). Neither proteins are able to refold as demonstrated by the loss in signal for post-denaturation spectra. EXAMPLE 4 – mDHFR activity assay The activity of cyclic mDHFR was next assayed by monitoring the reduction in absorbance at 340 nm due to the consumption of NADPH cofactor during the DHFR catalysed reduction of dihydrofolate (DHF) into tetrahydrofolate (THF) (Figure 7). No significant differences in specific activity were observed between the linear and cyclic protein at 20 °C (1.65 U / mg – Figure 8). To validate the apparent improved thermal stability observed in CD studies, mDHFR was incubated at 45 °C for 15 minutes, then cooled on ice before repeating the activity assay at 20 °C. Although 45 °C is above the observed Tm for cyclic mDHFR by CD analysis, the temperature was selected to achieve complete denaturation of the linear protein while retaining a proportion (approx.40% cyclic vs.0% linear according to CD) of folded cyclic protein (Figure 7). In agreement with the CD thermal denaturation analysis, no reduction in absorbance at 340 nm was observed when linear mDHFR (pre-incubated at 45 °C) was used, demonstrating complete thermal denaturation of the enzyme (Figure 8). On the other hand, NADPH consumption was detected when using the heat treated cyclic mDHFR. The lower specific activity observed (Figure 8, 0.70 U / mg for heat- treated vs.1.65 U / mg for ambient) can be attributed to the lower abundance of active enzyme due to partial denaturation of the cyclic protein. EXAMPLE 5 – Cyclisation of Kalata B1 To demonstrate that the method of intracellular ligation described in Examples 1 to 4 is applicable to a broad range of peptides, the inventors recombinantly produced Kalata B1 precursor peptide (see SEQ ID NO: 12) and cyclised the peptide as described in Example 1 above. Mature kalata B1 (SEQ ID NO: 8) is a cystine knotted cyclotide (29 amino acids, head-to-tail cyclic, and 3 pairs of disulfide bonds). Biological activities of Kalata B1 include hemolytic,antimicrobial, anti-HIV, anti- helmintic, and insecticidal. Importantly, bioactive epitopes can be grafted onto cyclotides to generate stable peptide therapeutic leads. (Wong and Craik, 2018.; Muratspahić et al., 2020). The cyclic and Cys-knotted nature means the cyclotide is rigid, exhibits a range of biological activities and is highly stable (Wang et al., 2011). However, this topology also makes its production challenging, expensive and environmentally harmful, as chemical synthesis of this peptides relies on toxic solvents and reagents such as DMF. In addition, with six Cys residues forming three disulfide pairs, there are 15 possible pairing options. Therefore, oxidative folding of this peptide is inherently low yielding. The method of intracellular ligation described herein overcomes these limitations of prior art methods and shows efficient formation of cyclised Kalata B1, as demonstrated in Figure 10. The method of intracellular ligation is thus applicable to a wide variety of peptides, including those that pose challenges with traditional chemical production techniques, such as Kalata B1. EXAMPLE 6 – Production of optimised oaAEP enzyme In order to identify further variants of the oaAEP1 enzyme used in examples 1 to 5, the inventors carried out a surface library screen in which certain positions in the oaAEP enzyme were changed to polar or charged residues. These are surface charges that are expected to improve solubility, which means more enzyme is available in solution to perform catalysis, and / or improve catalytic rate. The library read-out used mDHFR as split enzyme where the activity is only regained if the oaAEP1 variant ligates the N- and C-terminal pieces. Without a functional oaAEP1 enzyme, the N- and C-terminal pieces of the split mDHFR do not ligate, resulting in cell death. This screen identified the oaAEP1 enzyme variant according to SEQ ID NO: 6 as a functional oaAEP1 enzyme variant. The variant was isolated using a His-tag as shown in SEQ ID NO: 7, as shown below with changes underlined: ARDGDYLHLPSEVSRFFRPQETNDDHGEDSVGTRWAVLIAGSKGYANYRHQAGVCHAYQILKRGGLKD ENIVVFMYDDIANNESNPRPGVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKSAITGGSGKVVDS GPNDHIFIYYTDHGAAGVIGMPSKPNLNADELNDALKKKHASGTYKSLVFYLEACESGSMFEGNLPEDLNI YALTSTNTTESSWAYYCPAQENPPPPEYNVCLGDLFSVAWLEDSDEQNSKDETLNQQYHHVDKRISHAS HATQYGNLKLGEEGLFVYMGSNPANDHHHHHH (SEQ ID NO: 7) Figure 11 shows that this variant has intracellular activity in E. Coli using the mDHFR system described in Examples 1 to 4. Figure 11A that this variant (“Hit 4”) is able to cyclise mDHFR, whereas other variants identified using a library screen (“Hit 1” and “Hit 3”) were not. This was confirmed by mass-spec which showed a peak at around 22991.01 amu, indicating the presence of cyclic mDHFR. Figure 12 further demonstrates the in vitro activity of this variant. Figure 12A shows that the AEP variant can be purified for in vitro cyclisation of a peptide, such as the substrate peptide of SEQ ID NO: 20 (Figure 12B). This substrate was incubated with the OaAEP1 variant or a buffer only control in a buffer containing 50 mM MES (pH 6.0), 100 mM NaCl, 1 mM EDTA, 1 mM TCEP. Test reactions were quenched with 1 M HCl. Figure 12B shows the presence of the desired cyclised product (see peak around 1448.74 Da) after incubating the substrate peptide with the variant OaAEP1 enzyme. EXAMPLE 7 – auto-catalysis / cyclisation of OaAEP1 Cyclic proteins tend to be more stable than linear proteins and this allows the enzyme to accumulate at higher levels in the cell. This has at least the following two advantages: (1) more enzyme can provide greater catalytic activity for the intracellular cyclisation reaction, and (2) improved expression yield for protein production for in vitro applications. To improve the method of intracellular ligation described in the above examples, the inventors set out to generate an auto-catalytic / cyclising OaAEP1 enzyme. This is expected to improve conversion rates, reduce proteolysis and minimise ligation scars. To do this, they appended the C-terminal and N-terminal recognition sequences of the OaAEP1 catalytic core domain (C247A) as set out in SEQ ID NO: 21, reproduced below with C-terminal and N-terminal recognition sequences underlined. MGRNGLHHHHHHGTRWAVLIAGSKGYANYRHQAGVCHAYQILKRGGLKDENIVVFMYDDIAYNESNPR PGVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKSAITGGSGKVVDSGPNDHIFIYYTDHGAAGVI GMPSKPYLYADELNDALKKKHASGTYKSLVFYLEACESGSMFEGILPEDLNIYALTSTNTTESSWAYYCP AQENPPPPEYNVCLGDLFSVAWLEDSDVQNSWYETLNQQYHHVDKRISHASHATQYGNLKLGEEGLFV YMGSNPANGL This sequence was expressed in a E. Coli Shuffle T7 express strain and induced with 0.4 mM IPTG at an OD600= 0.6. Cells were incubated overnight at 18ºC or 25ºC. Protein expression was assessed using SDS-page and the results are shown in Figure 13. Figure 13 shows significant protein over-expression with molecular weight corresponding to cyclised OaAEP1 (31.0 kDa) compared with original linear enzyme under the same expression conditions. These data indicate the successful auto-catalysis and cyclisation OaAEP1 enzyme. References A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein. Reference Full citation J. M. Antos, J. Ingram, T. Fang, N. Pishesha, M. C. Truttmann and H. L. Ploegh, Antos et al., 2017 Current Protocols in Protein Science, 2017, 89, 15.13.11-15.13.19. M. L. Colgrave and D. J. Craik , Thermal, chemical, and enzymatic stability of Colgrave et al., the cyclotide kalata B1: the importance of the cyclic cystine knot, Biochemistry, 2004 2004, 43 , 5965 —5975. Dall E, Brandstetter H. Mechanistic and structural studies on legumain explain its Dall et al., 2013 zymogenicity, distinct activation pathways, and regulation. Proc Natl Acad Sci U S A.2013;110(27):10940-10945. Gaglione et al., R. Gaglione, K. Pane, E. Dell’Olmo, V. Cafaro, E. Pizzo, G. Olivieri, E. Notomista 2019 and A. Arciello, New Biotechnology, 2019, 51, 39-48. I. Hara-Nishimura , Y. Takeuchi and M. Nishimura , Molecular characterization of Hara-Nishimura et a vacuolar processing enzyme related to a putative cysteine proteinase of al., 1993 Schistosoma mansoni, Plant Cell, 1993, 5 , 1651 Harmand et al., T. J. Harmand, N. Pishesha, F. B. H. Rehm, W. Ma, W. B. Pinney, Y. J. Xie and 2021 H. L. Ploegh, ACS Chemical Biology, 2021, 16, 1201-1207. K. S. Harris, T. Durek, Q. Kaas, A. G. Poth, E. K. Gilding, B. F. Conlan, I. Saska, Harris et al., 2015 N. L. Daly, N. L. van der Weerden, D. J. Craik and M. A. Anderson, Nature Communications, 2015, 6, 10199-10199. H. C. Hayes, L. Y. P. Luk and Y.-H. Tsai, Organic & Biomolecular Hayes et al., 2021 Chemistry, 2021, 19, 3983-4001. Huynh, K., & Partch, C. L. (2015). Analysis of protein stability and ligand Huynh et al., 2015 interactions by thermal shift assay. Current protocols in protein science, 79, 28.9.1–28.9.14. L. Y. P. Luk, J. J. Ruiz-Pernía, A. S. Adesina, E. J. Loveridge, I. Tuñón, V. Luk et al., 2015 Moliner and R. K. Allemann, Angewandte Chemie International Edition, 2015, 54, 9016-9020. Muge Ma, Simon T.M. Tang, Matthew T. Dickerson, Louis Y.P. Luk, Preparing Ma et al., 2023 recombinant “Split AEP” for protein labeling, Methods in Enzymology, Academic Press, 2023. K. M. Mikula, L. Krumwiede, A. Plückthun and H. Iwaï, Journal of Biomolecular Mikula et al., 2018 NMR, 2018, 71, 225-235. K. M. Mikula, I. Tascón, J. J. Tommila and H. Iwaï, FEBS Letters, 2017, 591, Mikula et al., 2017 1285-1294. Muratspahić E, Koehbach J, Gruber CW, Craik DJ. Harnessing cyclotides to Muratspahić et al., design and develop novel peptide GPCR ligands. RSC Chem Biol. 2020 2020;1(4):177-191. Published 2020 Jul 22. doi:10.1039 / d0cb00062k G. K. T. Nguyen , S. J. Wang , Y. B. Qiu , X. Hemu , Y. L. Lian and J. P. Tam , Nguyen et al., 2014 Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis, Nat. Chem. Biol., 2014, 10 , 732 —738 G. K. T. Nguyen , Y. Qiu , Y. Cao , X. Hemu , C.-F. Liu and J. P. Tam , Butelase- Nguyen et al., 2016 mediated cyclization and ligation of peptides and proteins, Nat. Protoc., 2016, 11 , 1977 —1988 Z. Qian, C. A. Rhodes, L. C. McCroskey, J. Wen, G. Appiah-Kubi, D. J. Wang, Qian et al., 2017 D. C. Guttridge and D. Pei, Angewandte Chemie (International ed. in English), 2017, 56, 1525-1529. F. B. H. Rehm, T. J. Harmand, K. Yap, T. Durek, D. J. Craik and H. L. Ploegh, Rehm et al., 2019 Journal of the American Chemical Society, 2019, 141, 17388-17393. F. Rigoldi, S. Donini, A. Redaelli, E. Parisini and A. Gautieri, APL Bioeng, 2018, Rigoldi et al., 2018 2, 011501. Reissmann S. Cell penetration: scope and limitations by the application of cell- Reissmann, 2014 penetrating peptides. J Pept Sci.2014;20(10):760-784. doi:10.1002 / psc.2672 C. P. Scott, E. Abel-Santos, M. Wall, D. C. Wahnon and S. J. Benkovic, Scott et al., 1999 Proceedings of the National Academy of Sciences, 1999, 96, 13638-13643. Si et al., 2016 M. Si, Q. Xu, L. Jiang and H. Huang, PLOS ONE, 2016, 11, e0162318. N. H. Shah, G. P. Dann, M. Vila-Perelló, Z. Liu and T. W. Muir, Journal of the Shah et al., 2012 American Chemical Society, 2012, 134, 11338-11341. Schechter et al., I. Schechter and A. Berger , On the size of the active site in proteases. I. Papain, 1967 Biochem. Biophys. Res. Commun., 1967, 27 , 157 —162 C. Sohrabi, A. Foster and A. Tavassoli, Nature Reviews Chemistry, 2020, 4, 90- Sohrabi et al., 2020 101. Solberg, R., Lunde, N. N., Forbord, K. M., Okla, M., Kassem, M., & Jafari, A. Solberg et al., 2022 (2022). The Mammalian Cysteine Protease Legumain in Health and Disease. International journal of molecular sciences, 23(24), 15983. Song M. The Asparaginyl Endopeptidase Legumain: An Emerging Therapeutic Song et al.,2022 Target and Potential Biomarker for Alzheimer's Disease. Int J Mol Sci. 2022;23(18):10223. Published 2022 Sep 6. K. Strijbis, E. Spooner and H. L. Ploegh, Traffic (Copenhagen, Denmark), 2012, Strijbis et al., 2012 13, 780-789. T. M. S. Tang, D. Cardella, A. J. Lander, X. Li, J. S. Escudero, Y.-H. Tsai and L. Tang et al., 2020 Y. P. Luk, Chemical Science, 2020, 11, 5881-5888. T. M. S. Tang and L. Y. P. Luk, Organic & Biomolecular Chemistry, 2021, 19, Tang et al., 2021 5048-5062. Vinogradov et al., A. A. Vinogradov, Y. Yin and H. 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Claims

Claims:

1. A method of intracellular peptide ligation, the method comprising the steps of: a) providing a cell comprising: i. a first peptide sequence comprising P1-P1’- P2’-Xn-COOH / CONH2, wherein P1 is Asn; P1’ is any amino acid except Pro; P2’ is an amino acid selected from the group Phe, Ile, Leu, Met, and Trp; X is any amino acid and n is an integer of 0 or 1 or more, ii. a second peptide sequence comprising H2N-P1”-P2” wherein P1” and P2” are any amino acid; and iii. an asparaginyl endopeptidase (AEP) catalytic domain construct, b) culturing the cell comprising the first and second peptide sequences and the AEP catalytic domain construct, such that the AEP catalytic domain construct hydrolyses the bond between P1 and P1’ of the first peptide sequence and ligates the resulting Asn-containing sequence to the N- terminus of the second peptide sequence such that the first peptide sequence is ligated to the second peptide sequence.

2. The method of claim 1, wherein the AEP catalytic domain construct comprises P1-P1’- P2’-Xn- COOH / CONH2 at its C terminus, wherein P1 is Asn; P1’ is any amino acid except Pro; P2’ is an amino acid selected from the group Phe, Ile, Leu, Met, and Trp; X is any amino acid and n is an integer of 0 or 1 or more, and H2N-P1”-P2” at its N terminus, wherein P1” and P2” are any amino acid.

3. The method of claim 1 or 2, wherein the first peptide sequence and the second peptide sequence are part of the same peptide.

4. The method of claim 1, wherein the first peptide sequence, the second peptide sequence, and the AEP catalytic domain construct are part of the same peptide.

5. The method according to any preceding claim, wherein a) P1’ is any naturally occurring amino acid except Pro; and / or b) P2’ is a naturally occurring amino acid selected from the group Phe, Ile, Leu, Met, and Trp; and / or c) X is any naturally occurring amino acid; and / or d) P1” is any naturally occurring amino acid, and / or e) P2” is a naturally occurring amino acid, optionally a naturally occurring amino acid selected from Val, Leu, or Ile.

6. The method according to any preceding claim, wherein P1’ is Cys or Gly; and P2’ is Leu; P1” is Gly; and P2” is Leu.

7. The method according to any preceding claim, wherein the AEP catalytic domain construct comprises the catalytic domain of Oldenlandia affinis, oaAEP1, or a mutant thereof.

8. The method according to claim 7, wherein the oaAEP1 mutant comprises a C247A mutation (“C247 mutant”).

9. The method according to any preceding claim, wherein the AEP catalytic domain construct is as set out in SEQ ID NO: 5 (“C247A mutant”) or a variant thereof.

10. The method according to claim 9, wherein the variant has at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5, optionally wherein the variant has at least 95% sequence identity to SEQ ID NO:

5.

11. The method according to claim 9, wherein the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions, optionally wherein the variant has no more than 10 substitutions.

12. The method according to claims 9 – 11, wherein the variant comprises or consists of a sequence as set out in SEQ ID NO:

6.

13. The method according to claims 9 - 12, wherein the variant has one or more functional properties that are at least the same compared to the C247A mutant, optionally wherein the one or more functional properties comprise activity, stability, kinetic properties and / or solubility.

14. The method according to claim 13, wherein the variant has at least the same activity compared with the C247A mutant, optionally wherein the activity is determined by measuring the cyclisation of a substrate peptide, optionally wherein the substrate peptide is GLPVSTKPVATRNGL (SEQ ID NO: 9) or GLPYSTKPYATRNGL (SEQ ID NO: 20), optionally wherein measuring the cyclisation of substrate peptide is carried out by liquid chromatography-mass spectrometry (LC–MS).

15. The method according to claim 13, wherein the variant has at least the same stability compared with the C247A mutant, optionally wherein the stability is determined by a thermal shift assay.

16. The method according to claim 13, wherein the variant has at least the same solubility compared with the C247A mutant, optionally wherein the solubility is determined by measuring the absorbance of the variant at 280 nm, or by using a biuretic methods (e.g. BCA assay, Lowry assay), colorimetric dye based method (e.g. Bradford (Coomassie) assay), or fluorescent dye method.

17. The method according to claim 13, wherein the variant has at least the same kinetic properties compared with the C247A mutant, optionally wherein the kinetic properties are determined by fluorescence energy transfer (FRET), surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC).

18. The method according to claims 1 -17, wherein the AEP catalytic domain construct is cyclic.

19. The method according to any preceding claim, wherein the first peptide fragment and / or second peptide fragment comprises a labelling moiety.

20. The method according to claim 19, wherein the labelling moiety is an affinity tag, e.g. a His tag, and / or a detectable label, e.g. a GFP tag, and / or degradation tag, e.g. a SSRA tag.

21. The method according to any preceding claim, wherein the cell is a bacterial cell, optionally an Escherichia coli cell.

22. The method according to any preceding claim, wherein the method is performed at a temperature lower than 50 ºC, lower than 40 ºC, or lower than 35 ºC.

23. The method according to any preceding claim, wherein the method is performed at a temperature of between about 30 ºC and about 37 ºC.

24. The method according to any preceding claim, wherein the method is performed at a temperature of about 30 ºC.

25. The method according to any preceding claim, wherein the method is carried out in the absence of amide coupling reagents, and / or in the absence of organic solvents.

26. The method according to any preceding claim, wherein the method further comprises isolating the ligated peptide from the cell.

27. The method according to any preceding claim, wherein providing in the cell the first and second peptide sequence comprises delivering one or more nucleic acid(s) encoding the first and second peptide sequences to the cell, such that the cell expresses the first and second peptide sequences.

28. The method according to any preceding claim, wherein providing in the cell the AEP catalytic domain construct comprises delivering one or more nucleic acid(s) encoding the AEP catalytic domain construct, such that the cell expresses the AEP catalytic domain construct.

29. A ligated peptide and / or cells obtainable according to any of the preceding claims.

30. A method for screening peptide libraries for transcription factor antagonists, the method comprising; a) providing a cell, wherein the cell comprises a peptide ligated according to the method of claims 1- 28, and first and second candidate binding partners, andb) assaying whether the ligated peptide is able to inhibit association between the first and second candidate binding partner.

31. The method according to claim 30, wherein assaying for whether the ligated peptide is able to inhibit association between the first and second candidate binding partners comprises determining whether the ligated peptide is able to modulate expression and / or activity of a reporter protein.

32. The method according to claim 31, wherein association of the first and second candidate binding partners forms a DNA-binding complex that binds one or more binding site(s) in a nucleic acid encoding the reporter protein, wherein binding of the DNA-binding complex to the binding site inhibits expression of the reporter protein, optionally wherein some or all of the binding sites are located in the transcribed sequence of the nucleic acid encoding the reporter protein.

33. The method according to claims 30 - 33, wherein the first and second candidate binding partners are transcription factors.

34. The method according to claim 33, wherein the transcription factor is a bZIP transcription factor, optionally wherein the bZIP transcription factor is c-Jun, CREB, and / or BZLF1.

35. An AEP catalytic domain construct for use in a method of intracellular peptide ligation.

36. The AEP catalytic domain construct of claim 35, wherein the AEP catalytic domain comprises the catalytic domain of Oldenlandia affinis, oaAEP1, or a mutant thereof.

37. The AEP catalytic domain construct of claim 36, wherein the oaAEP1 mutant comprises a C247A mutation (“C247 mutant”).

38. The AEP catalytic domain construct of claims 35 - 37, wherein the AEP catalytic domain construct is as set out in SEQ ID NO: 5 (“C247A mutant”) or a variant thereof.

39. The AEP catalytic domain construct of claims 35 - 38, wherein the variant has at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5, optionally wherein the variant has at least 95% sequence identity to SEQ ID NO:

5.

40. The AEP catalytic domain construct of claims 35 - 39, wherein the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions, optionally wherein the variant has no more than 10 substitutions.

41. The AEP catalytic domain construct of claims 35 - 40, wherein the variant comprises or consists of a sequence as set out in SEQ ID NO: 6.

42. The AEP catalytic domain construct of claims 35 -41, wherein the AEP catalytic domain construct is cyclic.

43. A kit for use in any of the methods of claims 1 to 34, wherein the kit comprises an AEP catalytic domain construct.

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