Purification of recombinant fusion protein

Abstract

The invention relates to methods of enriching target polypeptides from a sample. Further, the invention relates to self-cleaving inteins useful for this method.

Description

[0001] NOVEL METHOD

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods of enriching target polypeptides from a sample. Further, the invention relates to self-cleaving inteins useful for this method.

[0004] BACKGROUND OF THE INVENTION

[0005] Peptides have many advantages that make them highly suitable for development in a wide variety of sectors, including pharmaceutical, agrochemical and materials (Chen et al., 2021 ; Wang et al., 2022). Their flexibility in terms of primary, secondary and potentially even tertiary structure enables a huge number of potential structures and uses (Peptide Applications in Biomedicine, Biotechnology and Bioengineering, ScienceDirect, 2018). Most short peptides are generated by synthetic chemistry, generally using solid phase synthesis. This is effective and cost efficient for peptides up to about 10-15 amino acids, but the cost increases significantly as the length of the peptide chain increases beyond this (Reddy, 2017). Meanwhile, recombinant (DNA encoded) peptide production methods have started to gain increased industrial use, especially for longer peptides (Wegmuller and Schmid, 2014).

[0006] However, it usually slower to set up biotechnological methods for recombinant production of peptides and limitations in terms of N-terminal sequence and production costs can still be a major restriction to development. The main costs of recombinant peptide production are usually associated with the downstream part of the process, in particular generation of isolated peptide with quality sufficient for its ultimate use (Gaglione et al., 2019).

[0007] Numerous methods have been developed to improve the route to isolated peptides with a selectable N-terminal amino acid. Numerous affinity purification methods have been developed, which rely on adding an ‘affinity tag’ to the peptide (such as the common hexahistidine tag) and using this to bind e.g., to a column, washing to remove other components that do not bind the affinity matrix, then releasing the peptide via a number of methods. However, the cost of the reagents and materials required for affinity purification is also high, can lead to difficulties with scale and the requirement for heavy metals can lead to waste stream and safety issues (Rauwolf et al., 2021).

[0008] There is therefore a need to develop improved methods for purifying recombinant proteins away from contaminants. SUMMARY OF THE INVENTION

[0009] According to a first aspect of the invention, there is provided a method for enriching a target polypeptide in a sample, comprising the steps:

[0010] (i) providing a sample containing the target polypeptide in the form of a recombinant fusion protein in which it is tagged with a cleavable polypeptide tag;

[0011] (ii) separating components of the sample on the basis of a first molecular weight cut-off such that components of the sample having a molecular weight greater than the first molecular weight cut-off are retained in a first retained fraction such that the tagged target polypeptide is in the first retained fraction;

[0012] (iii) cleaving the cleavable tag from the target polypeptide in the first retained fraction; and thereafter

[0013] (iv) separating components of the first retained fraction on the basis of a second molecular weight cut-off such that components of the first retained fraction having a molecular weight less than the second molecular weight cut-off are retained in a second retained fraction such that the target polypeptide is in the second retained fraction and is enriched relative to the sample.

[0014] According to a further aspect of the invention, there is provided a mutant self-cleaving intein wherein the mutant intein is a mutant Mtu RecA intein comprising an amino acid substitution at a position selected from the group consisting of: His429, His439 and Asn440.

[0015] In certain embodiments, the mutant intein comprises an amino acid substitution selected from the group consisting of: H439V, H439W, H439L, H439G, H439M, H439I, N440Q, N440T, N440S, N440Y and H429F, preferably a substitution selected from the group consisting of: H439V, H439W, H439L, H439G, N440S and N440Q.

[0016] In a further aspect, there is provided a recombinant fusion protein comprising a target polypeptide and the intein described herein, optionally further comprising a sequence which directs expression of the recombinant fusion protein to inclusion bodies.

[0017] According to a yet further aspect of the invention, there is provided a use of the intein as described herein as a component of a cleavable tag in a method for enriching a target polypeptide in a sample as described herein.

[0018] BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1 : Representative embodiment of the tagged peptide and released peptide showing cleavage to produce the final desired peptide. The target peptide is tagged with a cleavable polypeptide tag comprising an inclusion body (IB) tag, which directs expression of the tagged target polypeptide to inclusion bodies, and a self cleaving intein. After cleavage, the tag is removed, leaving only the target peptide.

[0020] Figure 2: Representative embodiment of the working principle of the method. The cells expressing the tagged target peptide of Figure 1 are fermented and the fermentation broth is harvested and lysed. The inclusion bodies are isolated and subjected to a first molecular weight filter which separates the tagged peptide and larger contaminants from smaller contaminants. The fraction comprising the tagged peptide and larger contaminants is subjected to conditions which result in intein cleavage, separating the target peptide from the tag. The cleaved sample is then subjected to a second molecular weight filter which separates the target protein from the tag and the larger contaminants.

[0021] Figure 3: Representative schematic showing example conditions and process for use in the claimed method. Schematic of the process described in Example 3. Solid boxes represent material retained. Dotted boxes represent materials discarded. The pellet from the Cleavage step (H) and the filtrate from the Purification 2 step (J) are both retained as they contain uncleaved peptide, which can be recovered and passed through the process again.

[0022] Figure 4: Example Mtu RecA inteins and variants showing critical residues. Inteins based on the Mycobacterium tuberculosis RecA intein include the wild-type (WT) RecA intein, reduced size variants with the endonuclease domain removed: Al, AI-CM, AI-SM (168 amino acids) and even smaller variants where an additional peptide loop is replaced: AAIhh, AAlhh-SM and AAlhh-CM (139 amino acids) (from Van Roey et al., 2007).

[0023] Figure 5: Schematic to show the genetic components of the generated plasmids pSIM238, pSIM245, p068-009, pJP178, p068-021, p068-023. (A) Plasmids pSIM238, pSIM 245 and p068-009 contain a T7 promoter, RBS, and a CDS encoding a transcript for a 6xHIS- tag, thrombin site, Ketosteroid isomerase insolubility tag, AI-CM mini intein (parental sequence in pSIM238, H439V mutation in pSIM245 and p068-009) and His-tagged GLP-1 (7-37) peptide sequence. Plasmid p068-009 also contained a lac operator binding site. (B) Plasmids pJP178, p068-021 and p068-023 contain a T7 promoter, RBS, and a CDS encoding a transcript for a 6xHIS-tag, thrombin site, Ketosteroid isomerase insolubility tag, AI-CM mini intein (parental sequence in pJP178, H439V in p068-021 and N440Q in p068-023) and untagged GLP-1 (7- 37).

[0024] Figure 6: Schematic to compare the amino acid substitutions in plasmids pJP178, p068-021 and p068-023. Amino acid mutations are shown in the context of the C- terminal sequence region of the AI-CM mini intein. pJP178 contains the parental AI-CM sequence, p068-021 contains an H439V mutation and p068-023 contains an N440Q mutation. Figure 7: SDS PAGE gel comparing supernatant and pellet following homogenisation for mutant inteins. Lanes as follows: 1. Marker, 2. p068-021 supernatant, 3. p068-021 pellet, 4. p068-023 supernatant, 5.p068-023 pellet, 6. pJP178 supernatant, 7. pJP178 pellet, 8. Marker. pJP178 contains the parental AI-CM sequence, p068-021 contains an H439V mutation and p068-023 contains an N440Q mutation.

[0025] Figure 8: SDS PAGE gel showing material at each stage of a representative process (from BL21(DE3) p068-021 fermentation). Lanes as follows: 1. Marker, 2. Homogenate supernatant, 3. Homogenate pellet, 4. Buffer exchange 1 filtrate, 5. Buffer exchange 1 retentate supernatant, 6. Buffer exchange 1 retentate precipitate, 7. Cleavage supernatant, 8. Cleavage pellet, 9. Cleavage pellet, 10. Buffer exchange 2 supernatant, 11. Buffer exchange 2 precipitate, 12. Purification 2 retentate, 13. Purification 2 filtrate, 14. Solid peptide, 15. solid peptide after protein precipitation.

[0026] Figure 9: HPLC trace showing material (expressed from fermentation of BL21(DE3) containing p068-021) relating to Example 3. Indicated are Stage G (Buffer Exchange 1), Stage H (Cleavage) and the GLP-1 peptide standard. Cleaved and Uncleaved protein along with released GLP-1 peptide are indicated with arrows.

[0027] Figure 10: Representative schematic showing example conditions and process for use in the claimed method. Schematic of the process described in Example 5. Solid boxes represent material retained. Dotted boxes represent materials discarded. The retentate from the Purification 2 step (I) are both retained as they contain uncleaved peptide, which can be recovered and passed through the process again.

[0028] Figure 11 : HPLC trace showing material (expressed from fermentation of BL21(DE3) containing p068-021) relating to Example 5. Indicated are Stage G (Dilution - addition of solubilised partially purified inclusion bodies to phosphate buffer) Stage H (Cleavage of the intein), Stage I (Purification 2 - Removal of large contaminants by concentration using Tangential Flow Filtration (TFF)), and Stage J (Desalt - Removal of buffer salts using C18). Cleaved and uncleaved protein along with released GLP-1 peptide are indicated with arrows.

[0029] Figure 12: Schematic to show the genetic components of the generated plasmids p086-001, p086-010, p086-011, p086-045, p086-046, p086-047, p086-048, p086- 049, p086-050, p086-051. All plasmids contain a T7 promoter, RBS, and a CDS encoding a transcript for a 6xH I S-tag, AI-CM mini intein (parental sequence in p086-001 , H439V mutation in p086-010, N440Q mutation in p086-011 , H439I mutation in p086-045, H439L mutation in p086-046, N440S mutation in p086-047, N440T mutation in p086-048, H439G mutation in p086-049, H439W mutation in p086-050, H439M mutation in p086-051), a short linker of sequence GGGSGGGS (SEQ ID NO: 88) or HAGSGGGS (SEQ ID NO:90) as indicated and His-tagged GFP11 peptide sequence. AI-CM mini intein and mutants cleavage site is indicated by the cross mark.

[0030] BRIEF DESCRIPTION OF SEQ IDs

[0031] SEQ ID NO 1 : Complete plasmid sequence of pJP178

[0032] SEQ ID NO 2: KSI protein sequence

[0033] SEQ ID NO 3: AI-CM mini intein protein sequence

[0034] SEQ ID NO 4: Peptide sequence of GLP-1 (7-37)

[0035] SEQ ID NO 5: Complete peptide sequence expressed in pJP178

[0036] SEQ ID NO 6: Complete plasmid sequence of pSIM238

[0037] SEQ ID NO 7: GLP-1 (7-37) with PV linker and 6x HIS-tag

[0038] SEQ ID NO 8: Complete peptide sequence expressed in pSIM238

[0039] SEQ ID NO 9: lacZ reporter sequence

[0040] SEQ ID NO 10 Oligonucleotide OMSIM420

[0041] SEQ ID NO 11 Oligonucleotide OMSIM421

[0042] SEQ ID NO 12 Oligonucleotide OMSIM423

[0043] SEQ ID NO 13 Oligonucleotide OMSIM422

[0044] SEQ ID NO 14 Complete plasmid sequence of pSIM239

[0045] SEQ ID NO 15 Oligonucleotide OMSIM434

[0046] SEQ ID NO 16 Oligonucleotide OMSIM435

[0047] SEQ ID NO 17 Complete plasmid sequence of pSIM245

[0048] SEQ ID NO 18 AI-CM mini intein protein sequence (H439V)

[0049] SEQ ID NO 19 Complete peptide sequence expressed in pSIM245

[0050] SEQ ID NO 20 pET28a(+) (plasmid DNA)

[0051] SEQ ID NO 21 p068_009

[0052] SEQ ID NO 22 Oligonucleotide oJLOOl

[0053] SEQ ID NO 23 Oligonucleotide oJL002

[0054] SEQ ID NO 24 Complete plasmid sequence of p068-021

[0055] SEQ ID NO 25 Complete peptide sequence expressed in p068-021

[0056] SEQ ID NO 26 Complete plasmid sequence of p068-023

[0057] SEQ ID NO 27 Complete peptide sequence expressed in p068-023

[0058] SEQ ID NO 28 AI-CM mini intein protein sequence (N440Q)

[0059] SEQ ID NO 29 wild-type Mtu RecA intein

[0060] SEQ ID NO 30 Al mini intein

[0061] SEQ ID NO 31 AI-CM mini intein

[0062] SEQ ID NO 32 AI-SM mini intein

[0063] SEQ ID NO 33 AAlhh mini intein SEQ ID NO 34 AAlhh-CM mini intein

[0064] SEQ ID NO 35 AAlhh-SM mini Intein

[0065] SEQ ID NO 36 Complete plasmid sequence of p086-001

[0066] SEQ ID NO 37 GGGSGGGS linker and GFP11 6x HIS-tagged released peptide

[0067] SEQ ID NO 38 Complete protein sequence expressed in p086-001

[0068] SEQ ID NO 39 Complete plasmid sequence of p068-010

[0069] SEQ ID NO 40 Complete protein sequence expressed in p086-010

[0070] SEQ ID NO 41 Complete plasmid sequence of p068-011

[0071] SEQ ID NO 42 Complete protein sequence expressed in p086-011

[0072] SEQ ID NO 43 Complete plasmid sequence of p086-036

[0073] SEQ ID NO 44 Complete plasmid sequence of p086-037

[0074] SEQ ID NO 45 Complete plasmid sequence of p086-039

[0075] SEQ ID NO 46 Complete protein sequence expressed in p086-039

[0076] SEQ ID NO 47 Al-CM mini intein protein sequence (H429F)

[0077] SEQ ID NO 48 Complete plasmid sequence of p086-040

[0078] SEQ ID NO 49 Complete protein sequence expressed in p086-040

[0079] SEQ ID NO 50 Al-CM mini intein protein sequence (H429V)

[0080] SEQ ID NO 51 Complete plasmid sequence of p086-041

[0081] SEQ ID NO 52 Complete protein sequence expressed in p086-041

[0082] SEQ ID NO 53 Al-CM mini intein protein sequence (H439A N440Q)

[0083] SEQ ID NO 54 Complete plasmid sequence of p086-042

[0084] SEQ ID NO 55 Complete protein sequence expressed in p086-042

[0085] SEQ ID NO 56 Al-CM mini intein protein sequence (G435D)

[0086] SEQ ID NO 57 Complete plasmid sequence of p086-043

[0087] SEQ ID NO 58 Complete protein sequence expressed in p086-043

[0088] SEQ ID NO 59 Al-CM mini intein protein sequence (H439A)

[0089] SEQ ID NO 60 Complete plasmid sequence of p086-044

[0090] SEQ ID NO 61 Complete protein sequence expressed in p086-044

[0091] SEQ ID NO 62 Al-CM mini intein protein sequence (H439F)

[0092] SEQ ID NO 63 Complete plasmid sequence of p086-045

[0093] SEQ ID NO 64 Complete protein sequence expressed in p086-045

[0094] SEQ ID NO 65 Al-CM mini intein protein sequence (H439I)

[0095] SEQ ID NO 66 Complete plasmid sequence of p086-046

[0096] SEQ ID NO 67 Complete protein sequence expressed in p086-046

[0097] SEQ ID NO 68 Al-CM mini intein protein sequence (H439L)

[0098] SEQ ID NO 69 Complete plasmid sequence of p086-047 SEQ ID NO 70 Complete protein sequence expressed in p086-047

[0099] SEQ ID NO 71 AI-CM mini intein protein sequence (N440S)

[0100] SEQ ID NO: 72 Complete plasmid sequence of p086-048

[0101] SEQ ID NO: 73 Complete protein sequence expressed in p086-048

[0102] SEQ ID NO: 74 AI-CM mini intein protein sequence (N440T)

[0103] SEQ ID NO 75 Complete plasmid sequence of p086-049

[0104] SEQ ID NO 76 Complete protein sequence expressed in p086-049

[0105] SEQ ID NO 77 AI-CM mini intein protein sequence (H439G)

[0106] SEQ ID NO 78 Complete plasmid sequence of p086-050

[0107] SEQ ID NO 79 Complete protein sequence expressed in p086-050

[0108] SEQ ID NO 80 AI-CM mini intein protein sequence (H439W)

[0109] SEQ ID NO 81 Complete plasmid sequence of p086-051

[0110] SEQ ID NO 82 Complete protein sequence expressed in p086-051

[0111] SEQ ID NO 83 AI-CM mini intein protein sequence (H439M)

[0112] SEQ ID NO 84 Complete plasmid sequence of p086-052

[0113] SEQ ID NO 85 Complete protein sequence expressed in p086-052

[0114] SEQ ID NO 86 AI-CM mini intein protein sequence (N440Y)

[0115] SEQ ID NO 87 HAGSGGGS linker and GFP11 6x HIS-tagged released peptide

[0116] SEQ ID NO 88: GGGSGGGS linker in front of GFP-11

[0117] SEQ ID NO 89 PVHHHHHH linker downstream of GLP1 (7-37)

[0118] SEQ ID NO 90 HAGSGGGS linker in front of GFP-11

[0119] DETAILED DESCRIPTION OF THE INVENTION

[0120] The present inventors have developed a novel method for scalable cost-efficient enrichment of a target polypeptide in a sample, which relies on the target polypeptide being tagged with a cleavable polypeptide tag. The tagged polypeptide is enriched from a sample using a method that separates the tagged polypeptide from most other solutes based on their relative molecular weights. This first filtration step is followed by cleavage of the tag from the target polypeptide, resulting in the target peptide and the released tag. Lastly, the target polypeptide is separated from the released tag and any remaining contaminants in a second filtration step that separates them based on their, now changed, relative molecular weights. Figure 2 provides a representative embodiment of the working principle of this method.

[0121] Therefore, according to a first aspect of the invention, there is provided a method for enriching a target polypeptide in a sample, comprising the steps: (i) providing a sample containing the target polypeptide in the form of a recombinant fusion protein in which it is tagged with a cleavable polypeptide tag;

[0122] (ii) separating components of the sample on the basis of a first molecular weight cut-off such that components of the sample having a molecular weight greater than the first molecular weight cut-off are retained in a first retained fraction such that the tagged target polypeptide is in the first retained fraction;

[0123] (iii) cleaving the cleavable tag from the target polypeptide in the first retained fraction; and thereafter

[0124] (iv) separating components of the first retained fraction on the basis of a second molecular weight cut-off such that components of the first retained fraction having a molecular weight less than the second molecular weight cut-off are retained in a second retained fraction such that the target polypeptide is in the second retained fraction and is enriched relative to the sample.

[0125] Reference herein to “enriching” relates to increasing the proportion of the target polypeptide in the sample relative to other components of the sample, such as contaminants, and therefore increasing the purity of the target polypeptide in the sample. In one embodiment, the purity of the target polypeptide in the second retained fraction is at least 80% e.g., at least 85% e.g., at least 90%. In one embodiment the purity of the target polypeptide in the second retained fraction is greater than 90%.

[0126] For efficient separation of the tag and the target polypeptide in step (iv), the tag needs to be significantly larger than the target peptide. Therefore, in one embodiment, the molecular weight of the cleavable tag is at least 2 times e.g., at least 2.5, 3, 3.5 or 4 times the molecular weight of the target polypeptide. In a further embodiment, the molecular weight of the cleavable tag is at least 3 times the molecular weight of the target polypeptide.

[0127] Separation of the components is carried out on the basis of a molecular weight cut-off (MWCO). The sample is subjected to a first molecular weight filter which separates the tagged peptide and larger contaminants from smaller contaminants. Therefore, it will be clear to the person skilled in the art that the MWCO of the first molecular weight filter should be chosen such that the MWCO is smaller than the molecular weight of the tagged peptide. For maximum recovery of proteins during filtration it is advantageous select a MWCO at least 50% smaller e.g., at least 55%, 60%, 65%, 70% or 75% smaller than the molecular weight of the tagged peptide. The fraction comprising the tagged peptide and larger contaminants is then cleaved, separating the target peptide from the tag. The cleaved sample is subjected to a second molecular weight filter which separates the target protein from the tag and the larger contaminants. Therefore, it will be clear to the person skilled in the art that the MWCO of the second molecular weight filter should be chosen such that the MWCO is larger than the molecular weight of the cleaved peptide, but smaller than the molecular weight of the cleaved tag.

[0128] The methods of the invention have the advantage that target polypeptides can be purified at reduced cost of goods due to no requirement for costly column chromatography purification. Therefore, in one embodiment, the method does not comprise the separation of components using column chromatography. Separation of the components on the basis of a molecular weight cut-off can be performed by numerous other methods known in the art. For example, the method is compatible with various filtration systems including spin or gravity flow columns. Therefore, in one embodiment the components are separated by filtration.

[0129] Tangential Flow Filtration (TFF) is a cost effective and scalable method for removing contaminants from products by virtue of their relative molecular weights and for concentrating samples. Whilst highly valuable in terms of downstream processing options, it is unable to purify products away from components of the same or similar molecular weight (Pires and Palmer, 2021). For example, typically for retention, a molecular weight cut-off 3 to 6-fold smaller than the size of the component is required (e.g., a 5-10kDa filter to reliably capture a 30kDa component) (Schwartz and Seely, 2022). This barrier is overcome by the methods of the invention as the relative molecular weight of the target peptide is varied by cleavage from the peptide tag. Therefore, in one embodiment, the components are separated by tangential flow filtration.

[0130] Peptide Production

[0131] The present invention provides methods for enriching target polypeptides produced by both cell free and cellular methods.

[0132] In one embodiment, the tagged target polypeptide in the form of a recombinant fusion protein has been produced by cell-free protein synthesis and the sample is a sample of cell-free reaction mixture comprising the tagged target polypeptide. In an alternative embodiment, the tagged target polypeptide in the form of a recombinant fusion protein has been produced by fermentation and the sample is a sample of fermentation broth comprising the tagged target polypeptide.

[0133] Overexpressed recombinant proteins may exist in various forms: soluble properly folded forms, partially folded intermediates, and insoluble aggregates. Approximately 70% of recombinant proteins naturally form insoluble aggregates, known as inclusion bodies, when over-expressed (Yang et al (2011)). Therefore, in one embodiment, the tagged target polypeptide in the form of a recombinant fusion protein has been produced by fermentation and the sample is a sample comprising homogenised and solubilised inclusion bodies comprising the tagged target polypeptide.

[0134] Further, partially folded intermediates are the form most susceptible to proteolytic degradation. Therefore, encouraging recombinant proteins to form inclusion bodies can protect the protein from proteolysis and enhance protein production. Therefore, in one embodiment the cleavable polypeptide tag comprises a sequence which directs expression of the tagged target polypeptide to inclusion bodies. Examples of such sequences include, but are not limited to, ssTorA, TrpE, PurF, PagP, ketosteroid isomerase (KSI), GFIL16, Alpha helical 18A, Beta strand ELK16 or Surfactant L6KD.

[0135] References herein to a “tagged polypeptide” refers to a recombinant protein comprising the target polypeptide fused to an additional polypeptide sequence which allows the target polypeptide to be purified or detected (i.e. , the polypeptide ‘tag’). Polypeptide tags are known in the art, and may include, but are not limited to, affinity tags for use in affinity purification (e.g., Glutathione-S-transferase (GST), His-tags, Biotin and Strep-tags) and solubilisation tags to improve protein solubility (e.g., Maltose Binding Protein (MBP), NusA, thioredoxin, GST, and Small Ubiquitin-like Modifier (SUMO)).

[0136] The polypeptide tag may be fused onto the N-terminal or C-terminal of the target polypeptide sequence. In one embodiment, the polypeptide tag is N-terminal to the target polypeptide sequence. In an alternative embodiment, the polypeptide tag is C-terminal to the target polypeptide sequence.

[0137] References herein to a “cleavable polypeptide tag” relate to a polypeptide tag that comprises a polypeptide sequence that is susceptible to cleavage. In one embodiment, the polypeptide tag sequence is susceptible to chemical cleavage. For example, the sequence may comprise methionine residues susceptible to cleavage by cyanogen bromide, tryptophan residues susceptible to cleavage by BNPS-skatole, aspartic acid-proline peptide bonds susceptible to cleavage by formic acid, asparagine-glycine peptide bonds susceptible to cleavage by hydroxylamine, and / or cysteine residues susceptible to cleavage by 2-nitro-5- thiocyanobenzoic acid. In another embodiment, the polypeptide tag sequence is susceptible to enzymatic cleavage. Examples of enzymes suitable for the removal of polypeptide tags by cleavage include, but are not limited to: Enteropeptidase, Thrombin, Factor Xa, TEV protease, Rhinovirus 3C protease, Carboxypeptidase A, Carboxypeptidase B and DAPase.

[0138] One alternative chemical or enzymatic cleavage for tag removal is self cleaving inteins. Inteins are naturally occurring proteins that excise themselves from a protein through self-cleavage and ligation of their flanking peptide bonds. Therefore, in one embodiment, the cleavable tag comprises a self-cleaving intein.

[0139] A number of self-cleaving inteins have been described. Those which are suitable for peptide synthesis using an N-terminal tag are inteins where there is controllable C-terminal cleavage to release the peptide. Therefore, in one embodiment the cleavable tag comprises a selfcleaving intein and cleaving the cleavable tag comprises adjusting one or more sample conditions such that the intein self-cleaves from the target polypeptide in the adjusted sample conditions. For example, adjusting the sample conditions may comprise an adjustment of pH, an adjustment of temperature, or the addition of one of more compounds. In a particular embodiment, the sample condition is pH. In a further embodiment, the intein is capable of being cleaved at a pH of less than 6.8. In another embodiment, the sample condition is temperature. In one example embodiment, the intein is capable of being cleaved at a temperature equal to or greater than 28°C, such as a temperature equal to or greater than 30°C, 32°C, 34°C or 36°C. In particular, the intein is capable of being cleaved at a temperature equal to or great than 37°C, such as at 37°C.

[0140] One of the best described inteins is the Mycobacterium tuberculosis RecA (Mtu RecA) intein and the range of variant inteins based on Mtu RecA. References herein to “variant” inteins refer to inteins which are truncated versions of wild type inteins or have sequence modifications relative to the wild type intein designed to adapt (and especially improve) the properties of the intein, not including amino acid substitutions that result in reduced premature C-terminal cleavage. For example, variants may be designed to improve the stability of the intein. Figure 4 lists the most commonly used Mtu RecA variants, including reduced size variants with the endonuclease domain removed: Al, AI-CM, AI-SM (168 amino acids) and even smaller variants where an additional peptide loop is replaced: AAlhh, AAlhh-SM and AAlhh- CM (139 amino acids) (Hiraga et al., 2005). Therefore, in one embodiment the intein is wild-type Mtu RecA (SEQ ID NO: 29) or a variant and / or mutant thereof.

[0141] In a further embodiment, the variant Mtu RecA intein is selected from the group consisting of: Al mini intein (SEQ ID NO: 30), AI-CM mini intein (SEQ ID NO: 31), AI-SM mini intein (SEQ ID NO: 32), AAlhh mini intein (SEQ ID NO: 33), lhh-CM mini intein (SEQ ID NO: 34) or AAlhh- SM mini intein (SEQ ID NO: 35), or a mutant thereof. In a particular embodiment, the variant intein is AI-CM mini intein (SEQ ID NO: 31) or a mutant thereof.

[0142] Other self-cleaving inteins of use in in the present invention include, but are not limited to, HutMCM2 (WO2016174311) and Mxe GyrA (WO2014191455).

[0143] Improved Self-Cleaving Inteins

[0144] Inteins, in particular C-terminally cleaving inteins are notoriously difficult to control, leading to premature or partial cleavage. Solutions have focused on controlling intein function through the reassembly of frans-cleaving inteins, as well as the engineering of disulfide bonds into existing intein systems (Wood and Camarero, 2014). Some authors and inventors have described variant Mtu RecA inteins with improved controllability (Lin et al., 2020), including US11 ,136,360 and the paper by the same authors (Lin et al., 2020).

[0145] When referring to amino acid residue positions within variant Mtu RecA inteins, different numbering systems have been used in the prior art. Some references use the relative numbers in the AI-CM 168 amino acid mini-intein (e.g., H is 167), whilst other references use the relative numbers for the 440 amino acid wild-type intein (e.g., His 439). For the purpose of this description, the relative numbering for the AI-CM mini-intein will be placed in square brackets when the WT numbering is used, and vice versa.

[0146] In US11 ,136,360, the AI-CM intein was mutated with the aim to obtain a Mtu AI-CM mutant strain with reduced cleavage efficiency in E. coli cells (pH 7.4-7.8), but still capable of efficient cleavage at pH 6.0 in vitro. Amino acids within the range of 5 A around H157 [H429] and H167 [H439] were selected for site-specific saturated mutations, mainly aiming at amino acids which may form hydrogen bonds or have a charge effect. In particular, the inventors found that the mutations H73Y, T158V and T158S led to some improvement in controllability.

[0147] It is notable in the Lin et al. 2020 paper that they first mutated H429 [H157] and H439 [H167] into alanine and glycine individually and found that these variants lost the capacity of pH- induced cleavage. In addition, they created a C-terminal cleavage-silenced variant N440A [N168A], based on their understanding that the hydrogen bond between N440 [N168] and H439 [H167] is critical for activity. Therefore, someone skilled in the art would not be directed to continue mutating these residues if their aim was to generate an intein which could cleave more controllably. Other authors have also cited the importance of His429 [H157] and His439 [H167], such as Du et al. (2009).

[0148] Therefore, regarding where to mutate the Mtu AI-CM intein or related inteins to improve controllability, the patent and journal literature would suggest to avoid the two ‘key’ histidine residues H429 [H157] and H439 [H167] and the C-terminal amino acid N440 [N 168].

[0149] Surprisingly, the present inventors have found that these three residues can be mutated (e.g., in the Mtu AI-CM intein) to certain amino acids and still retain cleavage activity following pH shift to -pH 6.0, and that mutation of these residues results in further reduced premature cleavage. This is especially the case when the first amino acid of the released peptide is histidine. Thus, in certain embodiments the first amino acid of the released C-terminal peptide is Histidine. In a further embodiment, the amino acid immediately C-terminal of the cleavage site is Histidine. In a yet further embodiment, the first amino acid of the target polypeptide is Histidine, such as wherein the target polynucleotide sequence starts with Histidine. In further certain embodiments, the mutant inteins described herein comprise and / or retain self-cleaving ability. Said retention of self-cleaving ability is possessed by the mutant inteins wherein they comprise mutations of what have previously been considered the ‘key’ amino acid residues.

[0150] Therefore, according to a further aspect of the invention, there is provided mutant Mtu RecA inteins where premature C-terminal cleavage is reduced. Reference herein to “mutant inteins” refers to inteins having one or a small number of (e.g., 2 or 3) amino acid substitutions compared to the wild-type or variant intein sequence which results in reduced premature C- terminal cleavage. In one embodiment, the mutant Mtu RecA intein comprises an amino acid substitution at a position selected from the group consisting of: His429, His439 and Asn440. Suitably a mutation has only one amino acids substitution relative to the reference wildtype or variant intein sequence. When referring to amino acid residue positions within Mtu RecA inteins of the invention, amino acid numbering is relative to the 440 amino acid wild-type Mtu RecA intein.

[0151] Premature cleavage may be measured by a ratio of uncontrolled to controlled cleavage (exemplified herein as conditions A and B, respectively). In some embodiments, the mutant inteins described herein demonstrate a ratio of uncontrolled to controlled cleavage (uncontrolled : controlled) of between about 1 : 2 to 1 : 12. In a further embodiment, the ratio is between about 1 : 2.5 to 1 : 10, such as between about 1 : 2.6 to 1 : 9.8 or between about 1 : 9 to 1 : 9.8. In one embodiment, the ratio is about 1 : 2.5. In a further embodiment, the ratio is about 1 : 2.6. The amount of cleavage may also be measured by a ratio of cleaved to uncleaved protein. In some embodiments, the mutant inteins described herein demonstrate a ratio of cleaved to uncleaved protein (cleaved : uncleaved) of between about 1 : 0 to 1 : 10. In a further embodiment, the ratio is between about 1 : 0 to 1 : 4, such as between about 1 : 0 to 1 : 0.5 or between about 1 : 0 to 1 : 0.2. In one embodiment, the ratio is about 1 : 0.1. In a further embodiment, the ratio is about 1 : 0.2. In a yet further embodiment, the ratio is about 1 : 0.6. The ratio representing the amount of cleaved to uncleaved protein may also be compared between conditions where cleavage is desired (i.e. controlled cleavage as in condition B herein) and where cleavage is not desired (i.e. uncontrolled cleavage as in condition A herein). In some embodiments, the mutant inteins described herein demonstrate a difference between the cleaved : uncleaved ratio in conditions A and B herein of at least about 4-fold. In further embodiments, said difference is at least about 6-fold. In one particular embodiment, said difference is at least about 25-fold, such as about 28-fold. In a further embodiment, said difference is at least about 55-fold, such as about 56-fold. In a yet further embodiment, said difference is about 65-fold, such as about 68-fold.

[0152] In a further embodiment, the Mtu RecA mutant intein comprises an amino acid substitution selected from the group consisting of: H439V and N440Q. Preferably, the Mtu RecA mutant intein comprises the amino acid substitution H439V.

[0153] In one embodiment, the mutant intein is a mutant of wild-type Mtu RecA, i.e., the amino acid substitution is in the wild-type Mtu RecA intein sequence (SEQ ID NO: 29).

[0154] In an alternative embodiment, the mutant intein is a mutant of a variant of wild-type Mtu RecA intein (SEQ ID NO: 29), i.e., the amino acid substitution is made to a variant Mtu RecA sequence. As previously described the most commonly used variants include the reduced size variants with the endonuclease domain removed: Al (SEQ ID NO: 30), AI-CM (SEQ ID NO: 31), AI-SM (SEQ ID NO: 32) (168 amino acids) and even smaller variants where an additional peptide loop is replaced: AAIhh (SEQ ID NO: 33), AAIhh-CM (SEQ ID NO: 34) and AAIhh-SM (SEQ ID NO: 35) (139 amino acids). Therefore, in one embodiment the mutant intein is a mutant of a variant intein selected from the group consisting of Al mini intein (SEQ ID NO: 30), AI-CM mini intein (SEQ ID NO: 31), AI-SM mini intein (SEQ ID NO: 32), AAlhh mini intein (SEQ ID NO: 33), AAIhh-CM mini intein (SEQ ID NO: 34) or AAIhh-SM mini intein (SEQ ID NO: 35). It will be appreciated that the mutant inteins would be valuable in the claimed methods for enriching target polypeptides. The improved, more controllable self-cleaving inteins allow for increased control of the cleavage step. Therefore, in one embodiment of the methods as described herein, the mutant Mtu RecA intein comprises an amino acid substitution at a position selected from the group consisting of: His429, His439 and Asn440. In a further embodiment, the amino acid substitution results in an amino acid other than Glycine and Alanine at a position selected from the group consisting of: His429, His439 and Asn440. In a further embodiment, the mutant Mtu RecA intein comprises an amino acid substitution selected from the group consisting of: H439V and N440Q. Preferably, the mutant Mtu RecA intein comprises the amino acid substitution H439V. Still preferably, the mutant Mtu RecA intein comprises the amino acid substitution H439W. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitution H439L. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitution H439G. Alternatively, the mutant Mtu RecA intein comprises the amino acid substitution H439M. Still alternatively, the mutant Mtu RecA intein comprises the amino acid substitution H439I. Preferably, the mutant Mtu RecA intein comprises the amino acid substitution N440Q. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitution N440S. Alternatively, the mutant Mtu RecA intein comprises the amino acid substitution N440T. Further alternatively, the mutant Mtu RecA intein comprises the amino acid substitution N440Y. Alternatively, the mutant Mtu RecA intein comprises the amino acid substitution H429F.

[0155] Any of said preferable substitutions at position His439 may be made alone or in combination with one or more substitution at positions His429 and Asn440, in particular the preferable substitutions at positions His429 and Asn440, and vice versa. Preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439V and N440Q. Still preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439W and N440Q. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitution H439L and N440Q. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439G and N440Q. Still preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439V and H429F. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439W and H429F. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439L and H429F. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439G and H429F. Also preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H429F and N440Q. Still preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439V, H429F and N440Q. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439W, H429F and N440Q. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439L, H429F and N440Q. Still further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439G, H429F and N440Q. Preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439V and N440S. Still preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439W and N440S. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitution H439L and N440S. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439G and N440S. Also preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H429F and N440S. Still preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439V, H429F and N440S. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439W, H429F and N440S. Further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439L, H429F and N440S. Still further preferably, the mutant Mtu RecA intein comprises the amino acid substitutions H439G, H429F and N440S.

[0156] Control of other self-cleaving inteins may be enhanced by mutation in a similar way, where the terminal two amino acids of the wild type intein are HN. Examples of such self-cleaving inteins include Mxe GyrA and HutMCM2.

[0157] As described hereinbefore, the mutant inteins will find particular utility in enriching target polypeptides by virtue of their ability to self-cleave under controlled conditions, thus acting as a cleavable tag as described herein. Therefore, in a further aspect of the invention there is provided a use of the inteins described herein, in particular the mutant self-cleaving inteins, in a method for enriching a target polypeptide in a sample as described herein. As will be readily appreciated, in certain embodiments said use and enrichment method are performed in vitro.

[0158] Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

[0159] The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). All publications, patents, patent applications, internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number / database sequence were specifically and individually indicated to be so incorporated by reference.

[0160] EXAMPLES

[0161] Materials

[0162] Buffer recipes

[0163] Lysis buffer and wash buffer 2, 20 mM tris buffer, pH 8.5, 1 L (Example 3 and 5):

[0164] Adjust volume to 1 L with reverse osmosis (RO) water

[0165] Adjust pH to 8.5 with 6 M hydrochloric acid (Fisher)

[0166] Wash buffer 1, 0.1 M urea buffer, pH 8.5, 1 UExample 3 and 5):

[0167] Adjust volume to 1 L with RO water

[0168] Adjust pH to 8.5 with 1 M sodium hydroxide (Fisher)

[0169] Solubilisation buffer

[0170] 8 M urea buffer, pH 8.5, 1 L (Example 3)'.

[0171] Adjust volume to 1 L with RO water

[0172] Adjust pH to 8.5 with 1 M sodium hydroxide (Fisher)

[0173] 4 M urea buffer, pH 8.5, 1 L (Example 5):

[0174] Adjust volume to 1 L with RO water

[0175] Adjust pH to 8.5 with 1 M sodium hydroxide (Fisher) Purification buffer 1 , 2 M urea buffer, pH 8.5, 1 L (Example 5

[0176] Adjust volume to 1 L with RO water

[0177] Adjust pH to 8.5 with 1 M sodium hydroxide (Fisher)

[0178] Refold and dilution buffer, 50 mM phosphate buffer, pH 7.5, 1 L (Example 3 and 5): Adjust volume to 1 L with RO water

[0179] Adjust with 1 M hydrochloric acid (Fisher) or 1 M sodium hydroxide as required

[0180] Cleavage buffer, 50 mM phosphate buffer, pH 4, 1 (.(Example 3Y

[0181] Adjust volume to 1 L with RO water Adjust with 6 M hydrochloric acid as required

[0182] Purification buffer 2, 50 mM phosphate buffer, pH 6.2, 1 L (Example 5):

[0183] Adjust volume to 1 L with RO water

[0184] Adjust with 6 M hydrochloric acid as required Towbin buffer:

[0185] Adjust volume to 1 L with RO water

[0186] PBST solution:

[0187] Adjust volume to 0.5 L with RO water

[0188] PBST-milk solution:

[0189] Adjust volume to 0.1 L with RO water

[0190] MOPS buffer: To 1 L RO water add:

[0191] Stir with a magnetic stirrer bar and stirrer plate (IKA) until solution clear 50 mM Tris buffer (as used in example 6):

[0192] Adjust volume to 0.1 L with RO water.

[0193] Adjust to pH 8.5 or pH 6.2 as required

[0194] 50 mM phosphate buffer (as used in example 8):

[0195] Adjust volume to 0.1 L with RO water.

[0196] Adjust to pH 8.5 or pH 6.2 as required Media

[0197] 2TY:

[0198] Plates are poured (~30 mL) and dried extensively in a laminar flow hood before use

[0199] Vegetable peptone broth: Post autoclave conditions:

[0200] VLB broth:

[0201] Basal medium pH adjusted to 7.2 with 30% ammonia

[0202] Post autoclave additions:

[0203] Citric acid / lron chloride II solution:

[0204] Calcium chloride solution: Trace elements solution:

[0205] General Methods Protein precipitation

[0206] Proteins were precipitated from a complex mixture, e.g., clarified lysate or cell-free protein expression reaction mixture, as follows. 4 volume equivalents of cold acetone (Fisher) were added to the mixture (e.g., 800 pL acetone added to 200 pL solution). The sample was thoroughly mixed by a vortex (MS2 minishaker (I KA)) for a few seconds and then the sample was incubated on ice for 15 minutes. The sample was spun by centrifugation in a microfuge for 10 minutes at 4 °C. After centrifugation, the solvent layer was removed, and any residual solvent was removed via speed vacuum (Savant SpeedVac SPD1030, Thermofisher) for 3 minutes. 9 volume equivalents of water (e.g., 900 pL) was added to the precipitate solubilized in 8 M urea buffer (e.g., 100 pL) for one hour.

[0207] Protein concentration quantification

[0208] Protein concentration was quantified using a NanoPhotometer NP80 (Implen) on the protein UV setting to record protein concentrations at 280 nm absorbance. The instrument was calibrated against bovine serum albumin (BSA). Additionally, samples were quantified using a Qubit 3.0 fluorometer (Invitrogen) with Qubit Protein Assay Kit (ThermoFisher). This instrument was calibrated using the standards including within the assay kit.

[0209] Optical density

[0210] The optical density of samples was measured by use of a NanoPhotometer NP80 (Implen) on QD600 setting.

[0211] Bacterial strains and growth conditions

[0212] Escherichia coli DH10B (GibcoBRL) and Escherichia coli BL21 (DE3) (LMBP 01455, BCCM) were grown in 2TY liquid medium or 2TY semi-solid agar media, as described by Sambrook et al. (1989), supplemented with kanamycin (50 pg / ml) or ampicillin (100 pg / ml). E. coli transformants were typically selected for with either 50 pg / ml kanamycin or 100 mg / mL ampicillin, depending on resistance marker.

[0213] DNA manipulation and sequencing

[0214] DNA manipulations, PCR and electroporation procedures were carried out as described in Sambrook et al. (1989). PCR amplifications were carried out using Q5 2x Master Mix (NEB) within a SimpliAmp Thermal Cycler (Applied Biosystems). SLiCE mediated DNA assembly is based on the method described in Zhang, Werling and Edelmann (2012). Golden Gate Assembly is based on the method of Engler et al. (2009). Automated DNA sequencing and DNA synthesis was carried out at a contract service provider, such as Azenta Genewiz or Eurofins. pET28a(+) (SEQ ID NO: 20) is a DNA plasmid supplied by Novagen. SDS-PAGE and western blot analysis of proteins

[0215] Sample preparation varied depending on the sample to be analysed by SDS-PAGE and

[0216] Western blot:

[0217] The protein concentration of all samples was quantified using the NanoPhotometer NP80 or Qubit 3.0 as previously described. To prepare samples for loading onto a gel, the following was aliquoted into a clean 0.5 mL Eppendorf:

[0218] The samples were boiled for 5 minutes at 95 °C on a myblock mini digital dry bath heater block (Benchmark Scientific). The samples were then spun in a microfuge for 10 seconds, 14000 rpm, room temperature.

[0219] To run the gel, the well comb was removed from a pre-cast gel (FastGene PAGE Gel 12%, 10 x 8 cm, 12 wells (NIPPON Genetics), or mPAGE 12% Bis-Tris Precast gel 10 x 8 cm, 15 wells (Merck Millipore)) and the gel was secured on a running module (Mini-Protean companion, BioRad) and inserted into a gel tank (Mini PROTEAN tetra system, BioRad).

[0220] MOPS buffer was added to the running module and tank. 5 pL pre-stained protein ladder midrange, molecular weight 10-180 kDa (Abeam), was aliquoted to the first lane of the gel, and 20 pL of the prepared samples was added to the remaining wells using gel saver II tip 1-200 pL (Starlab). The tank was topped up with MOPS buffer. The lid was secured onto the gel tank and the electrodes connected to PowerPac Basic (BioRad). Migration was performed at 120 V for 70-90 minutes.

[0221] To stain the migrated gel, the gel was transferred into a clear plastic container (10.5 x 13 cm). 20 mL PageBlue protein staining solution (ThermoFisher) was added for 1 hour with gentle agitation on a platform shaker (Stuart Scientific). The staining solution was removed, and RO water (20 mL) was added to the gel. The container was covered with plastic wrap and incubated at room temperature for 16 hours with gentle agitation.

[0222] To transfer the gel onto a nitrocellulose membrane for western blotting, the gel was removed from the cast and transferred onto a nitrocellulose blotting membrane, Amersham Protran 0.45 pL (Cytiva) using a trans-blot SD semi-dry transfer unit (BioRad). All components were washed with towbin buffer and transfer was completed with the following assembly: Western blotting filter paper x2, 8 x 10.5 cm (ThermoFisher), nitrocellulose membrane, SDS-PAGE gel, western blotting filter paper x2. Positive pressure was applied to remove excess liquid and air bubbles. The trans-blot SD semi-dry transfer unit was connected to the power, PowerPac Basic, and run at 11 for 35 minutes. The set up was then disassembled when transfer was complete.

[0223] For western blot generation, the nitrocellulose membrane was placed in PBST-milk solution for 1 hour with gentle agitation. After 1 hour the PBST-milk solution was removed. In a 50 mL centrifuge tube, 3 pL primary antibody - anti-6* His, Histag mouse McAb (Proteintech) - was added to 30 mL PBST-milk solution. The membrane was incubated for 1 hour in this solution with gentle agitation. After 1 hour the solution was removed and the membrane was washed three times with PBST-milk solution. In a 50 mL centrifuge tube, 6 pL conjugated secondary antibody - Goat Anti-Mouse IgG H&L, HRP (Abeam) - was added to 30 mL PBST-milk solution. The membrane was incubated in this solution for 1 hour with gentle agitation. After 1 hour the solution was removed and the membrane was washed three times with PBS solution. The following solution: 1 mL Opti-4CM diluent (BioRad), 0.2 mL Opti-4CM substrate (BioRad), 9 mL RO water, was added to the membrane. Protein bands on the membrane became visible between 1 and 20 minutes after substrate solution was added.

[0224] High-performance liquid chromatography (HPLC) Analysis of Recombinant Peptide and Tagged Protein

[0225] HPLC grade acetonitrile and water, and LC-MS grade water were purchased from Thermo Fisher Scientific. Analytical grade trifluoracetic acid and formic acid were purchased from Sigma. Analytical column and guard (BioResolve polyphenyl, 450 A, 2.7 pm) was purchased from Waters.

[0226] Sample preparation varied depending on the sample to be analysed by HPLC:

[0227] Analysis of all prepared samples was performed on an Agilent HP1100 HPLC system, consisting of a quaternary pump, autosampler, column oven and diode array detector, as described below:

[0228] • Injection volume: 5 pL

[0229] • Column 150 x 4.6 mm column, base-deactivated reversed

[0230] • Stationary phase: phase superficially-porous-silica, 450 A, 2.7 pm particle size (BioResolve polyphenyl).

[0231] • Column guard 3.9 x 5 mm cartridge, base-deactivated reversed

[0232] • Stationary phase: phase superficially-porous-silica, 450 A, 2.7 pm particle size (BioResolve polyphenyl).

[0233] • Mobile phase A: 5% acetonitrile:95% water, containing 0.1% trifluoracetic acid

[0234] • Mobile phase B: 95% acetonitrile:5% water, containing 0.1% trifluoracetic acid

[0235] • Mobile phase gradient: T = 0 min, 5% B; T = 1 min, 30% B; T = 9 min, 55% B;

[0236] • T = 9.1 min, 95% B; T = 12.0 min, 95% B; T = 12.1 min, 5% B, T = 15 min, 5% B.

[0237] • Flow rate: 1 .2 mL / min.

[0238] • Temperature: 60 °C

[0239] • Detection: UV at 214, 230, 280, 200 nm (DAD acquisition over 190-600 nm);

[0240] Liquid chromatography-mass spectrometry (LCMS) Analysis of Recombinant Peptide and Tagged protein

[0241] Sample preparation varied depending on the sample to be analysed by LCMS:

[0242] Analysis of all prepared samples was performed on an Agilent 1200 HPLC system, consisting of a quaternary pump, autosampler, column oven and diode array detector with a Thermo Finnigan LTQ mass spectrometer, as described below:

[0243] • Injection volume: 5 pL

[0244] • Column 150 x 4.6 mm column, base-deactivated reversed stationary phase: phase superficially-porous-silica, 450 A, 2.7 pm particle size (BioResolve polyphenyl).

[0245] • Column guard 3.9 x 5 mm cartridge, base-deactivated reversed stationary phase: phase superficially-porous-silica, 450 A, 2.7 pm particle size (BioResolve polyphenyl).

[0246] • Mobile phase A: 5% acetonitrile:95% water, containing 0.1% formic acid.

[0247] • Mobile phase B: 95% acetonitrile:5% water, containing 0.1% formic acid

[0248] • Mobile phase gradient: T = 0 min, 5% B; T = 0.5 min, 5% B; T = 1 min, 20% B; T = 9 min, 60% B; T = 10 min, 95% B; T = 13 min, 95% B; T = 13.5 min, 5% B, T = 15 min,

[0249] 5% B.

[0250] • Flow rate: 1 .2 mL / min, 0.24 mL / min directed to MS using a splitter

[0251] • Temperature: 60 °C

[0252] • Detection: DAD acquisition over 210-400 nm

[0253] • MS run time: 16 min

[0254] • Source mode: Positive-ion mode electrospray ionisation

[0255] • Capillary temperature: 270 °C

[0256] • Sheath gas flow: 5.00

[0257] • Source Voltage: 5.50 kV

[0258] • Source current: 100 uA • Tube lens: 105.00 V

[0259] • Capillary Voltage: 7.00 V

[0260] • Normalised collision energy: 35 V

[0261] • Mass analyser: ion trap • Mass scan range: 600-2000 m / z

[0262] • Divert valve: 0.1-3.5 mins, 15.5-16 mins

[0263] Assessment of purity

[0264] Isolated peptides were analysed using HPLC method 1 and LCMS method 1 described above. Purity was assessed by at multiple wavelengths (e.g., 214, 230, 280, 200 nm) and confirmed by inspection of Mass Spectrometry analysis.

[0265] Examples

[0266] Example 1 : Generation of peptide expression vectors with mutant inteins Composition of the generated plasmids

[0267] The components of the generated plasmids are set out in Table 1 below and Figure 5.

[0268] Table 1: List of expression plasmids generated

[0269] Generation ofpJP178 (an expression vector for AI-CM mini intein) pJP178 is a plasmid of sequence (SEQ ID NO: 1) containing a T7 promoter, lac operator binding site, RBS, T7 terminator and a CDS encoding a transcript for a 6xHIS-tag, thrombin site, Ketosteroid isomerase insolubility tag (KSI, genbank M22749.1 , SEQ ID NO: 2), AI-CM mini intein ((Zhao et a / ., 2017), SEQ ID NO: 3) and GLP-1 (7-37, SEQ ID NO: 4) peptide sequence. The complete protein sequence encoded from this transcript is SEQ ID NO: 5. Generation ofp068-021 (an expression vector for intein mutant H 439V) pSIM238 is a plasmid of sequence (SEQ ID NO: 6) containing a T7 promoter, RBS, and a CDS encoding a transcript for a 6xHIS-tag, thrombin site, Ketosteroid isomerase insolubility tag (KSI, genbank M22749.1 , SEQ ID NO: 2), AI-CM mini intein ((Zhao et al., 2017), SEQ ID NO: 3) and 6x His-tagged GLP-1 (7-37 with Pro+Val linker, SEQ ID NO: 7) peptide sequence. The complete protein sequence encoded from this transcript is SEQ ID NO: 8.

[0270] A dsDNA fragment containing a lactose promoter, lactose regulatory sequence, RBS sequence, lacZ CDS and transcriptional terminator (SEQ ID NO: 9) was amplified by PCR using oligos OMSIM420 (SEQ ID NO: 10) and OMSIM421 (SEQ ID NO: 11). Plasmid pSIM238 (SEQ ID NO: 6) was PCR amplified with oligos OMSIM423 (SEQ ID NO: 12) and OMSIM422 (SEQ ID NO: 13). The two fragments were ligated using SLICE based assembly and transformed in to DH10B cells. This formed plasmid pSIM239 (SEQ ID NO: 14).

[0271] Lyophilised ssDNA oligos OMSIM434 (SEQ ID NO: 15) and OMSIM435 (SEQ ID NO: 16) were rehydrated in dH2O to 100 pM concentration. 5’ phosphorylation occurred in a 50 pl reaction consisting of 5 pl 10x T4 ligase buffer (NEB), 1 pl T4 PNK (NEB), 42 pl dH2O and 2 pM of each oligonucleotide. The reaction was incubated at 37°C for 30 mins, with enzyme heat inactivation at 65°C for 20 mins. The phosphorylated ssDNA oligos were annealed together with the addition of 2.5 pl of 1M NaCI to the phosphorylation reaction, incubation in a thermocycler and incubation at 95 °C for 5 mins. The reaction was cooled from 95 °C to 22 °C in 1 °C decrements with 1 min at each intermediate temperature over a total of 73 mins. This reaction formed a dsDNA fragment with ssDNA 5’ ends of sequences 5’-tggc-3’ and 5’-ctag- 3’. The annealed and phosphorylated oligonucleotide mix was then diluted ten-fold in dH2O.

[0272] Plasmid pSIM245 (SEQ ID NO: 17) was formed in a ‘Golden-Gate Assembly’ one-pot digestion and ligation reaction. A 10 pl reaction consisting of 1 pl of the ten-fold diluted annealed-oligonucleotides (SEQ ID NO: 15 and SEQ ID NO: 16), 1 pl plasmid pSIM239 (SEQ ID NO: 14), 1 pl 10x T4 ligase buffer (NEB), 0.5 pl T4 DNA ligase (NEB), 1 pl BsmBI HF (NEB) and 5.5 pl dH2O. The reaction was placed in a thermocycler programme with 45 cycles of 42 °C for DNA digestion and 16 °C for ligation, followed by 10 mins at 50 °C, and heat inactivation at 80 °C for 5 mins. The inserted oligonucleotide duplex adjusted the amino acid sequence of the AI-CM mini intein to contain a mutation of H439V, as shown in SEQ ID NO: 18. The complete protein coding sequence of the transcript in pSIM245 is SEQ ID NO: 19. The assembled DNA construct was transformed in to DH10B cells. Plasmids pET28a(+) (SEQ ID NO: 20) and pSIM245 (SEQ ID NO: 19) were digested with Xbal and Hindlll-HF (NEB). The 1.1kbp fragment from pSIM245 was ligated in to pET28a(+) background, forming plasmid p068-009 (SEQ ID NO: 21). Plasmid p068-009 was PCR amplified using oligos oJLOOl (SEQ ID NO: 22) and oJL002 (SEQ ID NO: 23) and the PCR amplified product re-circularised and mutagenized using SLICE reaction assembly mix. This assembly removed a 24bp DNA sequence from the plasmid, thus removing an amino acid sequence of PVHHHHHH (SEQ ID NO: 89) from the 3’ sequence of 6x His-tagged GLP-1 (7- 37, SEQ ID NO: 7), thereby forming untagged GLP-1 (7-37) amino acid sequence (SEQ ID NO: 4). This mutagenesis formed the final plasmid p068-021 (SEQ ID NO: 24). The complete protein coding sequence of the transcript in p068-021 is SEQ ID NO: 25.

[0273] Generation of p068-023 (an expression vector for intein mutant N440Q) p068-023 is a plasmid derived from p068-021 , generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 26. The protein sequence expressed from p068-023 is SEQ ID NO: 27. The sequence of Al- CM (N440Q) is SEQ ID NO: 28.

[0274] Generation of p086-001 (an expression vector for AI-CM mini intein with GFP11 and GGGSGGGS linker) p086-001 is a plasmid of sequence (SEQ ID NO: 36) containing a T7 promoter, lac operator binding site, RBS, T7 terminator and a CDS encoding a transcript for a 6xHIS-tag, AI-CM mini intein ((Zhao et al., 2017), SEQ ID NO: 3), and a peptide sequence of an eight residue linker sequence of GGGSGGGS (SEQ ID NO: 88) and a sequence of split Green Fluorescent Protein S65T fragment 11 (GFP11 , SEQ ID NO: 37). The complete protein sequence encoded from this transcript is SEQ ID NO: 38. The released peptide cleaved via AI-CM mini Intein is SEQ ID NO: 37.

[0275] Generation of p086-010 (an expression vector for AI-CM mini Intein mutant H439V with GFP11 and GGGSGGGS linker) p086-010 is a plasmid derived from p086-001 , generated using standard molecular biology methods including PCR and ‘Site Directed Mutagenesis’ (SDM). It has a DNA sequence of SEQ ID NO: 39. The protein sequence expressed from p086-010 is SEQ ID NO: 40. The sequence of AI-CM (H439V) is SEQ ID NO: 18. The complete protein sequence encoded from this transcript is SEQ ID NO: 38. The released peptide cleaved via AI-CM (H439V) mini Intein is SEQ ID NO: 37. Generation of p086-011 (an expression vector for AI-CM mini Intein mutant N440Q with GFP11 and GGGSGGGS linker) p086-011 is a plasmid derived from p086-001 , generated using standard molecular biology methods including PCR and ‘Site Directed Mutagenesis’ (SDM). It has a DNA sequence of SEQ ID NO: 41. The protein sequence expressed from p086-010 is SEQ ID NO: 42. The sequence of AI-CM (N440Q) is SEQ ID NO: 28. The released peptide cleaved via AI-CM (N440Q) mini Intein is SEQ ID NO: 37.

[0276] Generation of p086-036 (a cloning vector for creating further AI-CM mini intein mutant variants with GFP11 and HAGSGGGS linker) p086-036 is a cloning plasmid derived from p086-001 , generated using standard molecular biology methods including PCR and SLiCE assembly. The plasmid contains a lacZ reporter cassette flanked by PaqCI restriction sites to allow for Golden Gate cloning and insertion of nucleotide sequences for scarless and in-frame mutation of AI-CM mini intein between G422 and N440Q. It has a DNA sequence of SEQ ID NO: 43.

[0277] Generation of p086-037 (a cloning vector for creating further AI-CM mini intein mutant variants with GFP11 and HAGSGGGS linker) p086-037 is a cloning plasmid derived from p086-001 , generated using standard molecular biology methods including PCR and SLiCE assembly. The plasmid contains a lacZ reporter cassette flanked by PaqCI restriction sites to allow for Golden Gate cloning and insertion of nucleotide sequences for scarless and in-frame mutation of AI-CM mini intein between V438 and N440Q. It has a DNA sequence of SEQ ID NO: 44.

[0278] Generation of p086-039 (an expression vector for intein mutant H429F) p068-039 is a plasmid derived from p068-036, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 45. The protein sequence expressed from p086-039 is SEQ ID NO: 46. The sequence of AI- CM (H429F) is SEQ ID NO: 47. The released peptide cleaved via AI-CM (H429F) mini Intein is SEQ ID NO: 87.

[0279] Generation of p086-040 (an expression vector for intein mutant H429V) p068-040 is a plasmid derived from p068-036, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 48. The protein sequence expressed from p086-040 is SEQ ID NO: 49. The sequence of Al- CM (H429V) is SEQ ID NO: 50. The released peptide cleaved via AI-CM (H429V) mini Intein is SEQ ID NO: 87.

[0280] Generation of p086-041 (an expression vector for intein mutant H439A N440Q) p068-041 is a plasmid derived from p068-036, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 51. The protein sequence expressed from p086-041 is SEQ ID NO: 52. The sequence of AI- CM (H439A N440Q) is SEQ ID NO: 53. The released peptide cleaved via AI-CM (N440Q) mini Intein is SEQ ID NO: 87.

[0281] Generation of p086-042 (an expression vector for intein mutant G435D) p068-042 is a plasmid derived from p068-036, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 54. The protein sequence expressed from p086-042 is SEQ ID NO: 55. The sequence of AI- CM (G435D) is SEQ ID NO: 56. The released peptide cleaved via AI-CM (G435D) mini Intein is SEQ ID NO: 87.

[0282] Generation ofp086-043 (an expression vector for intein mutant H439A) p068-043 is a plasmid derived from p068-036, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 57. The protein sequence expressed from p086-043 is SEQ ID NO: 58. The sequence of AI- CM (H439A) is SEQ ID NO: 59. The released peptide cleaved via AI-CM (H439A) mini Intein is SEQ ID NO: 87.

[0283] Generation of p086-044 (an expression vector for intein mutant H439F) p068-044 is a plasmid derived from p068-036, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 60. The protein sequence expressed from p086-044 is SEQ ID NO: 61. The sequence of AI- CM (H439F) is SEQ ID NO: 62. The released peptide cleaved via AI-CM (H439F) mini Intein is SEQ ID NO: 87.

[0284] Generation of p086-045 (an expression vector for intein mutant H439I) p068-045 is a plasmid derived from p068-037, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 63. The protein sequence expressed from p086-045 is SEQ ID NO: 64. The sequence of Al- CM (H439I) is SEQ ID NO: 65. The released peptide cleaved via AI-CM (H439I) mini Intein is SEQ ID NO: 87.

[0285] Generation of p086-046 (an expression vector for intein mutant H439L) p068-046 is a plasmid derived from p068-037, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 66. The protein sequence expressed from p086-046 is SEQ ID NO: 67. The sequence of AI- CM (H439L) is SEQ ID NO: 68. The released peptide cleaved via AI-CM (H439L) mini Intein is SEQ ID NO: 87.

[0286] Generation of p086-047 (an expression vector for intein mutant N440S) p068-047 is a plasmid derived from p068-037, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 69. The protein sequence expressed from p086-047 is SEQ ID NO: 70. The sequence of AI- CM (N440S) is SEQ ID NO: 71. The released peptide cleaved via AI-CM (N440S) mini Intein is SEQ ID NO: 87.

[0287] Generation of p086-048 (an expression vector for intein mutant N440T) p068-048 is a plasmid derived from p068-037, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 72. The protein sequence expressed from p086-048 is SEQ ID NO: 73. The sequence of AI- CM (N440T) is SEQ ID NO: 74. The released peptide cleaved via AI-CM (N440T) mini Intein is SEQ ID NO: 87.

[0288] Generation ofp086-049 (an expression vector for intein mutant H439G) p068-049 is a plasmid derived from p068-037, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 75. The protein sequence expressed from p086-049 is SEQ ID NO: 76. The sequence of AI- CM (H439G) is SEQ ID NO: 77. The released peptide cleaved via AI-CM (H439G) mini Intein is SEQ ID NO: 87.

[0289] Generation ofp086-050 (an expression vector for intein mutant H439W) p068-050 is a plasmid derived from p068-037, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 78. The protein sequence expressed from p086-050 is SEQ ID NO: 79. The sequence of Al- CM (H439W) is SEQ ID NO: 80. The released peptide cleaved via AI-CM (H439W) mini Intein is SEQ ID NO: 87.

[0290] Generation ofp086-051 (an expression vector for intein mutant H439M) p068-051 is a plasmid derived from p068-037, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 81. The protein sequence expressed from p086-051 is SEQ ID NO: 82. The sequence of AI- CM (H439M) is SEQ ID NO: 83. The released peptide cleaved via AI-CM (H439M) mini Intein is SEQ ID NO: 87.

[0291] Generation ofp086-052 (an expression vector for intein mutant N440Y) p068-052 is a plasmid derived from p068-037, generated using standard molecular biology methods including PCR and ‘Golden Gate Assembly’. It has a DNA sequence of SEQ ID NO: 84. The protein sequence expressed from p086-052 is SEQ ID NO: 85. The sequence of AI- CM (N440Y) is SEQ ID NO: 86. The released peptide cleaved via AI-CM (N440Y) mini Intein is SEQ ID NO: 87.

[0292] Example 2: Comparison of cleavage of mutant inteins

[0293] Plasmids pJP178, p068-009, p068-021 and p068-023 were introduced into E. COH B H (DE3) cells by heat shock transformation, plated onto 2TY + kanamycin agar and incubated overnight at 37 °C. A single kanamycin resistant colony was inoculated in to 5 ml_ of 2TY liquid media + kanamycin and grown overnight at 37 °C with shaking at 250 rpm, 2.5 cm orbital throw.

[0294] The following morning, the QD600 of the overnight bacterial growth was measured, and the culture diluted back to an QD600 of 0.05 in 50 ml of fresh 2TY + Kanamycin media, in 250ml non-baffled glass flasks, bunged with a foam bung. The flasks were shaken at 250 rpm, 2.5 cm orbital throw at 37 °C until the bacterial cultures reached an QD600 of 0.1. 1 mM IPTG was added to remove repression of the lacl / lac operator and allow expression of the intein- peptide construct.

[0295] Cultures were then incubated at 30 °C for a further ~5 hrs with 250 rpm shaking and 2.5 cm orbital throw. Cultures were then spun at 4500 rpm, 4 °C, 10 minutes by centrifugation. The growth media supernatants were discarded, and the bacterial pellets were resolubilized in 20 mM tris buffer, pH 8.5, stirring for 1 hour at room temperature. The solubilized pellet was passed through the homogenizer (A vestin C3, Biopharma group) three times at 1000 bar. The optical density of the homogenate decreased more than 80%. An aliquot (1 mL) of each culture was transferred to a 2 ml_ microfuge tube and spun by centrifugation, 4 °C, 13200 rpm, 15 minutes. The supernatant was aliquoted into a fresh 2 mL microfuge tube, and the pellet was resolubilised in 8 M urea buffer (1 mL) for 16 hours. The supernatant and solubilised pellet protein samples were analysed by SDS-PAGE gels and western blot. As shown in Figure 7, AI-CM inteins with H439V or N440Q mutations led to much less (early or premature) cleaved product when compared to the parental AI-CM intein.

[0296] The homogenates from a subset of cultures was spun at 4500 rpm, 4 °C, 45 minutes by centrifugation. The pellets were resolubilized for 16 hours in urea buffer (5 mL). One volume equivalent (5 mL) of phosphate buffer, pH 7.5 (general methods), was slowly added to each solubilized pellet and stored at 4 °C. After 2 hours 10 mL of phosphate buffer, pH 7.5, was slowly added to the solubilized pellets and stored at 4 °C. After 2 hours 20 mL of phosphate buffer, pH 7.5, was slowly added to the solubilized pellets and stored for 16 hours at 4 °C.

[0297] One volume equivalent of acetate buffer pH 4.8 (general methods) was added to the retentate material. The solution was stored at 37 °C in static conditions for 24 hours. The solution was shaken, and an aliquot (1 mL) was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. The solution was spun at 3500 rpm, 4 °C, 30 minutes by centrifugation. The supernatant was decanted. The precipitate was re-solubilised in 8 M urea buffer for 24 hours with stirring using a magnetic stirrer plate and magnetic stirrer bar. An aliquot of the urea solubilized sample was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. This later analysis confirmed that all tested inteins led to cleaved peptide (AI-CM intein with H439V or N440Q mutations and the parental AI-CM intein).

[0298] Example 3: Generation of GLP1 peptide using mutant intein and TFF

[0299] Figure 3 provides a representative schematic of Steps A-K described in this Example.

[0300] A: Fermentation and Recovery of BL21 (DE3) p068-021

[0301] 50 mL VLB containing 25 pg / mL kanamycin in a 250-mL baffled Erlenmeyer flask was inoculated with E. coli BL21(DE3) containing plasmid p068-021 and incubated at 37 °C with shaking at 250 rpm (2.5 cm throw) for 16 hours, to be used as the first seed stage culture.

[0302] 400 mL peptone vegetable broth containing 25 pg / mL kanamycin in a 2-L Erlenmeyer flask was inoculated with 8 mL of the first seed stage culture broth and incubated at 37 °C with shaking at 250 rpm (2.5 cm throw) for 8 hours, to be used as the second seed stage culture. 2 L of Basal medium, supplemented with Feed medium in a 7-L Stirred Tank Reactor was inoculated with the second seed stage culture to a final OD595 of 0.2. The culture was grown with the following controls in place: culture temperature was kept at 30 °C, filtered air was sparged through at 2 L / min, 2 Rushton impellors were used at 400-1000 rpm (stepped increase), culture pH was kept at 7.2 (+ / - 0.1) using 30% ammonium hydroxide and 2 M phosphoric acid solutions. Foaming was suppressed using AF204 antifoam and a foam detector probe. Addition of Feed media was as follows. At the start of the fermentation, Feed medium was added at a linearly increasing rate, starting at 0.32 mL / min and increasing to 1.72 mL / min over the first 24 hours. Then, upon a dissolved oxygen spike above a 10% threshold, Feed medium was automatically added as a 10 mL bolus. After 22 hours, IPTG was added to a final concentration of 1 mM, followed by further cultivation at pH 7.6, 30 °C for 18 hours. The culture was then harvested.

[0303] 1 L of fermentation whole broth was spun at 3500 rpm, 4 °C, 45 minutes by centrifugation (refrigerated floor centrifuge, Sorvall). The supernatant was discarded, and the mass of the wet cell weight was recorded.

[0304] B: Homogenisation of inclusion bodies from BL21(DE3) p068-021

[0305] The pellet collected in Step A was re-dissolved in 0.5 L tris buffer and stirred using a magnetic stirrer bar and plate (IKA) for 1 hour at room temperature.

[0306] The optical density of the tris solubilised pellet was measured as described in general methods. The solubilized pellet was passed through the homogenizer (Avestin C3, Biopharma group) three times at 1000 bar. The optical density of the homogenate had decreased more than 75%. An aliquot of the homogenate was taken for SDS-PAGE sample preparation and analysis.

[0307] The homogenised material was spun at 3500 rpm, 4 °C, 50 minutes by centrifugation. The supernatant was decanted.

[0308] C: Wash 1 of inclusion bodies from BL21(DE3) p068-021

[0309] The pellet was re-suspended in 0.3 L wash buffer 1 , 0.1 M urea (general methods) and spun at 3500 rpm, 4 °C, 50 minutes. The supernatant was decanted, an aliquot was taken for SDS- PAGE sample preparation and analysis.

[0310] D: Wash 2 of inclusion bodies from BL21(DE3) p068-021 The pellet was re-suspended in 0.3 L wash buffer 2, 20 mM tris buffer and spun at 3500 rpm, 4 °C, 50 minutes. The supernatant was decanted, an aliquot was taken for SDS-PAGE sample preparation and analysis.

[0311] E: Resolubilisation of inclusion bodies from BL21(DE3) p068-021

[0312] The pellet was solubilised in 8 M urea buffer (in a volume to yield 20% cell solids) and stirred using a magnetic stirrer bar and plate for 16 hours at room temperature. An aliquot (1 ml_) of the solubilized homogenate pellet was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods.

[0313] F: Purification 1 - Removal of small contaminants by discontinuous diafiltration using Tangential Flow Filtration (TFF)

[0314] The Minimate™ EVO TFF System (Pall) was set up with a Centramate™ LV cassette holder (Pall) and a T-series Centramate™ Cassette, Omega PES membrane, 30 kDa molecular weight cut-off (MWCO), 0.02 m2effective filtration area (Pall). The system was stored in 0.1 M sodium hydroxide and washed with water until the pH of the retentate and filtrate lines reached pH 7.0. The water was then removed from the TFF system and 8 M urea buffer (50 mL) was added to the TFF reservoir. The urea was circulated around the TFF system for 5 minutes then removed. The reservoir was placed on a stirrer plate (I KA) with a magnetic stirrer bar placed in the reservoir.

[0315] The urea solubilized pellet was spun at 3500 rpm, 4 °C, 20 minutes by centrifugation. The supernatant was transferred to the TFF reservoir, and the volume was doubled with 8 M urea buffer. The pellet was discarded. The sample was circulated round the TFF system with the peristaltic pump set to 50 rpm and 1.5 bar external pressure applied using a small clamp to force small contaminants through the membrane. Once the TFF retentate was concentrated back to its starting volume, one volume equivalent of 8 M urea buffer was added to the reservoir. The sample was circulated round the TFF system with the peristaltic pump set to 50 rpm and 1.5 bar external pressure. The retentate was concentrated back to its starting volume and two diafiltration volumes of filtrate were collected (material that had permeated through the 30 kDa MWCO membrane). An aliquot (1 mL) of the retentate and filtrate were taken for HPLC and SDS-PAGE sample preparation and analysis as described in general methods. The urea solubilised inclusion body remains in the TFF reservoir.

[0316] G: Buffer exchange 1 - Continuous diafiltration of urea to phosphate buffer using Tangential Flow Filtration (TFF) Prior to continuous diafiltration the refold buffer (general methods) was stored for 16 hours at 4 °C. A circular cold pack was secured around the TFF reservoir, and the refold buffer was stored on ice.

[0317] The lid was secured on the TFF reservoir. One end of tubing was placed in the phosphate buffer and the other end secured to the leur lock connection on the reservoir lid. The reservoir was put under vacuum using a 20 ml_ syringe. The sample was circulated round the TFF system with the peristaltic pump set to 10 rpm and no external pressure applied. The refold buffer was added to the sample feed reservoir at the same flowrate as the filtrate was generated. Three diafiltration volumes (three volume equivalents of the starting volume in the TFF reservoir) were performed yielding 95% buffer exchange.

[0318] The retentate (solution in reservoir) was collected through the retentate line in a 1 L duran and stored at 4 °C for 16 hours. An aliquot (1 mL) of the retentate was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. An aliquot (1 mL) of the filtrate was taken for HPLC (See Figure 9) and SDS-PAGE sample preparation and analysis as described in general methods. The filtrate was discarded.

[0319] Optimal conditions for buffer exchange to minimise protein precipitation can vary dependent on the specific peptide. Buffer exchange may be improved by varying the buffer conditions within the following ranges pH 7.0 - 8.0, L-arginine hydrochloride concentration 0 - 0.4 M, sodium chloride concentration 0 - 0.5 M.

[0320] H: Cleavage of the intein

[0321] One volume equivalent of cleavage buffer (general methods) was added to the retentate. The pH was adjusted to pH 6.2 with 1 M HCI. The solution was stored at 37 °C, 250 rpm for 24 hours. Using a wide bore pipette tip, an aliquot of the solution was taken for HPLC (See Figure 9), LCMS and SDS-PAGE sample preparation and analysis as described in general methods. The solution was spun at 3500 rpm, 4 °C, 30 minutes by centrifugation. The supernatant contains most of the recombinant peptide.

[0322] Optimal conditions for cleavage can vary dependent on the specific peptide. Cleavage may be improved by operating within the following ranges pH 6.0 - 6.8 (direct pH adjustment with 1 HCI), imidazole concentration 0 - 50 mM, sodium chloride concentration 0 - 0.5 M, temperature 28 - 37 °C, 14 - 24 hours. Residual untagged peptide may precipitate into the pellet, this material can be resolubilised in 8 M urea and the process repeated from step E to recover additional peptide.

[0323] I: Buffer exchange 2 - Continuous diafiltration to remove buffer salts using TFF

[0324] Continuous diafiltration was employed to remove at least 95% of buffer components and salts from the sample without increasing the sample volume.

[0325] The Minimate™ EVO TFF System (Pall) was set up with a Centramate™ LV cassette holder (Pall) and a T-series Centramate™ Cassette, Omega PES membrane, 1 kDa molecularweight cut-off (MWCO), 0.02 m2effective filtration area (Pall). The system was stored in 0.1 M sodium hydroxide and washed with water until the pH of the retentate and filtrate lines reached pH 7.0. The reservoir was placed on a stirrer plate (I KA) with a magnetic stirrer bar placed in the reservoir.

[0326] The cleaved material was transferred into the TFF reservoir and circulated around. The lid was secured on the reservoir. One end of tubing was placed in RO water (diafiltration buffer 2) and the other end secured to the leur lock connection on the reservoir lid. The reservoir was put under vacuum using a 20 mL syringe. The sample was circulated round the TFF system with the peristaltic pump set to 120 rpm and 1 .5 bar external pressure applied with a clamp. The transmembrane pressure fluctuated between 0 - 0.3 bar. Diafiltration buffer 2 was added to the sample feed reservoir at the same flowrate as the filtrate was generated (material that had permeated through the 1 kDa MWCO membrane). Five diafiltration volumes were performed, yielding greater than 95% buffer exchange. The retentate (solution in reservoir) was collected in a 1L duran and stored at 4 °C for 16 hours. An aliquot (1 mL) of the retentate was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. The filtrate was discarded.

[0327] J: Purification 2 - Removal of large contaminants by continuous diafiltration using Tangential Flow Filtration (TFF)

[0328] The Minimate™ EVO TFF System (Pall) was set up with a Centramate™ LV cassette holder (Pall) and a T-series Centramate™ Cassette, Omega PES membrane, 30 kDa molecular weight cut-off (MWCO), 0.02 m2effective filtration area (Pall). The system was stored in 0.1 M sodium hydroxide and washed with RO water until the pH of the retentate and filtrate lines reached pH 7.0.

[0329] The buffer exchanged material was transferred into the TFF reservoir to be concentrated. The sample was circulated around the TFF system with the peristaltic pump set to 100 rpm and 2 bar external pressure applied with a clamp. The transmembrane pressure was 0 - 0.1 bar. The filtrate was collected. The retentate continued to be concentrated until the volume in the TFF reservoir was 10 ml_. Once 10 mL was reached, diafiltration was performed. One end of tubing was placed in RO water (diafiltration buffer 2) and the other end secured to the leur lock connection on the reservoir lid. The reservoir was put under vacuum using a 20 ml_ syringe. The sample was circulated around the TFF system with the peristaltic pump set to 100 rpm and 2 bar external pressure applied with a clamp. The diafiltration buffer was added to the reservoir at the same flowrate as the filtrate was generated. Ten diafiltration volumes were performed. The filtrate was collected and transferred into a 1 L round bottom flask. An aliquot (1 or 8 ml_) of the filtrate sample was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. An aliquot (1 mL) of the retentate sample was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. Residual untagged peptide may remain in the retentate, this material can be resolubilised in 8 M urea and the process repeated from step E to recover additional peptide.

[0330] K: Lyophilisation - Isolation of the cleaved target peptide

[0331] The 30 kDa TFF membrane filtrate was frozen and placed on the freeze dryer (BenchTop Pro, SP Scientific) for 24 hours. The condenser was -100 °C and the vacuum was 15 mbar. A known mass of the lyophilized power sample was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. This material is shown to be of high purity (Figure 8).

[0332] As can be seen from this work, a cost-effective method is described to generate high purity specific recombinant peptides, which does not require costly purification methods such as preparative HPLC, which are typically used to purify peptides. This is due to the use of a first molecular weight cutoff filter, intein cleavage, then a second molecular weight cutoff filter.

[0333] Example 4: Optional further purification of peptides from the process

[0334] Additional purification of the cleaved target peptide

[0335] Residual buffer salts may remain in the sample after lyophilisation. Additional purification of the peptide can be achieved by continuous diafiltration and or peptide precipitation.

[0336] Dissolve the lyophilized powder in minimum volume 10 mL RO water. Transfer the sample to the TFF reservoir with a 1 kDa MWCO centramate membrane fitted. Perform continuous diafiltration using the same method as described in example 3 (Buffer exchange 2). Prepare and analyse the isolated peptide by HPLC, LCMS and SDS-PAGE as described in general method.

[0337] Peptide precipitation

[0338] Precipitate the peptide by dissolving the remaining lyophilized powder in a minimal volume of RO water. Add four volume equivalents of acetone and incubate on ice for 30 minutes. Spin the sample at 4500 rpm, 4 °C, 30 minutes by centrifugation. Discard the supernatant and place the precipitate on the freeze dryer for 24 hours with the condenser set at -100 °C and vacuum below 150 mbar. Prepare and analyse the isolated peptide by HPLC and LCMS as described in general methods.

[0339] Example 5: Generation of GLP1 peptide using mutant intein and TFF

[0340] Figure 10 provides a representative schematic of Steps A-K described in this Example.

[0341] A: Fermentation and Recovery of BL21(DE3) p068-021

[0342] 50 mL VLB containing 25 pg / mL kanamycin in a 250-mL baffled Erlenmeyer flask was inoculated with E. coli BL21(DE3) containing plasmid p068-021 and incubated at 37 °C with shaking at 250 rpm (2.5 cm throw) for 16 hours, to be used as the first seed stage culture.

[0343] 400 mL peptone vegetable broth containing 25 pg / mL kanamycin in a 2-L Erlenmeyer flask was inoculated with 8 mL of the first seed stage culture broth and incubated at 37 °C with shaking at 250 rpm (2.5 cm throw) for 8 hours, to be used as the second seed stage culture.

[0344] 2 L of Basal medium, supplemented with Feed medium in a 7-L Stirred Tank Reactor was inoculated with the second seed stage culture to a final OD595 of 0.2. The culture was grown with the following controls in place: culture temperature was kept at 30 °C, filtered air was sparged through at 2 L / min, 2 Rushton impellors were used at 400-1000 rpm (stepped increase), culture pH was kept at 7.2 (+ / - 0.1) using 30% ammonium hydroxide and 2 M phosphoric acid solutions. Foaming was suppressed using AF204 antifoam and a foam detector probe. Addition of Feed media was as follows. At the start of the fermentation, Feed medium was added at a linearly increasing rate, starting at 0.32 mL / min and increasing to 1.72 mL / min over the first 24 hours. Then, upon a dissolved oxygen spike above a 10% threshold, Feed medium was automatically added as a 10 mL bolus. After 22 hours, IPTG was added to a final concentration of 1 mM, followed by further cultivation at pH 7.6, 30 °C for 18 hours. The culture was then harvested. B: Homogenisation of inclusion bodies from BL21(DE3) p068-021

[0345] The optical density of harvested material was measured as described in general methods. The harvested material was passed straight through the homogenizer (GEA Pony) once at 1500 bar. The optical density of the homogenate had decreased more than 75%. An aliquot of the homogenate was taken for SDS-PAGE sample preparation and analysis.

[0346] The homogenised material was spun at 16000 g, 4 °C, 50 minutes by centrifugation (refrigerated floor centrifuge, Sorvall). The supernatant was decanted.

[0347] C: Wash 1 of inclusion bodies from BL21(DE3) p068-021

[0348] The pellet was re-suspended in wash buffer 1 , 0.1 M urea, (general methods) in a volume to yield 15% cell solids, and spun at 16000 g, 4 °C, 50 minutes. The supernatant was decanted, an aliquot was taken for SDS-PAGE sample preparation and analysis.

[0349] D: Wash 2 of inclusion bodies from BL21(DE3) p068-021

[0350] The pellet was re-suspended in wash buffer 2, 20 mM tris buffer, (general methods) in a volume to yield 15% cell solids, and spun at 16000 g, 4 °C, 50 minutes. The supernatant was decanted, an aliquot was taken for SDS-PAGE sample preparation and analysis.

[0351] E: Resolubilisation of inclusion bodies from BL21(DE3) p068-021

[0352] The pellet was solubilised in 4 M urea buffer, (general methods) in a volume to yield 15% cell solids and stirred using an overhead stirrer for 3.5 hours at room temperature, then frozen at -20 °C for 20 hours. Once thawed an aliquot (1 mL) of the solubilized homogenate pellet was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods.

[0353] F: Purification 1 - Removal of small contaminants by concentration using Tangential Flow Filtration (TFF)

[0354] The Sartocon Slice Ultrafiltration set (Sartorius) was set up with a SartoJet pump, connected with sanitary Tri Clamp adapters to a Sartocon Slice holder fitted with a Sartocon Slice Hydro Cassette, Omega PES membrane, 30 kDa molecular weight cut-off (MWCO), 0.1 m2effective filtration area (Sartorius). The system was stored in 0.1 M sodium hydroxide and washed with water until the pH of the retentate and filtrate lines reached pH 7.0. The water was then removed from the TFF system and 4 M urea buffer (300 mL) was added to the TFF reservoir, a 2 L aspirator bottle with hose outlet O.D. 10 mm (Aldrich). The 4 M urea was circulated around the TFF system for 5 minutes then removed. The reservoir was placed on a stirrer plate (I KA) with a magnetic stirrer bar placed in the reservoir. Dependent on the size of the inclusion body, the size of the MWCO filter may vary and different sized cassettes (i.e. 5 or 10 kDa) may be installed.

[0355] The thawed urea solubilized pellet was spun at 16000 g, 4 °C, 20 minutes by centrifugation. The supernatant was filtered via Buchner filtration and then transferred to the TFF reservoir. The pellet was discarded. The sample was circulated round the TFF system with the peristaltic pump set to 1.2% and 1 bar external pressure applied. TFF retentate was diafiltered with purification buffer 1 (general methods). The diafiltration buffer was drip-fed into the TFF reservoir via peristaltic pump at 10 mL / min, equivalent to the filtrate flow rate. Diafiltration continued until three diafiltration volumes of filtrate were achieved. The retentate was collected. An aliquot (1 mL) of the concentrated retentate and filtrate were taken for HPLC and SDS-PAGE sample preparation and analysis as described in general methods. The urea solubilised inclusion body remains in the TFF retentate.

[0356] G: Dilution - Addition of solubilised partially purified inclusion bodies to phosphate buffer Prior to dilution the refold buffer (general methods) was stored for 16 hours at 4 °C. The refold buffer was nine times the volume of the urea solubilised inclusion body. The refold buffer was stored in a 5 L duran. Via a peristaltic pump, the urea solubilised protein, stored in a separate Duran on ice was drip-fed at 1 mL / min via tubing in to the ice-cold refold buffer with overhead stirring, set to 300 rpm (IKA).

[0357] The diluted material was stored at 4 °C for 16 hours. An aliquot (1 mL) was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods.

[0358] Optimal conditions for dilution to minimise protein precipitation can vary dependent on the specific peptide. This may be improved by varying the buffer conditions within the following ranges pH 7.0 - 8.0, L-arginine hydrochloride concentration 0 - 0.4 M, sodium chloride concentration 0 - 0.5 M.

[0359] H: Cleavage of the intein

[0360] The diluted material was pH adjusted to pH 6.2 with 6 M HCI. The solution was stored at 42 °C, 80 rpm for 24 hours. Using a wide bore pipette tip, an aliquot of the solution was taken for HPLC (See Figure 9), LCMS and SDS-PAGE sample preparation and analysis as described in general methods. The solution was spun at 3500 rpm, 4 °C, 30 minutes by centrifugation. The supernatant contains most of the recombinant peptide. Optimal conditions for cleavage can vary dependent on the specific peptide. Cleavage may be improved by operating within the following ranges pH 6.0 - 6.8 (direct pH adjustment with 1M HCI), imidazole concentration 0 - 50 mM, sodium chloride concentration 0 - 0.5 M, temperature 28 - 42 °C, 14 - 24 hours.

[0361] Residual untagged peptide may precipitate into the pellet, this material can be resolubilised in 2 M urea and the process repeated from step E to recover additional peptide.

[0362] / : Purification 2 - Removal of large contaminants by concentration using Tangential Flow Filtration (TFF)

[0363] The Sartocon Slice Ultrafiltration set (Sartorius) was set up with a SartoJet pump, connected with sanitary Tri Clamp adapters to a Sartocon Slice holder fitted with a Sartocon Slice Hydro Cassette, Omega PES membrane, 10 kDa molecular weight cut-off (MWCO), 0.1 m2effective filtration area (Sartorius). The system was stored in 0.1 M sodium hydroxide and washed with water until the pH of the retentate and filtrate lines reached pH 7.0. The water was then removed from the TFF system and purification buffer 2, 300 mL (general methods) was added to the TFF reservoir, a 2 L aspirator bottle with hose outlet O.D. 10 mm (Aldrich). The buffer was circulated around the TFF system for 5 minutes then removed. The reservoir was placed on a stirrer plate (IKA) with a magnetic stirrer bar placed in the reservoir. Dependent on the size of the peptide, the size of the MWCO filter may vary and different sized cassettes (i.e. 5 or 30 kDa) may be installed.

[0364] The cleaved material was spun at 16000 g, 4 °C, 20 minutes by centrifugation. The supernatant was filtered via Buchner filtration. The clarified supernatant was transferred into the TFF reservoir. The sample was circulated around the TFF system with the SartoJet pump set to 0.2% and 1.5 bar external pressure applied. Following this discontinuous diafiltration was conducted. The volume of the retentate volume was recorded and an equivalent volume of purification buffer 2 was added to the TFF reservoir. This was repeated, collecting an additional two diafiltration volumes of filtrate. The filtrate containing the cleaved peptide was collected.

[0365] An aliquot (1 or 8 mL) of the filtrate sample was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. An aliquot (1 mL) of the retentate sample was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. Residual untagged peptide may remain in the retentate, this material can be resolubilised in 4 M urea and the process repeated from step E to recover additional peptide. J: Desalt - Removal of buffer salts using C18

[0366] A 10 g C18 SPE cartridge (Waters) is equilibrated with 3 column volumes (CV’s) acetonitrile, 3 CV’s water, 3 CV 5% acetonitrile. The filtrate is loaded onto the SPE cartridge under vacuum, the flowthrough collected and 1 mL sample taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis. The peptide was eluted off the SPE cartridge using the following method. 3 CV 5% acetonitrile, 6 CV 20% acetonitrile, 3 CV 30% acetonitrile, 6 CV 40% acetonitrile, 3 CV 50% acetonitrile and 3 CV 100% acetonitrile. The majority of peptide was collected in the 40% acetonitrile fraction. An aliquot of all fractions was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis.

[0367] An alternate desalting method is HP20 resin. 2% w / v of activated (in methanol) HP20 was added to the filtrate and stirred overnight with an overhead stirrer at room temperature. The supernatant was analysed to ensure peptide has adsorbed to the resin (a 1 mL aliquot for HPLC analysis). The mixture was spun, supernatant discarded and the resin slurry transferred to a 70 g empty DLV cartridge (Biotage). Using the vacuum pump the peptide was eluted off the resin using the following method. 3 CV of water, 3 CV 5% acetonitrile + 0.1% formic acid, 6 CV 20% acetonitrile + 0.1 % formic acid, 3 CV 30% acetonitrile + 0.1% formic acid, 6 CV 40% acetonitrile + 0.1% formic acid, 3 CV 50% acetonitrile + 0.1% formic acid and 3 CV 100% acetonitrile + 0.1% formic acid. The majority of peptide was collected in the 40% acetonitrile fraction. An aliquot of all fractions was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis.

[0368] An alternate desalting method is Sephadex G-10 (Cytiva). This could be used instead of C18 or HP20 or as an additional purification step. The resin is packed into a polypropylene column. The column is equilibrated in three column volumes of an equilibration buffer and the flowthrough discarded. Load the filtrate onto the column and then an additional 0.7 mL of the equilibration buffer. Allow the sample and equilibration buffer to pass through the column. Collect the flowthrough.

[0369] K: Lyophilisation - Isolation of the cleaved target peptide

[0370] The desalted material was shell frozen using isopropanol and dry ice prior to being placed on the freeze dryer (BenchTop Pro, SP Scientific) for 24 hours. The condenser was -100 °C and the vacuum was 15 mbar. A known mass of the lyophilized power sample was taken for HPLC, LCMS and SDS-PAGE sample preparation and analysis as described in general methods. This material is shown to be of high purity (Figure 8). As can be seen from this work, a cost-effective method is described to generate high purity specific recombinant peptides, which does not require costly purification methods such as preparative HPLC, which are typically used to purify peptides. This is due to the use of a first molecular weight cutoff filter, intein cleavage, then a second molecular weight cutoff filter.

[0371] Example 6: Expression of constructs containing new intein sequences

[0372] Plasmids p086-001, p086-010, p086-011, p086-039, p086-040, p086-041, p086-042, p086- 043, p086-044, p086-045, p086-046, p086-047, p086-048, p086-049, p086-050 and p086- 051 , and p086-052 were introduced into E. coli BL21 (DE3) cells by heat shock transformation, plated onto 2TY + kanamycin agar and incubated overnight at 37 °C. A single kanamycin resistant colony was inoculated in to 5 mL of 2TY liquid media + kanamycin and grown overnight at 37 °C with shaking at 250 rpm, 2.5 cm orbital throw.

[0373] The following morning, the OD600 of the overnight bacterial growth was measured, and the culture diluted back to an OD600 of 0.05 in 50 ml of fresh 2TY + Kanamycin media, in 250 ml non-baffled glass flasks, bunged with a foam bung. The flasks were shaken at 250 rpm, 2.5 cm orbital throw at 37 °C until the bacterial cultures reached an OD600 of 0.1. 0.5 mM IPTG was added to remove repression of the lacl / lac operator and allow expression of the intein- peptide construct.

[0374] Cultures were then incubated at 18 °C for a further ~18 hrs with 250 rpm shaking and 2.5 cm orbital throw. Cultures were split in to 2x 25 ml volumes within 50 ml centrifuge tubes, then spun at 3900 g, 4 °C, 15 minutes by centrifugation. The growth media supernatants were discarded, and the bacterial pellets were resuspended by vortexing in 5 ml 50 mM tris buffer at either pH 8.5 or pH 6.2 and held on crushed ice.

[0375] The bacterial suspensions were sonicated using a QSonica Q500 with a four-probe horn with 1 / 4” tips. Sonication occurred within a sound enclosure chamber, with sample tubes stood within a chilled Coolrack 4x 50 ml tube rack (Corning) sat on a bed of crushed ice. Sonication was programmed for 6 minutes with 10 seconds pulsing on and off at 50% amplitude (total pulse “on” time was 3 minutes). Sonicated lysates were confirmed to remain at pH at 8.5 or 6.2 as desired and any adjustments made as required with 0.5 N HCI or 0.5 N NaOH. The lysates were centrifuged at 3900 g, 4 °C, 15 minutes and the supernatant containing soluble protein was aspirated from the pellet. 500 pl of lysate supernatant in 2 ml round bottom microfuge tubes was incubated at 4 °C without shaking in the case of lysates within 50 mM Tris buffer pH 8.5 (cleavage condition A), and at 42 °C with 150 rpm shaking, 2.5 cm orbital throw in the case of lysates within 50 mM Tris buffer pH 6.2 (cleavage condition B). Incubation in both cases occurred for approximately 18 hours.

[0376] Incubated samples were briefly vortexed to homogenise, and quantified using Qubit Protein Assay Kit (ThermoFisher) and with a Qubit 3.0 fluorometer (Invitrogen). 10 pg of total protein from each incubated lysate was loaded in to SDS-PAGE gel wells for electrophoresis and then transferred to nitrocellulose membrane for Western Blot analysis as described in general methods.

[0377] Example 7: Comparison of inteins

[0378] Following Western Blot, nitrocellulose membranes were imaged, protein bands detected and densitometry analysed using Image Lab 5.2.1 software (Biorad).

[0379] Table 1 : Comparison of inteins by ratio of protein cleavage in conditions A and B As can be seen from table 1 , the inteins H439L, N440S, N440T, H439G, H439W, H439I, H429F, N440Y have improved (higher) ratios of “controlled” cleaved intein following incubation at 42 °C, pH6.2 (cleavage condition B) as compared to “uncontrolled” cleavage at 4 ° C, pH8.5 (cleavage condition A) as compared to AI-CM. An improved ratio of controlled to uncontrolled cleavage is desirable for the reasons described hereinbefore, namely said improved (higher) ratio demonstrates a reduced premature C-terminal cleavage.

[0380] Table 2: Ratio comparison of inteins between Cleaved and Uncleaved protein and in each cleavage condition

[0381] As can be seen from table 2, the inteins H439L, N440S, N440T, H439G, H439W, H439M, H439I, have improved levels of total cleavage (shown as ratio of cleaved to uncleaved protein on Western Blot) following incubation at 42 °C, pH6.2 (cleavage condition B) when considering the percentage proportion of “controlled” cleaved intein of each mutant compared with the original AI-CM. Improved levels of total cleavage are desirable, for example, to maximise yield (i.e. enrichment) of the target polypeptide after separation according to a second molecular weight cut-off following cleavage as described herein. Example 8: Expression of constructs containing new intein sequences

[0382] Plasmids pJP178, p068-021 and p068-023 were introduced into E. coli BL21 (DE3) cells by heat shock transformation, plated onto 2TY + kanamycin agar and incubated overnight at 37 °C. A single kanamycin resistant colony was inoculated in to 5 mL of 2TY liquid media + kanamycin and grown overnight at 37 °C with shaking at 250 rpm, 2.5 cm orbital throw.

[0383] The following morning, the OD600 of the overnight bacterial growth was measured, and the culture diluted back to an OD600 of 0.05 in 50 ml of fresh 2TY + Kanamycin media, in 250ml non-baffled glass flasks, bunged with a foam bung. The flasks were shaken at 250 rpm, 2.5 cm orbital throw at 37 °C until the bacterial cultures reached an OD600 of 0.1. 0.2 mM IPTG was added to remove repression of the lacl / lac operator and allow expression of the intein- peptide construct.

[0384] Cultures were then incubated at 30 °C for a further ~18 hrs with 250 rpm shaking and 2.5 cm orbital throw. Cultures were split in to 4x 12.5 ml volumes within 50 ml centrifuge tubes, then spun at 3900 g, 4 °C, 15 minutes by centrifugation. The growth media supernatants were discarded, and the bacterial pellets were resuspended by vortexing in 10 ml 50 mM phosphate buffer at either pH 8.5 or pH 6.2 and held on crushed ice.

[0385] The bacterial suspensions were sonicated using a QSonica Q500 with a four-probe horn with 1 / 4” tips. Sonication occurred within a sound enclosure chamber, with sample tubes stood within a chilled Coolrack 4x 50 ml tube rack (Corning) sat on a bed of crushed ice. Sonication was programmed for 6 minutes with 10 seconds pulsing on and off at 50% amplitude (total pulse “on” time was 3 minutes). Sonicated lysates were confirmed to remain at pH at 8.5 or 6.2 as desired and any adjustments made as required with 0.5 N HCI or 1 N NaOH. The lysates were centrifuged at 3900 g, 4 °C, 15 minutes and the supernatant containing soluble protein was aspirated from the pellet.

[0386] 500 pl of lysate supernatant in 2 ml round bottom microfuge tubes was incubated at 4 °C without shaking in the case of lysates within 50 mM phosphate buffer pH 8.5 (cleavage condition A), and at 42 °C with 150 rpm shaking, 2.5 cm orbital throw in the case of lysates within 50 mM phosphate buffer pH 6.2 (cleavage condition B). Incubation in both cases occurred for approximately 18 hours.

[0387] Incubated samples were briefly vortexed to homogenise, and quantified using Qubit Protein Assay Kit (ThermoFisher) and with a Qubit 3.0 fluorometer (Invitrogen). 10 pg of total protein from each incubated lysate was loaded in to SDS-PAGE gel wells for electrophoresis and transferred to nitrocellulose membrane for Western Blot analysis as described in general methods.

[0388] Example 9: Comparison of inteins

[0389] Following Western Blot, nitrocellulose membranes were imaged, protein bands detected and densitometry using Image Lab 5.2.1 software (Biorad). Table 3: Comparison of inteins by ratio of protein cleavage in condition A (50 mM phosphate buffer, pH 8.5, 4 °C, no shaking) and condition B (50 mM phosphate buffer, pH 6.2, 42 °C, 150 rpm shaking). Incubation was for ~18 hrs.

[0390] As can be seen from table 3, the inteins H439V and N440Q have improved (higher) ratios of “controlled” cleaved intein following incubation at 42 °C, pH 6.2 (cleavage condition B) as compared to “uncontrolled” cleavage at 4 °C, pH 8.5 (cleavage condition A) as compared to AI-CM. An improved ratio of controlled to uncontrolled cleavage is desirable for the reasons described hereinbefore, namely said improved (higher) ratio demonstrates a reduced premature C-terminal cleavage.

[0391] Table 4: Ratio comparison of inteins between Cleaved and Uncleaved protein and in each cleavage condition A or B As can be seen from table 4, the inteins H439V and N440Q have improved ratios of cleaved to uncleaved protein (as determined by protein on Western Blot) following incubation at 42 °C, 150 rpm, pH6.2 (cleavage condition B) compared with “uncontrolled” cleavage present following incubation at 4 °C, pH 8.5 (cleavage condition A). Original AI-CM displays similar ratios of cleaved : uncleaved protein irrespective of condition A or B being used, exhibiting almost complete uncontrolled cleavage. Thus, the inteins H439V and N440Q demonstrate reduced premature cleavage (i.e. more controlled cleavage) compared to AI-CM when incubated in the conditions intended for cleavage.

[0392] REFERENCES

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Claims

CLAIMS1. A method for enriching a target polypeptide in a sample, comprising the steps:(i) providing a sample containing the target polypeptide in the form of a recombinant fusion protein in which it is tagged with a cleavable polypeptide tag;(ii) separating components of the sample on the basis of a first molecular weight cut-off such that components of the sample having a molecular weight greater than the first molecular weight cut-off are retained in a first retained fraction such that the tagged target polypeptide is in the first retained fraction;(iii) cleaving the cleavable tag from the target polypeptide in the first retained fraction; and thereafter(iv) separating components of the first retained fraction on the basis of a second molecular weight cut-off such that components of the first retained fraction having a molecular weight less than the second molecular weight cut-off are retained in a second retained fraction such that the target polypeptide is in the second retained fraction and is enriched relative to the sample.

2. The method of any of claim 1 , wherein the cleavable polypeptide tag is N-terminal to the target polypeptide sequence.

3. The method of claim 1 or claim 2, wherein the tagged target polypeptide in the form of a recombinant fusion protein has been produced by fermentation and the sample is a sample of fermentation broth comprising the tagged target polypeptide.

4. The method of claim 1 or claim 2, wherein the tagged target polypeptide in the form of a recombinant fusion protein has been produced by fermentation and the sample is a sample comprising inclusion bodies comprising the tagged target polypeptide that have been homogenised and solubilised.

5. The method of claim 1 or claim 2, wherein the tagged target polypeptide in the form of a recombinant fusion protein has been produced by cell-free protein synthesis and the sample is a sample of cell-free reaction mixture comprising the tagged target polypeptide.

6. The method of claim 5, wherein the cleavable polypeptide tag comprises a sequence which directs expression of the tagged target polypeptide to inclusion bodies.

7. The method of claim 6, wherein the sequence is selected from the group consisting of: ssTorA, TrpE, PurF, PagP, ketosteroid isomerase (KSI), GFIL16, Alpha helical 18A, Beta strand ELK16 or Surfactant L6KD.

8. The method of any of claims 1 to 7, wherein the molecular weight of the cleavable tag is at least 2 times the molecular weight of the target polypeptide.

9. The method of claim 8, wherein the molecular weight of the cleavable tag is at least 3 times the molecular weight of the target polypeptide.

10. The method of any of claims 1 to 9, wherein the components are separated by tangential flow filtration.

11. The method of any of claims 1 to 10, wherein the cleavable tag comprises a self cleaving intein.

12. The method of claim 11 , wherein the intein self-cleaves from the target polypeptide in one or more adjusted sample conditions, in particular wherein controllable C-terminal cleavage occurs to release the target polypeptide when one or more sample conditions are adjusted.

13. The method of claim 12, wherein adjusting the one or more sample conditions comprises adjustment of pH, adjustment of temperature or the addition of one or more compounds.

14. The method of claim 13, wherein adjustment of pH comprises adjustment to a pH of less than 6.8, or wherein adjustment of temperature comprises adjustment to a temperature equal to or greater than 28°C, in particular equal to or greater than 37°C.

15. The method of claim 14, wherein the intein does not self-cleave at a pH greater than or equal to 6.8 or at a temperature less than 28°C, in particular a temperature less than 37°C, and self-cleaves from the target polypeptide at a pH less than 6.8 or at a temperature equal to or greater than 28°C, in particular equal to or greater than 37°C.

16. The method of any of claims 11 to 15, wherein cleaving the cleavable tag comprises adjusting one or more sample conditions such that the intein self-cleaves from the target polypeptide in the adjusted sample conditions.

17. The method of claim 16, wherein the sample condition is pH.

18. The method of any of claims 11 to 17, wherein the intein is capable of being cleaved at a pH of less than 6.8.

19. The method of any of claims 11 to 18, wherein the intein is Mtu RecA, Mxe GyrA or HutMCM2, or a variant and / or mutant thereof.

20. The method of any of claims 11 to 19, wherein the intein is wild-type Mtu RecA (SEQ ID NO: 29) or a variant and / or mutant thereof.21 . The method of claim 20, wherein the variant intein is selected from the group consisting of: Al mini intein (SEQ ID NO: 30), AI-CM mini intein (SEQ ID NO: 31), AI-SM mini intein (SEQ ID NO: 32), AAIhh mini intein (SEQ ID NO: 33), AAIhh-CM mini intein (SEQ ID NO: 34) or AAIhh- SM mini intein (SEQ ID NO: 35), or a mutant thereof.

22. The method of claim 21 , wherein the variant intein is AI-CM mini intein (SEQ ID NO: 31) or a mutant thereof.

23. The method of any of claims 20 to 22, wherein the intein is a mutant Mtu RecA intein which comprises an amino acid substitution at a position selected from the group consisting of: His429, His439 and Asn440.

24. The method of claim 23, wherein the mutant intein comprises an amino acid substitution selected from the group consisting of: H439V, H439W, H439L, H439G, H439M, H439I, N440Q, N440T, N440S, N440Y and H429F, preferably wherein the substitution is selected from the group consisting of: H439V, H439W, H439L, H439G, N440Q, N440S and H429F.

25. The method of any of claims 20 to 24, wherein the mutant intein comprises or consists of a sequence selected from any of: SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NOs: 28-35, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 71 , SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 83 and SEQ ID NO: 86.

26. A mutant self-cleaving intein wherein the mutant intein is a mutant Mtu RecA intein comprising an amino acid substitution at a position selected from the group consisting of: His429, His439 and Asn440.

27. The intein of claim 26, wherein the mutant intein comprises an amino acid substitution selected from the group consisting of: H439V, H439W, H439L, H439G, H439M, H439I, N440Q, N440T, N440S, N440Y and H429F, preferably wherein the substitution is selected from the group consisting of: H439V, H439W, H439L, H439G, N440Q, N440S and H429F.

28. The intein of claim 26 or claim 27, wherein the mutant intein is a mutant of wild-type Mtu RecA (SEQ ID NO: 29).

29. The intein of any of claims 26 to 28, wherein the mutant intein is a mutant of a variant intein selected from the group consisting of Al mini intein (SEQ ID NO: 30), AI-CM mini intein (SEQ ID NO: 31), AI-SM mini intein (SEQ ID NO: 32), AAIhhmini intein (SEQ ID NO: 33), AAIhh- CM mini intein (SEQ ID NO: 34) or AAIhh-SM mini intein (SEQ ID NO: 35).

30. The intein of any of claims 26 to 29, comprising or consisting of a sequence selected from any of: SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NOs: 28-35, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 71 , SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 83 and SEQ ID NO: 86.31 . A recombinant fusion protein comprising a target polypeptide and the intein of any of claims 26 to 30, optionally further comprising a sequence which directs expression of the recombinant fusion protein to inclusion bodies.

32. Use of the intein of any of claims 26 to 30 as a component of a cleavable tag or the recombinant fusion protein of claim 31 in a method for enriching a target polypeptide in a sample as described in any one of claims 1 to 18.

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