Protein purification using cleavage intein systems

Abstract

The invention relates to protein purification using a cleavage intein system. The present invention relates to protein purification mainly in the field of chromatography. More specifically, the present invention relates to affinity chromatography using a cleavage intein system with improved C-intein tags and N-intein ligands wherein the target protein can be purified as a tagless end product with a native N-terminus.

Description

This is a divisional application. The Chinese application number of its parent application is 202080080416.2, the international application number is PCT / EP2020 / 082966, and the filing date is November 20, 2020. TECHNICAL FIELD

[0001] The present invention relates to protein purification, mainly in the field of chromatography. More precisely, the present invention relates to affinity chromatography using a split intein system with improved C-intein tags and N-intein ligands, where the target protein can be purified as a tag-free end product with a native N-terminus. BACKGROUND ART

[0002] Inteins are protein elements expressed as in-frame inserts that interrupt the enzyme sequence and catalyze the excision and ligation of their two flanking polypeptides, generating an active protein. Genetically, inteins are encoded in two different ways: as a full intein, interrupting two flanking extein sequences, or as a split intein, where each extein and part of the intein are encoded by two different genes. Although they hold great promise as biotechnological and protein purification tools, split inteins with fast kinetic properties found in nature rely on specific amino acids at the intein-extein junction, severely limiting the proteins that can be fused with the intein for affinity purification and recovery of native protein sequences. In particular, the prototype split intein DNAE from Nostoc punctiforme exhibits kinetic properties suitable for protein purification applications. However, its activity depends on phenylalanine at the +2 position in the C-extein. This dependence severely narrows and impairs its general applicability.

[0003] Inteins have been engineered to achieve several important functions in biotechnology, including applications as self-cleaving proteins for recombinant protein purification. Split inteins are particularly promising in this regard because they can provide both an affinity ligand and self-cleaving properties. In protein purification, the target protein to be purified can replace either extein. To date, the DNAE family of split inteins has shown the greatest promise for C-terminal cleavage protein purification methods.

[0004] WO2014 / 004336 describes proteins fused to a split intein N-fragment and a split intein C-fragment, which can be attached to a support. The solid support can be a particle, bead, resin, or slide.

[0005] WO2014 / 110393 describes a protein of interest fused to a split intein C-fragment, bringing it into contact with a split intein N-fragment and a purification tag. The N-fragment can be attached to a solid phase via the purification tag, and methods for affinity purification are discussed.

[0006] US10066027 describes a protein purification system and a method of using the system. Disclosed is a split intein comprising an N-terminal intein segment and a C-terminal intein segment, the N-terminal intein segment may be immobilized, and the C-terminal intein segment has self-cleavage properties and may be attached to a protein of interest. The N-terminal intein segment is provided with a motif that enhances sensitivity, which renders it more sensitive to external conditions.

[0007] US10308679 describes a fusion protein comprising an N-intein polypeptide and an N-intein solubilizing mate, and an affinity matrix comprising such a fusion protein.

[0008] WO 2018 / 091424 describes a method for producing an affinity chromatography resin, the affinity chromatography resin comprising an amino-terminal (N-terminal) split intein fragment as an affinity ligand, the method comprising the following steps: a) expressing the N-terminal split intein fragment protein as an insoluble protein in inclusion bodies in bacterial cells, preferably Escherichia coli; b) harvesting the inclusion bodies; c) solubilizing the inclusion bodies and releasing the expressed protein; d) binding the protein to a solid support; e) refolding the protein; f) releasing the protein from the solid support; and g) immobilizing the protein as a ligand onto a chromatography resin to form an affinity chromatography resin. This procedure enables immobilization of a ligand density of 2 - 10 mg / ml resin.

[0009] As described above, split inteins have been used for protein purification using a combination of affinity tags and tag cleavage mechanisms. However, the utility of such systems is limited by several factors. First, there are amino acid requirements at the splice junction of the desired product, i.e., a Phe is required at the +2 position of the C-extein for cleavage and purification of the tag-free protein. Recombinant protein production without foreign amino acids at the N-terminus is highly desirable. Second, the cleavage for releasing the protein must be fast enough and provide an acceptable yield. Third, there are solubility requirements for the N or C fragments of the split intein for its attachment to the solid support. Fourth, to date, there is no available split intein system suitable for large-scale purification of tag-free proteins. SUMMARY OF THE INVENTION

[0010] The present invention overcomes the drawbacks in the prior art and enables general purification of tag-free / native proteins in only one rapid affinity chromatography step using a split intein system.

[0011] The present invention provides a naturally occurring split intein or an N-intein protein variant sequence derived from a consensus sequence of a naturally occurring intein and a split intein, wherein the N-intein variant is modified compared to the natural sequence or consensus sequence to eliminate all asparagine (N) amino acid residues present in the sequence. Preferably, all such N-intein variant sequences are further modified to replace the cysteine (C) at position 1 with any other amino acid that is not cysteine.

[0012] The present invention provides an N-intein protein variant of a naturally occurring split intein or a consensus sequence derived from an intein / split intein, wherein the N-intein protein variant does not include asparagine (N) at position 36 of the variant sequence. This position is calculated starting from the initial catalytic cysteine numbered 1 according to a conventional alignment of the natural split intein. This position is conserved for N and natural N-intein sequences in the prior art, but the inventors have found that this position can be mutated to other amino acids that are less sensitive to deamidation, such as histidine (H or His) or glutamine (Q or Gln), and thereby achieve increased base stability, which is important as it confers tolerance to increased pH values during, for example, chromatography procedures. At least the N at position 36 must be mutated, but it is also contemplated that more Ns in the N-intein sequence can be mutated, preferably to H or Q.

[0013] The present invention also provides N-inteins and C-inteins that overcome the absolute requirement for phenylalanine at the +2 position of the target protein of interest (POI). The N-inteins and C-inteins of the present invention can be used to produce any recombinant protein. By using the N-inteins and C-inteins of the present invention, tag cleavage will occur at the exact junction between the tag intein and the POI, which means that the POI will be expressed in its native form, free of foreign amino acids encoded by the affinity tag. In addition, using the intein sequences of the present invention, the POI is produced in high yield and with rapid cleavage kinetics. The N-intein is coupled to a solid phase that can be regenerated under alkaline conditions.

[0014] The present invention provides N-inteins, C-inteins, split intein systems as defined in the appended claims and methods of using the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a graph showing the relative binding capacity of N-intein ligands (A40, A41, and A48) according to the present invention coupled to an SPR biosensor chip.

[0016] Figure 2shows a staple diagram of the relative binding capacities of the N-intein ligands (B72, B22, A48) and a comparative ligand (A53) according to the present invention conjugated to an SPR sensor chip.

[0017] Figure 3 Shows the static binding capacity of the N-intein ligands of the present invention. Amino acid analysis (AAA) was done by conventional methods. The A48 prototype was conjugated to porous agarose beads via epoxy chemistry.

[0018] Figure 4A Is a chromatogram of the purification results of Experiment 6.

[0019] Figure 4B Shows the SDS PAGE results from Experiment 6.

[0020] Figure 5 Is a graph showing the relative binding capacities of the N-intein ligands (A40 and A48) according to the present invention conjugated to an SPR biosensor chip. DETAILED DESCRIPTION

[0021] Definition As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a functional group", "an alkyl" or "a residue" includes mixtures of two or more such functional groups, alkyls or residues and the like.

[0022] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When expressing such ranges, further aspects include from one particular value and / or to another particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about", it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each range are significant both relative to the other endpoint and independently of the other endpoint. It should also be understood that there are many values disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It should also be understood that each integer between two particular integers is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0023] Unless specifically stated to the contrary, the weight percentages (wt%) of components are based on the total weight of the formulation or composition in which the component is included.

[0024] As used herein, the term "optional" or "optionally" means that the subsequent described event or circumstance may or may not occur, and the description includes instances where the described event or circumstance occurs and instances where it does not occur.

[0025] As used herein, the term "contact" refers to bringing two biological entities together in such a way that a compound can directly affect the activity of a target; i.e., by interacting with the target itself, or indirectly affect the activity of a target; i.e., by interacting with another molecule, cofactor, factor, or protein upon which the activity of the target depends. "Contact" can also mean facilitating the interaction of two biological entities such as peptides to bond covalently or otherwise.

[0026] As used herein, "kit" means a collection of at least two components that make up the kit. These components together form a functional unit for a given purpose. The individual member components may be physically packaged together or separately packaged. For example, a kit that includes instructions for using the kit may or may not physically include instructions for the other individual member components. Instead, the instructions may be supplied as a separate member component in paper form, electronic form, or as a recorded presentation, the electronic form of which may be supplied on a computer-readable storage device or downloaded from an Internet website.

[0027] As used herein, "instructions" means a document that describes the relevant materials or methods related to a kit. These materials may include any combination of the following: background information, a list of components and information about their availability (purchase information, etc.), a brief or detailed protocol for using the kit, troubleshooting, references, technical support, and any other relevant documents. The instructions may be supplied with the kit or as a separate member component in paper form, electronic form, or as a recorded presentation, the electronic form of which may be supplied on a computer-readable storage device or downloaded from an Internet website. The instructions may consist of one or more documents and are intended to include future updates.

[0028] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and include proteins and their fragments. Polypeptides are disclosed herein as sequences of amino acid residues. These sequences are written from left to right in the direction from the amino terminus to the carboxyl terminus. In accordance with standard nomenclature, sequences of amino acid residues are named by the three-letter or one-letter codes shown below: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V). Peptides include any oligopeptide, polypeptide, gene product, expression product or protein. Peptides are composed of contiguous amino acids and encompass molecules that are naturally occurring or synthetic.

[0029] In addition, as used herein, the term "peptide" refers to amino acids joined to each other by peptide bonds or modified peptide bonds, such as peptide isosteres, etc., and may contain modified amino acids in addition to the 20 genetically encoded amino acids. Peptides can be modified by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Modifications can occur at any position in the peptide, including the peptide backbone, amino acid side chains, and the amino or carboxyl terminus. The same type of modification can be present at several sites in a given polypeptide in the same or varying degrees. Additionally, a given peptide can have multiple types of modifications. Modifications include, but are not limited to, the attachment of different domains or motifs, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of riboflavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenylation, sulfation, and transfer-RNA-mediated addition of an amino acid to a protein such as arginylation. (See Proteins—Structure and Molecular Properties, 2nd ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, ed., Academic Press, New York, pp. 1-12 (1983)).

[0030] As used herein, "variant" refers to a molecule that retains the same or substantially similar biological activity as the original sequence. Variants can be from the same or different species, or can be synthetic sequences based on natural molecules or prior molecules. Additionally, as used herein, "variant" refers to a molecule that has a structure derived from the structure of a parent molecule (e.g., a protein or peptide disclosed herein), and whose structure or sequence is sufficiently similar to those disclosed herein such that, based on that similarity, one of ordinary skill in the art would expect the molecule to exhibit the same or similar activity and utility as the parent molecule. For example, substituting a specific amino acid in a given peptide can produce a variant peptide with similar activity to the parent.

[0031] In the context of the present invention, a substitution in a variant protein is indicated as: [original amino acid / position in the sequence / substituted amino acid]. For example, an asparagine (N) at position 36 in an amino acid sequence that has been mutated to histidine (H) can be alternatively indicated as "N36H" or "N36 to H".

[0032] As used herein, the term "protein of interest (POI)" includes any synthetic or naturally occurring protein or peptide. Thus, the term encompasses those compounds conventionally regarded as pharmaceuticals, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, and the like. Examples of therapeutic agents are described in well-known references such as the Merck Index (14th Edition), Physicians' Desk Reference (64th Edition), and The Pharmacological Basis of Therapeutics (1st Edition), and they include, but are not limited to, medicaments; substances for the treatment, prevention, diagnosis, cure, or alleviation of a disease or illness; substances that affect the structure or function of the body, or prodrugs, which become biologically active or more active after they have been placed in a physiological environment.

[0033] As used herein, "isolated peptide" or "purified peptide" is intended to mean a peptide (or a fragment thereof) that is substantially free of the materials with which the peptide is normally associated in nature, or free of the materials with which the peptide is associated in an artificial expression or production system, including but not limited to expression host cell lysates, growth medium components, buffer components, cell culture supernatants, or components of a synthetic in vitro translation system. The peptides or fragments thereof disclosed herein can be obtained, for example, by extraction from a natural source (such as mammalian cells), expression of a recombinant nucleic acid encoding the peptide (such as in cells or in a cell-free translation system), or chemical synthesis of the peptide. Additionally, peptide fragments can be obtained by any of these methods or by cleavage of full-length proteins and / or peptides.

[0034] As used herein, the word "or" means any one member of a particular list and also includes any combination of the members of that list.

[0035] As used herein, the phrase "nucleic acid" refers to naturally occurring or synthetic oligonucleotides or polynucleotides, whether DNA or RNA or DNA-RNA hybrids, single-stranded or double-stranded, sense or antisense, that are capable of hybridizing to a complementary nucleic acid through Watson-Crick base pairing. The nucleic acids of the present invention can also include nucleotide analogs (such as BrdU) and non-phosphodiester internucleoside linkages (such as peptide nucleic acids (PNA) or phosphorothioate linkages). In particular, nucleic acids can include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0036] As used herein, "isolated nucleic acid" or "purified nucleic acid" is intended to mean DNA that is free of the genes that flank the gene in the natural - occurring genome of the organism from which the DNA of the invention is derived. Thus, the term includes, for example, recombinant DNA incorporated into a vector, such as an autonomously - replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryote or eukaryote (e.g., a transgene); or existing as a separate molecule (e.g., a cDNA or genomic or cDNA fragment produced by PCR, restriction enzyme digestion, or chemical or in vitro synthesis). It also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence. The term "isolated nucleic acid" also refers to RNA, e.g., mRNA molecules encoded by isolated DNA molecules, or chemically synthesized, or separated from at least some cellular components or substantially free of at least some cellular components, such as other types of RNA molecules or peptide molecules.

[0037] As used herein, "extein" refers to a part of an intein - modified protein that is not part of the intein and can be spliced or cleaved after intein excision.

[0038] "Intein" refers to an in - frame insertion sequence within a protein. An intein can catalyze its own excision from the protein through a post - translational protein - splicing process to produce a free intein and a mature protein. An intein can also catalyze the cleavage of an intein - extein bond at either the N - terminus of the intein, or the C - terminus of the intein, or both the N - and C - termini of the intein - extein. As used herein, "intein" encompasses mini - inteins, modified or mutated inteins, and split inteins.

[0039] As used herein, the term "split intein" refers to any intein in which one or more peptide bonds are broken between the N - terminal intein segment and the C - terminal intein segment such that the N - terminal and C - terminal intein segments become separate molecules that can non - covalently re - associate or re - constitute an intein that is functional for a splicing or cleavage reaction. Any catalytically active intein or fragment thereof can be used to derive split inteins for use in the systems and methods disclosed herein. For example, in one aspect, a split intein can be derived from a eukaryotic intein. In another aspect, a split intein can be derived from a bacterial intein. In another aspect, a split intein can be derived from an archaeal intein. Preferably, the split intein so derived will have only the amino acid sequence necessary for catalyzing the splicing reaction.

[0040] As used herein, "N-terminal intein segment" or "N-intein" refers to any intein sequence that includes an N-terminal amino acid sequence, which is functional for a splicing and / or cleavage reaction when combined with a corresponding C-terminal intein segment. Thus, the N-terminal intein segment also includes the sequence that is spliced out when splicing occurs. The N-terminal intein segment can include a modified sequence that is the N-terminal portion of a naturally occurring (native) intein sequence. Non-intein residues can also be genetically fused to the intein segment to provide additional functionality, such as the ability for affinity purification or covalent immobilization.

[0041] As used herein, "C-terminal intein segment" or "C-intein" refers to any intein sequence that includes a C-terminal amino acid sequence, which is functional for a splicing or cleavage reaction when combined with a corresponding N-terminal intein segment. In one aspect, the C-terminal intein segment includes the sequence that is spliced out when splicing occurs. In another aspect, the C-terminal intein segment is cleaved from the peptide sequence fused to its C-terminus. The sequence cleaved from the C-terminus of the C-terminal intein is referred to herein as the "protein of interest (POI)" and is discussed in more detail below. The C-terminal intein segment can include a modified sequence that is the C-terminal portion of a naturally occurring (native) intein sequence. For example, the C-terminal intein segment can include additional amino acid residues and / or mutated residues, provided that the inclusion of such additional and / or mutated residues does not render the C-terminal intein segment non-functional for splicing or cleavage.

[0042] A consensus sequence is a DNA, RNA, or protein sequence that represents aligned related sequences. The consensus sequence of related sequences can be defined in different ways, but is typically defined by the most common nucleotide or amino acid residue at each position. An example of a consensus sequence of the present invention is the N-intein consensus sequence of SEQ ID NO:6.

[0043] As used herein, the term "splice" or "splices" means excising the central portion of a polypeptide to form two or more smaller polypeptide molecules. In some cases, splicing also includes the step of fusing two or more smaller polypeptides together to form a new polypeptide. Splicing can also refer to the joining of two polypeptides encoded on two separate gene products through the action of a cleaved intein.

[0044] As used herein, the term "cleave" or "cleaves" means to split a single polypeptide to form two or more smaller polypeptide molecules. In some cases, cleavage is mediated by the addition of an exogenous endopeptidase, which is often referred to as "proteolytic cleavage". In other cases, cleavage can be mediated by the intrinsic activity of one or both of the cleaved peptide sequences, which is often referred to as "self-cleavage". Cleavage can also refer to the self-cleavage of two polypeptides induced by the addition of a non-proteolytic third peptide, such as under the action of the split intein system described herein.

[0045] The term "fused" means covalently bonded thereto. For example, when two peptides (e.g., via a peptide bond) are covalently bonded to each other, the first peptide is fused to the second peptide.

[0046] As used herein, an "isolated" or "substantially pure" substance is a substance that has been separated from the components that naturally accompany it. Generally, a polypeptide is substantially pure when it is at least 50% by weight (e.g., 60%, 70%, 80%, 90%, 95% and 99%) free of other proteins and the naturally occurring organic molecules to which it is naturally bound.

[0047] In this document, "bind" or "binds" means that one molecule recognizes and adheres to another molecule in a sample, but substantially does not recognize or adhere to other molecules in the sample. If a molecule has a binding affinity greater than about 10 5 to 10 6 liters / mole for another molecule, then the molecule "specifically binds" to the other molecule.

[0048] The nucleic acids, nucleotide sequences, proteins or amino acid sequences mentioned herein can be isolated, purified, chemically synthesized or produced by recombinant DNA technology. All of these methods are well known in the art.

[0049] As used herein, terms such as "modified intein" or "mutated intein" where "modified" or "mutated" refer to one or more modifications in the nucleic acid sequence or amino acid sequence such as an intein when compared to the native or naturally occurring structure. Such modifications can be substitutions, additions or deletions. Modifications can occur in one or more amino acid residues or one or more nucleotides of the structure such as an intein being referred to.

[0050] As used herein, the terms "modified peptide", "modified protein" or "modified protein of interest" or "modified target protein" refer to a protein that has been modified.

[0051] As used herein, "operably linked" refers to the association of two or more biomolecules in such a configuration relative to each other that the normal functions of the biomolecules can be performed. With respect to nucleotide sequences, "operably linked" refers to the association of two or more nucleic acid sequences by enzymatic ligation or other means in such a configuration relative to each other that the normal functions of the sequences can be performed. For example, if a nucleotide sequence encoding a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to the nucleotide sequence of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, it is operably linked to the coding sequence; and if a ribosome binding site is positioned so as to facilitate the translation of a sequence, it is operably linked to the coding sequence.

[0052] "Sequence homology" can refer to the situation in which nucleic acid or protein sequences are similar because they have a common evolutionary origin. "Sequence homology" can indicate that sequences are very similar. Sequence similarity is observable; homology can be based on observation. "Very similar" can mean at least 70% identity, homology or similarity; at least 75% identity, homology or similarity; at least 80% identity, homology or similarity; at least 85% identity, homology or similarity; at least 90% identity, homology or similarity; for example at least 93% or at least 95% or even at least 97% identity, homology or similarity. The "Align" program by Myers et al. (1988) CABIOS 4:11-17 and available at NCBI can be used to determine the similarity or homology or identity of nucleotide sequences. Additionally or alternatively, amino acid sequence similarity or identity or homology can be determined using the BlastP program available at NCBI (Altschul et al. Nucl. Acids Res. 25:3389-3402). Alternatively or additionally, for example, the terms "similarity" or "identity" or "homology" with respect to nucleotide sequences are intended to indicate a quantitative measure of homology between two sequences.

[0053] Alternatively or additionally, "similarity" with respect to sequences refers to the number of positions having equivalent nucleotides divided by the number of nucleotides in the shorter of the two sequences, where the alignment of the two sequences can be determined according to the Wilbur and Lipman algorithm (1983) Proc. Natl. Acad. Sci. USA 80:726. For example, using a window size of 20 nucleotides, a word length of 4 nucleotides and a gap penalty of 4, and a commercially available program (e.g., Intelligenetics TMSuite, Intelligenetics Inc., CA), conveniently performs computer-aided analysis and interpretation of sequence data including alignments. When an RNA sequence is said to be similar to or have a certain degree of sequence identity with a DNA sequence, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. The following references also provide algorithms for comparing the relative identity or homology or similarity of amino acid residues of two proteins, and additionally or alternatively to the foregoing, the references can be used to determine the percentage of homology or identity or similarity. Needleman et al. (1970) J. Mol. Biol. 48:444-453; Smith et al. (1983) Advances App. Math. 2:482-489; Smith et al. (1981) Nuc. Acids Res. 11:2205-2220; Feng et al. (1987) J. Molec. Evol. 25:351-360; Higgins et al. (1989) CABIOS 5:151-153; Thompson et al. (1994) Nuc. Acids Res. 22:4673-480; and Devereux et al. (1984) 12:387-395. "Stringent hybridization conditions" is a term well known in the art; see, for example, Sambrook, "Molecular Cloning, A Laboratory Manual" Second Edition, CSH Press, Cold Spring Harbor, 1989; "Nucleic Acid Hybridization, A Practical Approach", edited by Hames and Higgins, IRL Press, Oxford, 1985; see also Figure 2 and its description herein, where there is a sequence comparison.

[0054] The terms "plasmid", "vector", and "cassette" refer to extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell and are usually in the form of circular double-stranded DNA molecules. Such elements can be self-replicating sequences, genomic integration sequences, phages, or nucleotide sequences of single-stranded or double-stranded DNA or RNA derived from any source, many of which have been ligated or recombined into unique constructs that are capable of introducing a promoter fragment and the DNA sequence of a selected gene product, along with appropriate 3' untranslated sequences, into a cell. Generally, a "vector" is a modified plasmid that contains additional multiple insertion sites for cloning and an "expression cassette" that contains the DNA sequence (i.e., transgene) of a selected gene product for expression in a host cell. Such an "expression cassette" typically includes a 5' promoter region, a transgene ORF, and a 3' terminator region, as well as all the necessary regulatory sequences required for transcription and translation of the ORF. Thus, integrating the expression cassette into a host allows expression of the transgene ORF in the cassette.

[0055] The term "buffer" or "buffer solution" refers to a solution that resists changes in pH through the action of its conjugate acid-base range.

[0056] The term "loading buffer" or "equilibration buffer" refers to a buffer that contains one or more salts and is mixed with a protein preparation for loading the protein preparation onto a column. This buffer is also used to equilibrate the column before loading and to wash the column after loading the protein.

[0057] The term "washing buffer" is used herein to refer to a buffer that passes through (e.g., a column) after a protein of interest (e.g., a protein conjugated to a C-terminal intein fragment) has been loaded and before the protein of interest is eluted. The washing buffer can serve to remove one or more contaminants without significantly eluting the desired protein.

[0058] The term "elution buffer" refers to a buffer used to elute a desired protein from a column. As used herein, the term "solution" refers to a buffered or unbuffered solution, including water.

[0059] The term "washing" means passing an appropriate buffer through or over a solid support, such as a chromatography resin.

[0060] The term "eluting" a molecule (e.g., a desired protein or a contaminant) from a solid support means removing the molecule from such material.

[0061] The terms "contaminant" or "impurity" refer to any foreign or harmful molecule, particularly a biopolymer such as DNA, RNA, or protein, other than the protein to be purified, that is present in a protein sample to be purified. Contaminants include, for example, other proteins from the cells that express and / or secrete the protein to be purified.

[0062] As used in connection with protein purification, the terms "separate" or "separation" mean separating a desired protein from a mixture containing both the desired protein and a second protein or other contaminant or impurity mixture such that at least a majority of the desired protein molecules are removed from that portion of the mixture that contains at least a majority of the second protein molecules or other contaminant or impurity mixture.

[0063] The terms "purify" or "purifying" a desired protein from a composition or solution containing the desired protein and one or more contaminants mean increasing the purity of the desired protein in the composition or solution by (fully or partially) removing at least one contaminant from the composition or solution.

[0064] N-intein protein variant The present invention relates to the use of a split intein system according to the present invention for affinity chromatography and affinity tag cleavage mechanisms in a single step, the split intein system cleaving with a broad amino acid tolerance to generate a tag-free protein of interest (POI) as the end product. The two halves of the intein are an affinity ligand (N-intein) and an affinity tag (C-intein), and they bind rapidly. Fixing one half (N-intein) to the chromatography resin enables capture of the other half (C-intein) conjugated to the POI from solution. In the presence of Zn 2+ ions, the cleavage reaction is inhibited, enabling formation of a stable complex while washing away impurities. After elimination of the impurities, a chelating agent or reducing agent is added and the cleavage reaction proceeds, enabling collection of the POI while the intein tag remains non-covalently bound to the cognate intein attached to the chromatography resin.

[0065] Preferably, the present invention provides a naturally occurring split intein or an N-intein protein variant sequence derived from a consensus sequence of a naturally occurring intein and a split intein, wherein the N-intein variant is modified compared to the natural sequence or consensus sequence to eliminate all asparagine (N) amino acid residues present in the sequence. Preferably, all such sequences do not include a cysteine (C) at position 1 of the N-intein variant sequence.

[0066] Preferably, the present invention provides an N-intein protein variant sequence that does not include asparagine (N) at position 36 of the variant sequence. This position is calculated starting from the initial catalytic cysteine numbered 1 according to a conventional alignment of the native split intein. This position is conserved for N in the prior art and native N-intein sequences, but the inventors have found that this position can be mutated to an amino acid that provides increased base stability compared to the native N-intein protein sequence, which is important because it confers tolerance to increased pH values during, for example, chromatography procedures. Preferably, the amino acid that provides increased base stability is histidine (H or His) or glutamine (Q or Gln).

[0067] Native inteins are known in the art. A list of inteins is found in Table 1 below. All inteins have the potential to be prepared as split inteins, and some inteins occur naturally in split form. All inteins found in the table either exist as split inteins or have the potential to be prepared as split inteins modified at position 36 according to the present invention such that the conserved N is replaced with another amino acid conferring base stability such as H or Q.

[0068] Table 1 - Naturally occurring inteins The split intein of the disclosed composition or a split intein that can be used in the disclosed method can be a modified or mutant intein. The modified intein can comprise modifications to the N-terminal intein segment, the C-terminal intein segment, or both. The modifications can include additional amino acids at the N-terminus, C-terminus of any part of the split intein, or the modifications can be within any part of the split intein. Table 2 shows a list of amino acids, their abbreviations, polarity, and charge.

[0069] Table 2 - List of Amino Acids Preferably, the present invention provides an N-intein protein variant of the native N-intein domain of Nostoc punctiforme (Npu), wherein the native N-intein domain has the following sequence: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRV (SEQ ID NO:1) wherein the protein variant comprises an amino acid substitution of asparagine (N) at position 36 of SEQ ID NO:1 with the following amino acid: compared with the base stability of the native N-intein of SEQ ID NO:1, the amino acid increases the base stability of the N-intein protein variant.

[0070] Preferably, the present invention provides an N-intein protein variant of SEQ ID NO:1, wherein said protein variant comprises an amino acid substitution in which cysteine (C) at position 1 of SEQ ID NO:1 is substituted with any other amino acid that is not cysteine, plus an amino acid substitution in which asparagine (N) at position 36 of SEQ ID NO:1 is substituted with an amino acid that increases the alkali stability of the N-intein protein variant as compared to the alkali stability of the native N-intein of SEQ ID NO:1.

[0071] The present invention also provides an N-intein protein variant of a reference protein, wherein said reference protein has at least about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1, and preferably wherein said reference protein has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1, and wherein the N-intein protein variant of the present invention comprises an amino acid substitution in which asparagine (N) at position 36 of the reference protein is substituted with an amino acid that increases the alkali stability of the N-intein protein variant as compared to the alkali stability of the native N-intein of SEQ ID NO:1.

[0072] In another embodiment, the N-intein comprises the amino acid sequence of SEQ ID NO:2, which is a consensus-derived sequence of N-inteins. The N-intein variant sequence based on SEQ ID NO:2 further comprises an amino acid other than N at position 36, which amino acid increases the alkali stability of the N-intein protein variant as compared to the alkali stability of the native N-intein of SEQ ID NO:1. Preferably, the amino acid that increases alkali stability is an amino acid that is less sensitive to deamidation as compared to asparagine (N). The amino acid sequence of SEQ ID NO:2 is as follows: ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLED GSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV (SEQ ID NO:2) wherein X at positions 20, 35, 70, 73 and 95 are each independently selected from K, R or A; X at position 28 is C, A or S; X at position 36 is N, H or Q; X at position 25 is N or R; X at position 59 is D or C; X at position 80 is E or Q; and X at position 90 is Q, R or K.

[0073] Preferred embodiments of the N-intein according to the invention are selected from the group of N-intein variants herein called A48, B22, B72 and A41, wherein: A48 has the sequence of SEQ ID NO:2, wherein: X at positions 20, 35, 70, 73 and 95 is R; X at position 28 is A; X at position 36 is H; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q; B22 has the sequence of SEQ ID NO:2, wherein: X at positions 20, 35, 70, 73 and 95 is A; X at position 28 is A; X at position 36 is H; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q; B72 has the sequence of SEQ ID NO:2, wherein: X at positions 20, 35, 70, 73 and 95 is K; X at position 28 is C; X at position 36 is H; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q A40 has the sequence of SEQ ID NO:2, wherein: X at positions 20, 35, 70, 73 and 95 is R; X at position 28 is A; X at position 36 is N; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q.

[0074] A41 has the sequence of SEQ ID NO:2, wherein: X at positions 20, 35, 70, 73 and 95 is K; X at position 28 is A; X at position 36 is N; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q; Comparing ligand A53, having the sequence of SEQ ID NO:2, wherein: X at positions 20, 35, 70, 73 and 95 is K; X at position 28 is C; X at position 36 is N; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q.

[0075] The N - inteins of the present invention can be coupled to a solid phase such as a membrane, fiber, granule, bead or chip. The solid phase can be a chromatography resin of natural or synthetic origin, such as a natural or synthetic resin, preferably a polysaccharide such as agarose. The solid phase such as a chromatography resin can be provided with embedded magnetic particles. In another embodiment, the solid phase is a non - diffusion - limiting resin / fiber material.

[0076] In this case, the solid phase can be formed from one or more polymer nanofiber substrates such as electrospun polymer nanofibers. The polymer nanofibers used in the present invention generally have an average diameter of 10 nm to 1000 nm. The length of the polymer nanofibers is not particularly limited. The polymer nanofibers can suitably be monofilament nanofibers and can have, for example, a circular, oval or substantially circular / oval cross - section. Generally, one or more polymer nanofibers are provided in the form of one or more non - woven sheets, each non - woven sheet containing one or more polymer nanofibers. The non - woven sheet containing one or more polymer nanofibers is a mat of the one or more polymer nanofibers, wherein each nanofiber is substantially randomly oriented, i.e., it is not manufactured such that one or more nanofibers adopt a specific pattern. The non - woven sheet generally has an areal density of 1 to 40 g / m2. The non - woven sheet generally has a thickness of 5 to 120 μm. The polymer should be a polymer suitable for use as a chromatography medium, i.e., an adsorbent, in a chromatography method. Suitable polymers include polyamides such as nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polysulfones such as polyethersulfone (PES), polycaprolactone, collagen, chitosan, polyethylene oxide, agarose, agarose acetate, cellulose, cellulose acetate and combinations thereof.

[0077] The N-intein according to the present invention can be immobilized on a solid support to a very high degree, with 0.2 - 2 μmole / ml of N-intein coupled per ml of resin (swollen gel).

[0078] The N-intein according to the present invention can be coupled to a solid phase via a Lys tail at the C-terminus, the Lys tail containing one or more, for example at least two, Lys. Alternatively, the N-intein is coupled to a solid phase via a Cys tail at the C-terminus.

[0079] C-intein protein variant Preferably, the present invention also provides a C-intein, which comprises the following sequence SEQ ID NO 3: VKIVSRKSLGVQNVYDIGVEKDHNFLLANGLIASN (SEQ ID NO:3) or a sequence having at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and preferably a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0080] It should be understood that the selection of the N-intein and the C-intein can be from the same wild-type split intein (e.g., both from Npu), or variants of the N-intein or C-intein, or alternatively can be selected from different wild-type split inteins or consensus split intein sequences, since it has been found that the affinity of the N-fragment for different C-fragments (e.g., the Npu N-fragment or its variants for the Ssp C-fragment or its variants) still maintains sufficient binding affinity for use in the disclosed methods.

[0081] Vector containing the intein variant of the present invention In a third aspect, the present invention relates to a vector comprising the C-intein of SEQ ID NO:3 above and a gene encoding a protein of interest (POI). Also disclosed herein are vectors comprising a nucleic acid encoding a C-terminal intein segment, and cell lines comprising said vectors. As used herein, a plasmid or viral vector is a reagent that transports the disclosed nucleic acids (such as those encoding a C-terminal intein segment and a peptide of interest) into cells without degradation, and includes a promoter that enables gene expression in the cells into which they can be delivered. In one example, the C-terminal intein segment and the peptide of interest are derived from a virus or retrovirus. Compared to other viral vectors, retroviral vectors are capable of carrying a larger genetic payload, i.e., a transgene or marker gene, and are therefore commonly used vectors. However, they are not as useful in non-proliferating cells. Adenoviral vectors are relatively stable and easy to manipulate, have a high titer, and can be delivered in an aerosol formulation, and can transfect non-dividing cells. Poxvirus vectors are large and have several sites for inserting genes; they are heat-resistant and can be stored at room temperature.

[0082] Split intein system Preferably, the present invention provides a split intein system for the affinity purification of a protein of interest (POI), which comprises an N-intein and a C-intein as described above.

[0083] Preferably, the N-intein comprises the N36H mutation for increased base stability.

[0084] Preferably, the N-intein is attached to a solid phase, and the C-intein is co-expressed with the POI and serves as a tag for the affinity purification of the POI. The reverse is also possible, i.e., attaching the C-intein to the solid phase and using the N-intein as a tag, but the former is preferred.

[0085] The base stability of the N-intein ligand in the split intein system according to the present invention enables regeneration under alkaline conditions such as 0.05 - 0.5 M NaOH after the POI is cleaved from the solid phase. The solid phase can be regenerated up to 100 times.

[0086] In one embodiment, the C-intein and an additional tag are co-expressed with the POI. The additional tag can be any conventional chromatography tag, such as an IEX tag or an affinity tag.

[0087] Method for purifying a protein of interest (POI) In a fifth aspect, the present invention relates to a method for purifying a protein of interest (POI) using the split intein system according to the present invention, which comprises binding a C-intein and an N-intein at a neutral pH, such as 6 - 8, and in the presence of a divalent cation that weakens spontaneous cleavage; washing the solid phase in the presence of the divalent cation; adding a chelating agent to allow spontaneous cleavage between the C-intein and the POI; collecting the tag-free POI; and regenerating the solid phase under alkaline conditions, such as 0.5 M NaOH.

[0088] This protocol is suitable for proteins that are not sensitive to Zn. The advantage is that it allows long contact times with the resin and the addition of large sample volumes. Sample loading can be carried out for a long time, such as up to 1.5 hours.

[0089] According to the present invention, a POI yield of more than 30%, preferably 50%, and most preferably more than 80% is obtained in a cleavage of less than 4 hours.

[0090] When the N-intein is immobilized on a solid phase, the present invention enables a high ligand density. Preferably, the N-intein is attached to a chromatography resin, such as agarose, or any other suitable resin for protein purification. According to the present invention, it is possible to achieve a static binding capacity of 0.2 - 2 μmole / ml of POI bound by the C-intein per ml of resin settled.

[0091] Affinity tag The present invention also relates to a method for purifying a protein of interest (POI), which comprises the following steps: co-expressing the POI with a C-intein according to the present invention and an additional tag; binding the additional tag to its binding partner on a solid phase; cleaving off the POI and the C-intein; binding the C-intein to an N-intein attached to the solid phase at a neutral pH, and cleaving off the bound C-intein and N-intein from the POI; and regenerating the solid phase under alkaline conditions, such as 0.5 M NaOH. The purpose of this dual-tag is: to increase purity (enabling dual-affinity purification), solubility, and detectability.

[0092] An affinity tag can be a peptide or protein sequence that is cloned in-frame with a protein coding sequence that alters the behavior of the protein. The affinity tag can be attached to the N or C terminus of the protein and can be used in methods for purifying the protein from cells. Cells expressing a peptide containing an affinity tag can be expressed in the supernatant / cell culture medium together with a signal sequence. Cells expressing a peptide containing an affinity tag can also be pelleted, lysed, and the cell lysate applied to a column, resin, or other solid support that displays a ligand for the affinity tag. The affinity tag and any fused peptide bind to the solid support, which can also be washed several times with buffer to remove unbound (contaminating) proteins. If attached to the affinity tag, the protein of interest can be eluted from the solid support via a buffer that promotes dissociation of the affinity tag from the ligand, resulting in a purified protein, or a soluble protease can be used to cleave the bound affinity tag. As disclosed herein, the affinity tag is cleaved by the self-cleavage mechanism of the C-intein segment in an active intein complex.

[0093] Examples of affinity include but are not limited to maltose binding protein, which can bind to immobilized maltose to facilitate purification of the fusion target protein; chitin binding protein, which can bind to immobilized chitin; glutathione S-transferase, which can bind to immobilized glutathione; polyhistidine, which can bind to immobilized chelated metal; and FLAG octapeptide, which can bind to immobilized anti-FLAG antibody.

[0094] Affinity tags can also be used to facilitate purification of the protein of interest using the disclosed modified peptides by various methods including but not limited to selective precipitation, ion exchange chromatography, binding to a ligand capable of precipitation, dialysis (by altering the size and / or charge of the target protein), and other highly selective separation methods.

[0095] In some aspects, an affinity tag can be used that does not actually bind to a ligand but rather selectively precipitates or acts as a ligand for a fixed corresponding binding domain. In these cases, the tag is more generally referred to as a purification tag. For example, the ELP tag selectively precipitates under specific salt and temperature conditions, allowing purification of the fusion peptide by centrifugation. Another example is the antibody Fc domain, which acts as a ligand for a fixed protein A or protein G binding domain.

[0096] Protein of interest The target proteins for all scenarios are: any recombinant protein, especially a protein that requires a native or near-native N-terminal sequence, such as a therapeutic protein candidate, biologic, antibody fragment, antibody mimetic, protein scaffold, enzyme, recombinant protein or peptide, such as growth factor, cytokine, chemokine, hormone, antigen (viral, bacterial, yeast, mammalian) production, vaccine production, cell surface receptor, fusion protein.

[0097] The present invention will now be described more specifically in connection with some non-limiting examples and the accompanying drawings. Examples

[0098] Experiment 1: Alkali stability of the N-intein ligand of the present invention The N-intein ligands A40, A41, and A48 according to the present invention were immobilized onto a Biacore TM CM5 sensor chip (Cytiva, Sweden) in an amount sufficient to give a immobilization level of about 450 response units (RU) or higher. To track the relative binding capacity of the POI tagged with a C-intein to the immobilized surface, 20 μg / ml of green fluorescent protein (GFP) tagged with a C-intein (SEQ ID NO: 3) was flowed over the chip for 1 minute, and the signal intensity was recorded. The surface was then cleaned in situ (CIP), i.e., rinsed with 100 mM NaOH, 4 M guanidine-HCl for 10 minutes at room temperature of 22 ± 3 °C. This was repeated for 50 cycles, and after each cycle, the base stability of the immobilized ligand was tracked as the relative loss of the relative binding capacity (signal intensity) of the GFP tagged with a C-intein.

[0099] The results are shown in Figure 1 and indicate that ligand A48 (with the N36H mutation) has improved base stability compared to ligands A41 and A40. The base stability is further improved compared to the native sequence. Additionally, compared to the wild-type Npu N-intein sequence, the N36H mutation significantly improves the base stability (A52 with the C1A mutation compared to SEQ ID NO: 1).

[0100] After 50 CIP cycles, the relative remaining binding capacity (%) of A40 and A41 was 55%, while for A48 it was 69%. The base stability using 0.5 M NaOH is shown in Figure 5 and

[0101] Figure 5 The results of A40 and A48 during 20 cycles are shown. Relative remaining binding capacity (%) CIP: 2 minutes. 100 mM NaOH, 4 M Gdn-HCl, then 2 minutes of 0.5 M NaOH.

[0102] Experiment 2: Alkali stability of the N-intein ligand of the present invention The purified N-intein ligands A53, B72, B22, and A48 were immobilized onto a Biacore TM CM5 sensor chip (Cytiva, Sweden) in an amount sufficient to give a immobilization level of about 450 response units (RU) or higher. To track the relative binding capacity of the uncleavable C-intein-tagged POI to the immobilized surface, 20 μg / ml of uncleavable C-intein (SEQ ID NO:3)-tagged IL-1b was flowed over the chip for 1 minute, and the signal intensity was recorded. The surface was then cleaned in situ (CIP), i.e., rinsed with 100 mM NaOH and 4 M guanidine-HCl for 10 minutes at room temperature of 22 ± 3 °C. This was repeated for 50 cycles, and after each cycle, the base stability of the immobilized ligand was tracked as the relative loss of the binding capacity (signal intensity) of the uncleavable C-intein-tagged IL-1b.

[0103] The results are shown in Figure 2 and indicate that all three ligands (A48, B22, and B72) with the N36H mutation have improved base stability compared to ligand A53. After 50 CIP cycles, the relative remaining binding capacity (%) of A53 was only 20%, while for B72 it was 28%, for B22 it was 30%, and for A48 it was 35%.

[0104] Experiment 3: Immobilization of N-intein ligand A48 onto agarose gel resin 5 ml of epoxy-activated cross-linked activated gel resin was added into a polypropylene test tube. 2.7 ml of the N-intein ligand A48 corresponding to 135 mg with a C-terminal Lys tail in phosphate buffer was added into the tube, followed by 1.3 ml of phosphate buffer (pH 12.1) to adjust the agarose resin slurry to about 50%, and then 2 g of sodium sulfate was added. The pH of the resulting reaction mixture was adjusted to 11.5. And the reaction mixture was heated on an orbital shaker until 33 °C and kept shaking at 33 °C for 4 hours. Then the slurry was transferred to a glass filter and washed 3 times with 10 ml of distilled water. After washing, the gel was transferred into a three-neck round bottom flask (RBF), and 5 ml of Tris buffer (pH 8.6) and 375 μl of thioglycerol were added. The reaction mixture was placed on an orbital shaker at 45 °C for 2 hours. After the reaction, the slurry was transferred to a glass filter. The gel was washed 3 times with 5 ml of alkaline wash buffer and then 3 times with 5 ml of acidic wash buffer. This base / acid wash was repeated 2 more times, with a total of 18 washes in this step. Then the gel resin was washed 10 times with 5 ml of distilled water. Before analysis, the washed and drained gel was kept in 20% ethanol in the refrigerator.

[0105] The dry weight of the gel resin was determined by measuring the weight of 1 mL of the gel. In sample preparation, 2 g of drained gel resin was thoroughly mixed with 2 g of water to give a resin slurry of approximately 50%, and then the slurry was added to a 1 mL Teflon cube. A vacuum was then applied to drain the gel in the cube, and thus 1 mL of gel was obtained. The gel was transferred to a dry weight balance. The weight was determined after 35 minutes, with the drying temperature set at 105 °C.

[0106] Amino acid analysis was measured after dry weight determination. From the corresponding dry weight and information on protein size and primary amino acid sequence, the ligand density could be derived in mg / mL of gel resin.

[0107] The results for the coupled agarose resin were a dry weight of 90.6 mg / ml and a ligand content of 18.4 mg / ml, corresponding to 1.38 umole / ml.

[0108] Experiment 4: Static binding capacity related to ligand density The proposed capacity method presented herein can measure the binding capacity of the resin in a test tube.

[0109] Reaction setup Briefly, prototype resins with immobilized A48 ligands of various ligand densities and the doubly tagged test protein A43 (SEQ ID NO:5) were diluted to 2.5% resin slurry and 0.4 mg / mL, respectively, in assay buffer (2x PBS). 50 μL of the 2.5% resin slurry was added to an ILLUSTRA TM microcentrifuge column, followed by 150 μL of diluted A43 (SEQ ID NO:5). The reaction was allowed to incubate with shaking at 22 °C at 1450 rpm for a fixed time point of 2 hours, and then centrifuged at 3000 rcf for 1 minute.

[0110] SDS-PAGE The centrifuged samples (containing cleaved protein and unbound uncleaved protein) were mixed 1:1 with 2x SDS-PAGE reducing sample buffer, boiled at 95 °C for 5 minutes, and then subjected to SDS-PAGE (loading 18 μL). A test protein A43 (SEQ ID NO:5) standard with a C-intein tag added (usually a five-point standard of 18.75 - 300 μg / mL) was added to enable calculation of the concentration based on the optical density volume. The gel was Coomassie stained for a total of 60 minutes (~100 mL / gel), followed by destaining with gentle stirring at room temperature for 120 - 180 minutes (until the background was completely transparent). Optical density quantification was performed on the uncleaved / unbound and cleaved test proteins using IQTL software. The optical density raw data was then exported to Microsoft Excel.

[0111] SBC Calculation Since the test protein input in the reaction is known, we can indirectly calculate the static binding capacity (SBC) using the following equation: Figure 3 The static binding capacity of the N-intein ligands of the invention is shown. Amino acid analysis (AAA) was performed by conventional methods. The A48 prototype was coupled to porous agarose particles by epoxide chemistry.

[0112] Experiment 5: Purification of elongation factor G with and without Zn protocol In this example, the elongation factor G (Ef-G) from Thermoanaerobacter tengcongensis was purified using a resin prototype with immobilized ligand A48. EfG tagged with a C-intein (SEQ ID NO 3) was expressed intracellularly in Escherichia coli strain BL21 (DE3).

[0113] The frozen cell pellet after fermentation harvest was thawed and resuspended with extraction buffer (20mMTris-HCl, pH 8.0) by magnetic agitation. DNase I (bovine pancreas) and 1mM MgSO4 were added, followed by lysozyme (egg). After stirring at room temperature for 30 minutes, the cell suspension resuspended and treated with lysozyme was heated to 70-75°C in a water bath and kept at this temperature for 5 minutes. After the extract was briefly cooled on ice, the extract was clarified by centrifugation.

[0114] During sample loading and washing, the flow rate was 2 ml / min, then 1 ml / min. Purification using the Zn-free protocol was performed on the Avant system. Using a 1 ml HiTrap TM Column. Equilibration and binding of the C-intein-tagged target protein was done in 20 mM MES buffer supplemented with 100 mM NaCl at pH 6.3, and the sample was adjusted to pH 6.3 using 2 M acetic acid. Column washing and subsequent elution after sample application was done with 20 mM Tris-HCl buffer supplemented with 400 mM NaCl at pH 8.0. After column washing, the flow was stopped for a 4-hour incubation at room temperature before elution of the cleaved EfG. A second flow stop was added to allow for a second elution, which was completed after an additional 16-hour incubation.

[0115] In HiTrap TMAfter 4 h incubation on column, 17.8 mg of pure, tag-free EfG was eluted. According to mass spectrometry analysis, the mass difference between the eluted protein and the protein treated with CIP was equal to the mass of the C-intein tag. The purity according to SDS-PAGE was as high as the SEC analysis on Superdex TM 200 Increase. The total protein amount was calculated based on the theoretical UV absorption coefficient at 280 nm and the UV signals of the diluted elution and CIP fractions.

[0116] Purification was repeated using the protocol with Zn ions included in the equilibration buffer and clarified sample. The final Zn concentration was 1.6 mM. The flow rate was reduced to 0.5 ml / min during sample application and then increased to 1 ml / min during washing and elution. Washing and elution were done with 50 mM Tris-HCl, 20 mM imidazole buffer pH 7.5. Only one elution peak was collected in this purification, and this was after 4 h incubation following column washing.

[0117] After 4 h incubation on the HiTrap TM column, 16.6 mg of pure, tag-free EfG was eluted. According to SEC analysis on Superdex TM 200 Increase, the purity was 92%. The total protein amount was calculated based on the theoretical UV absorption coefficient at 280 nm and the UV signal of the diluted elution fraction.

[0118] Experiment 6: Purification of IL-1β A 1 ml HiTrap column containing immobilized A48 ligand was used to purify the target protein IL-1β (SEQ ID NO: 5) plus a C-intein tag, which was intracellularly expressed in Escherichia coli BL21(DE3) and lysed by sonication. Soluble proteins were harvested by centrifugation and loaded onto a 1 mL HiTrap TM column with immobilized A48 ligand. During sample loading and washing, at 4 ml / min (600 cm / h linear flow rate), on the TM Avant system, a Zn-free protocol (as in Experiment 4) was used. Then the run was paused for 4 h and then the flow was restarted at 1 mL / min to elute the cleaved protein (4 h cleavage fraction). Then the run was paused again for an additional 12 h and then the flow was started at 1 mL / min to elute the protein that was not cleaved after 4 h. Binding and equilibration for washing and elution were performed with a single buffer. The chromatogram from the purification is shown in . The raw material, flow-through, wash fractions, 4 h and 16 h elution fractions were subjected to SDS-PAGE and Coomassie staining, and subsequent analysis using IQTL software ( Figure 4A ​Figure 4B )。

[0119] After 4 h incubation on a HiTrap TM column, 9.4 mg of cleaved IL-1β was eluted, followed by an additional 1.1 mg after 16 h. According to SDS-PAGE analysis, the purity was 99.5% (4 h) and 99.8% (16 h). The total protein amount was calculated based on the theoretical UV absorption coefficient of the cleaved protein at 280 nm.

[0120] Experiment 7: Purification of the receptor-binding domain of SARS-COV-2 The receptor binding domain (RBD) of SARS-COV-2 NCBI with a C-intein plus tag was expressed in ExpiHEK cells and secreted into the cell culture medium. Using an Avant FPLC system, approximately 210 mL of supernatant was loaded onto a 1 mL HiTrap column with immobilized A48 ligand, and no salts or other additives were added to the cell culture supernatant. Sample application and washing were performed at 4 mL / min (loading time ~52.5 min (linear flow rate of 600 cm / h)), followed by a wash of 6 column volumes, followed by a pause / hold step for a total of 4 h. The elution phase was performed at 1 mL / min. The column was left standing for an additional 68 h, followed by a second elution. A single 40 mM phosphate buffer pH 7.4 buffer supplemented with 300 mM NaCl was used for all chromatography steps.

[0121] Theoretical absorbance 0.1% coefficient was used to determine the protein concentration and yield within Unicorn TM software (Cytiva Sweden AB). Purity was determined by densitometric SDS-PAGE analysis. For this experiment, a total of 14.1 mg of cleaved protein was obtained with a purity greater than 96%. The theoretical molecular weight was ~25 kDa, while experimental SDS-PAGE analysis indicated a molecular weight of 33 kDa, which could be explained by two glycosylations and was also determined by mass spectrometry analysis.

[0122] The CCT-RBD protein has the following sequence: METDTLLLWVLLLWVPGSTGVKIVSRKSLGVQNVYDIGVEKDHN FLLANGLIASNRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKS NLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVK NKCVNFHHHHHH (SEQ ID NO:4) Signal sequence - bold underlined.

[0123] CCT-tag - dot underlined.

[0124] The RBD domain is double underlined.

[0125] His-tag - dotted underline The purity results from the cleaved protein are found in Table 3.

[0126] Table 3 Elution Cleavage time Purity Target protein yield 4 hours 4 hours 96.5％ 4.9 mg 72 hours 72 hours 99.4％ 9.2 mg Experiment 8: Tandem tagging and affinity purification on two columns E. coli BL21(DE3) was transformed with the A43 expression plasmid TwinStrep TM and IL-1β with an added C-intein (SEQ ID NO 3) tag, and plated on agar plates containing 50 μg / ml kanamycin. The next day, single colonies were picked and grown in 5 ml of Luria-Bertani (LB) broth to an OD600 of 0.6. The culture was transferred to 200 ml of LB broth containing the same antibiotic and grown at 37 °C until the OD600 was 0.6. Protein expression was induced at 22 °C for 16 h by adding isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.5 mM). After expression, the cells were harvested by centrifugation at 4,000 x g for 15 min and stored at -80 °C until use.

[0127] For purification, the cell pellet was resuspended at 10 ml per gram of wet weight in buffer A1 (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) and lysed by sonication (Sonics Vibracell, microtip, 30% amplitude, on for 2 s, off for 4 s, for a total of 3 min).

[0128] After centrifugation at 40,000 x g for 20 min at 4 °C, the supernatant containing the soluble fraction was collected and passed through a 5 ml HiTrap TM column, Streptactin TM XT (GE Healthcare, Sweden). The column was washed with the same buffer A1 until the UV absorbance at 280 nm was below 20 mAU. The bound IL-1β with the C-intein tag was eluted and collected in buffer B1 (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 50 mM biotin, pH 8.0).

[0129] The purified protein was immediately applied to a 1 ml HiTrap TM column packed with resin containing the immobilized N-intein ligand A48 without the addition of the inhibitor ZnCl2. The cleaved, tag-free IL-1β was collected in the flow-through.

[0130] MSAWSHPQFEKGGGSGGGSGGSAWSHPQFEKGGGSGGGS VKIVSRKSLGVQNVYDIGVEKDHNFLLAN GLIASNAFVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLS CVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDF TMQFVSS AAA(SEQ ID NO:5)TwinStrep - Underlined with dots CCT - Underlined in bold IL1b (Test protein) - The patents and scientific literature mentioned in this text underlined establish the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are hereby incorporated by reference.

[0131] Although the present invention has been particularly shown and described with reference to its preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as covered by the appended claims. It should also be understood that the embodiments described herein are not mutually exclusive, and features from each embodiment may be combined in whole or in part in accordance with the present invention. This application relates to the following embodiments: 1. An N - intein variant that comprises at least one amino acid substitution of a native split intein, wherein the N - intein protein variant sequence does not include asparagine (N) at least at position 36 as measured from the initial catalytic cysteine, and wherein the substituted amino acid provides increased base stability as compared to the native N - intein protein sequence or a consensus N - intein sequence. 2. The N - intein variant of embodiment 1, wherein the substituted amino acid that provides increased base stability is H or Q. 3. An N - intein protein variant of a wild - type N - intein domain of Nostoc punctiforme (Npu), wherein the wild - type Npu N - intein domain comprises the following sequence: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLED GSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRV(SEQ ID NO:1), wherein the protein variant comprises an amino acid substitution of asparagine (N) at least at position 36 of SEQ ID NO:1 with the following amino acid: the amino acid increases the base stability of the N - intein protein variant as compared to the base stability of the wild - type N - intein domain and the variant or the wild - type N - intein domain. 4. The N-intein protein variant according to embodiment 3, wherein the amino acid substitution that increases base stability is histidine (H) or glutamine (Q). 5. The N-intein protein variant according to embodiment 4, wherein the amino acid substitution that increases base stability is histidine (H). 6. An N-intein variant sequence comprising: ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLED GSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV (SEQ ID NO:2) wherein, X at positions 20, 35, 70, 73 and 95 are each independently selected from K, R or A; X at position 28 is C, A or S; X at position 36 is N, H or Q; X at position 25 is N or R; X at position 59 is D or C; X at position 80 is E or Q; and X at position 90 is Q, R or K; and wherein the base stability is increased compared to SEQ ID NO:1. 7. The N-intein variant sequence according to embodiment 6, wherein X at positions 20, 35, 70, 73 and 95 are R; X at position 28 is A; X at position 36 is H; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q. 8. The N-intein variant sequence according to embodiment 6, wherein X at positions 20, 35, 70, 73 and 95 are A; X at position 28 is A; X at position 36 is H; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q. 9. The N-intein variant sequence according to embodiment 6, wherein X at positions 20, 35, 70, 73 and 95 is K; X at position 28 is C; X at position 36 is H; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q. 10. The N - intein variant sequence according to embodiment 6, wherein X at positions 20, 35, 70, 73 and 95 is R; X at position 28 is A; X at position 36 is N; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q. 11. The N - intein variant sequence according to embodiment 6, wherein X at positions 20, 35, 70, 73 and 95 is K; X at position 28 is A; X at position 36 is N; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q. 12. The N - intein variant sequence according to one or more of the above embodiments, which is coupled to a solid phase such as a membrane, fiber, particle, bead or chip. 13. The N - intein variant sequence according to embodiment 12, wherein the solid phase is a chromatography resin of natural or synthetic origin. 14. The N - intein variant sequence according to embodiment 12 or 13, wherein the solid phase is a chromatography resin, such as a natural or synthetic resin, preferably a polysaccharide such as agarose. 15. The N - intein variant sequence according to embodiment 13, wherein the solid phase is provided with embedded magnetic particles. 16. The N - intein variant sequence according to embodiment 12, wherein the solid phase is a non - diffusion - limited resin / fiber material. 17. The N - intein variant sequence according to embodiment 12 or 13, wherein the N - intein is coupled to the solid phase via a Lys tail containing one or more Lys at the C - terminus. 18. An N-intein variant sequence according to embodiment 12 or 13, wherein the N-intein is coupled to a solid phase via a Cys tail at the C-terminus. 19. An N-intein variant sequence according to one or more of the above embodiments 12-18, wherein 0.2-2 μmole / ml of the N-intein is coupled per ml of the solid phase, preferably the chromatography resin (ml of swollen gel). 20. An N-intein sequence according to one or more of the above embodiments 1-19, wherein the N-intein is stable under alkaline conditions corresponding to 0.05M-0.5M, preferably 0.1-0.5M NaOH. 21. A C-intein variant sequence comprising the following amino acid sequence: VKIVSRKSLGVQNVYDIGVEKDHNFLLANGLIASN (SEQ ID NO:3) or a sequence having at least 85% identity thereto. 22. A vector comprising the C-intein according to embodiment 21 and a gene encoding a protein of interest (POI). 23. A split intein system for affinity purification of a protein of interest (POI), comprising an N-intein variant sequence of a native N-intein and a C-intein, wherein the N-intein variant sequence has an N36H or N36Q mutation compared to the native N-intein. 24. The split intein system according to embodiment 23, comprising an N-intein sequence variant of any one of embodiments 1-20 and a C-intein variant sequence of SEQ ID NO:3. 25. The split intein system according to embodiment 23 or 24, wherein the C-intein and an additional tag are co-expressed with the POI. 26. The split intein system according to embodiment 23, 24 or 25, wherein the N-intein is immobilized on a solid phase, and the solid phase is regenerated after the POI is cleaved from the solid phase. 27. The split intein system according to embodiment 26, wherein the solid phase is regenerated under alkaline conditions such as 0.05-0.5M NaOH. 28. The split intein system according to embodiment 26 or 27, wherein the solid phase is regenerated up to 100 cycles, such as up to 50 cycles. 29. A chromatography column comprising a chromatography resin, the chromatography resin comprising one or more N-intein variant sequence ligands, wherein the N-intein variant sequence is defined as in one or more of embodiments 1-20. 30. A method for purifying a protein of interest (POI) tagged with a C-intein by using a split intein system according to one or more of embodiments 23-29, wherein the N-intein is immobilized on a solid phase; the method includes contacting the C-intein and the N-intein at a neutral pH, such as 6-8, and in the presence of a divalent cation; washing the solid phase in the presence of a divalent cation; adding a chelating agent to allow spontaneous cleavage between the C-intein and the POI; collecting the tag-free POI; and regenerating the solid phase under alkaline conditions, such as 0.05-0.5 M NaOH. 31. A method for purifying a protein of interest (POI) tagged with a C-intein by using a split intein system according to one or more of embodiments 23-29, wherein the N-intein is immobilized on a solid phase; the method includes contacting the C-intein and the N-intein at a neutral pH, such as 6-8, preferably at a high flow rate; washing the solid phase; collecting the tag-free POI after cleavage between the C-intein and the POI; and regenerating the solid phase under alkaline conditions, such as 0.05-0.5 M NaOH. 32. A method for purifying a protein of interest (POI), which includes the following steps: co-expressing the POI with a C-intein according to SEQ ID NO 3 and an additional tag; binding the additional tag to its binding partner on a first solid phase; cleaving off the POI and the C-intein; binding the C-intein to an N-intein attached to a second solid phase at a neutral pH, and cleaving off the bound C-intein and N-intein from the POI; and regenerating the second solid phase under alkaline conditions, such as 0.05-0.5 M NaOH. 33. The method according to embodiment 32, wherein the additional tag is an affinity tag, ion exchange, hydrophobic interaction, solubility, multimodal. 34. The method according to any one of embodiments 30-33, wherein the alkaline conditions are combined with a chaotropic agent, such as guanidine or urea, and the solid phase can be regenerated up to 100 times. 35. The method according to one or more of embodiments 30-34, wherein the POI is: a protein that requires a native or near-native N-terminal sequence, such as a therapeutic protein candidate, a biologic, an antibody fragment, an antibody mimetic, an enzyme, a recombinant protein or peptide, such as a growth factor, a cytokine, a chemokine, a hormone, an antigen (viral, bacterial, yeast, mammalian) production, vaccine production, a cell surface receptor, a fusion protein. 36. The method according to one or more of embodiments 30-35, wherein a POI yield of more than 30%, preferably more than 50%, most preferably more than 80% is achieved within a cleavage of less than 4 hours. 37. A method according to any one or more of embodiments 30 - 36, wherein the N - intein is immobilized to a chromatography resin, and wherein the static binding capacity is 0.2 - 2 μmole / ml of C - intein - bound POI / ml of settled resin. 38. An N - intein variant according to one or more of embodiments 1 - 5, wherein all asparagine (N) amino acid residues are replaced by amino acid residues which provide increased base stability as compared to the native N - intein protein sequence. 39. An N - intein variant according to one or more of embodiments 1 - 5, wherein all asparagine (N) amino acid residues are replaced by amino acid residues which provide increased base stability, and wherein the cysteine at the first residue is replaced by any other amino acid.

Claims

1. An N-intein variant that comprises at least one amino acid substitution of a native split intein, wherein the N-intein protein variant sequence does not include asparagine (N) at least at position 36 as measured from the initial catalytic cysteine, and wherein the substituted amino acid provides increased base stability compared to the native N-intein protein sequence or a consensus N-intein sequence.

2. The N-intein variant of claim 1, wherein the substituted amino acid that provides increased base stability is H or Q.

3. An N-intein protein variant of the wild-type N-intein domain of Nostoc punctiforme (Npu), wherein the wild-type Npu N-intein domain comprises the following sequence: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLED GSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRV (SEQ ID NO:1), and wherein the protein variant comprises an amino acid substitution of asparagine (N) at least at position 36 of SEQ ID NO:1 with the following amino acid: the amino acid increases the base stability of the N-intein protein variant compared to the base stability of the wild-type N-intein domain and the variant or the wild-type N-intein domain.

4. The N-intein protein variant according to claim 3, wherein the amino acid substitution that increases base stability is histidine (H) or glutamine (Q).

5. The N-intein protein variant according to claim 4, wherein the amino acid substitution that increases base stability is histidine (H).

6. An N-intein variant sequence that comprises: ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLED GSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV (SEQ ID NO:2) wherein, X at positions 20, 35, 70, 73, and 95 are each independently selected from K, R, or A; X at position 28 is C, A, or S; X at position 36 is N, H, or Q; X at position 25 is N or R; X at position 59 is D or C; X at position 80 is E or Q; and X at position 90 is Q, R, or K; and wherein the base stability is increased compared to SEQ ID NO:

1.

7. The N-intein variant sequence according to claim 6, wherein X at positions 20, 35, 70, 73, and 95 is R; X at position 28 is A; X at position 36 is H; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q.

8. The N-intein variant sequence according to claim 6, wherein X at positions 20, 35, 70, 73 and 95 is A; X at position 28 is A; X at position 36 is H; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q.

9. The N-intein variant sequence according to claim 6, wherein X at positions 20, 35, 70, 73 and 95 is K; X at position 28 is C; X at position 36 is H; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q.

10. The N-intein variant sequence according to claim 6, wherein X at positions 20, 35, 70, 73 and 95 is R; X at position 28 is A; X at position 36 is N; X at position 25 is N; X at position 59 is D; X at position 80 is E; and X at position 90 is Q.

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