Protein purification using the split intein system

Abstract

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

Description

Technical Field

[0001] The present invention relates to protein purification, mainly in the field of chromatography. More specifically, the present invention relates to affinity chromatography using a split intein system with an improved C-intein tag and an N-intein ligand, wherein 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 an enzymatic sequence and catalyze the excision and ligation of its own two flanking polypeptides, generating an active protein. Genetically, inteins are encoded in two different ways: as a complete intein, interrupting the two flanking extein sequences, or as a split intein, where each extein and partial intein are encoded by two different genes. While they hold great promise as bioengineering and protein purification tools, the fast kinetic properties of split inteins found in nature are dependent on specific amino acids at the intein-extein junction, severely limiting the proteins that can be fused to the intein for affinity purification and recovery of native protein sequences. In particular, the prototype split intein DNAE from Nostoc punctata displays kinetic properties suitable for protein purification applications. However, its activity is dependent on the presence of a phenylalanine at the +2 position in the C-extein. This dependency severely narrows and compromises its general applicability.

[0003] Inteins have been engineered to perform 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 affinity ligands and self-cleavage properties. In protein purification, the target protein can replace any 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, a bead, a resin or a glass slide.

[0005] WO2014 / 110393 describes a protein of interest fused to a split intein C fragment, contacting it 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] US 10 066027 describes a protein purification system and methods of using the system. Disclosed are split inteins comprising an N-terminal intein segment, which can be immobilized, and a C-terminal intein segment, which has self-cleaving properties and can be attached to a protein of interest. The N-terminal intein segment is provided with a sensitivity-enhancing motif that renders it more sensitive to external conditions.

[0007] US 10 308 679 describes fusion proteins comprising an N-intein polypeptide and an N-intein solubilizing partner, and affinity matrices comprising such fusion proteins.

[0008] WO 2018 / 091424 describes a method for producing an affinity chromatography resin comprising an amino-terminal (N-terminal) split intein fragment as an affinity ligand, the method comprising the steps of: 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 on a solid support; e) refolding the protein; f) releasing the protein from the solid support; and g) immobilizing the protein as a ligand on a chromatography resin to form an affinity chromatography resin. This procedure enables the immobilization of a ligand density of 2-10 mg / ml resin.

[0009] As described above, combined affinity tags and tag cleavage mechanisms have been used to use split inteins for protein purification. However, the utility of such systems is limited by several factors. First, there is an amino acid requirement at the splice point of the intended product, namely a Phe in the +2 position of the C-extein, in order to perform the cleavage and achieve purification of the tag-free protein. Recombinant protein production without foreign amino acids on the N-terminus is highly desirable. Secondly, the cleavage that releases the protein must be fast enough and provide an acceptable yield. Third, there are solubility requirements for the split intein N or C fragments for their attachment to a 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 shortcomings of the prior art and enables universal purification of tag-free / native proteins in only one rapid affinity chromatography step using a split intein system.

[0011] The present invention provides a natural split intein or an N-intein protein variant sequence derived from a consensus sequence of a natural split intein and a split intein, wherein the N-intein variant is modified to eliminate all asparagine (N) amino acid residues present in the sequence compared to the natural sequence or the consensus sequence. Preferably, all such N-intein variant sequences are further modified to replace the cysteine ​​(C) at position 1 with any other amino acid other than cysteine.

[0012] The present invention provides N-intein protein variants of natural split inteins or derived from a consensus sequence of inteins / split inteins, wherein the N-intein protein variant does not include an asparagine (N) at position 36 of the variant sequence. This position is calculated from the initial catalytic cysteine ​​numbered 1 according to conventional cluster alignments with natural split inteins. This position is conserved for N in the prior art and natural N-intein sequences, 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 alkaline stability, which is important because 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 N 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 in 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 of the tag intein and the POI, which means that the POI will be expressed in its native form without the 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 fast cleavage kinetics. The N-inteins are coupled to a solid phase that can be regenerated under alkaline conditions.

[0014] The present invention provides N-inteins, C-inteins, split intein systems and methods of use thereof as defined in the appended claims. 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 a SPR biosensor chip.

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

[0017] 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.

[0018] Figure 4A This is a chromatogram of the purification result of Experiment 6.

[0019] Figure 4B The SDS PAGE results from Experiment 6 are shown.

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

[0021] definition

[0022] 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 a mixture of two or more such functional groups, alkyl groups, or residues, and so forth.

[0023] Ranges may be expressed herein as from "about" a particular value and / or to "about" another particular value. When such ranges are expressed, further aspects include from a particular value and / or to another particular value. Similarly, when values ​​are expressed as approximations, by using the antecedent "about", it will be understood that a particular value forms a further aspect. It will be further understood that the endpoints of each range are both important relative to the other endpoint and independently of the other endpoint. It will also be understood that there are many values ​​disclosed herein, and in addition to the value itself, each value is also disclosed herein as "about" the particular value. For example, if the value "10" is disclosed, "about 10" is also disclosed. It will also be understood that each integer between two specific integers is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0024] Unless specifically stated to the contrary, the weight percent (wt %) of a component is based on the total weight of the formulation or composition in which the component is included.

[0025] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0026] As used herein, the term "contacting" refers to bringing two biological entities together in such a way that the compound can directly affect the activity of the target; that is, by interacting with the target itself, or indirectly affect the activity of the target; that is, by interacting with another molecule, cofactor, factor or protein on which the activity of the target depends. "Contacting" can also mean facilitating the interaction of two biological entities, such as peptides, to bond covalently or otherwise.

[0027] As used herein, "kit" means a collection of at least two components that constitute a kit. These components together constitute a functional unit for a given purpose. The individual member components may be physically packaged together or separately. For example, a kit containing instructions for using the kit may or may not physically include instructions for other individual member components. Instead, the instructions may be supplied as separate member components in paper form or in electronic form or as a recorded presentation, which electronic form may be supplied on a computer-readable storage device or downloaded from an Internet website.

[0028] As used herein, "instructions" means a document describing the relevant materials or methods involved in a test kit. These materials may include any combination of the following: background information, a list of components and their availability information (purchase information, etc.), a brief or detailed scheme for using the test kit, troubleshooting, reference materials, technical support, and any other relevant documents. The instructions may be supplied with the test kit, or as a separate member component in paper form or electronic form or as a recorded presentation, which electronic form may be supplied on a computer-readable storage device or downloaded from an Internet website. The instructions may contain one or more files and are intended to include future updates.

[0029] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right in the direction from the amino terminal to the carboxyl terminal. According to standard nomenclature, amino acid residue sequences are named by three letters or single letter codes as 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 consecutive amino acids and encompass naturally occurring or synthetic molecules.

[0030] In addition, as used herein, the term "peptide" refers to amino acids connected to each other by peptide bonds or modified peptide bonds, such as peptide isosteres, etc., and may contain modified amino acids other than the 20 genetically encoded amino acids. The peptide may be modified by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Modifications may occur at any position in the peptide, including the peptide backbone, amino acid side chains, and the amino or carboxyl termini. The same type of modification may be present at several sites in a given polypeptide to the same or varying degrees. In addition, a given peptide may have a variety of types of modifications. Modifications include, but are not limited to, linkage 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 pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to proteins such as arginylation. (See Proteins—Structure and Molecular Properties 2nd edition, TE Creighton, WH Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, BC Johnson, ed., Academic Press, New York, pp. 1-12 (1983)).

[0031] 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 are synthetic sequences based on natural or previous molecules. In addition, as used herein, "variant" refers to a molecule that has a structure obtained 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, based on the similarity, a person skilled in the art would expect that the molecule would show the same or similar activity and utility compared to the parent molecule. For example, replacing a specific amino acid in a given peptide can produce a variant peptide with similar activity to the parent.

[0032] In the context of the present invention, substitutions in variant proteins are indicated as: [original amino acid / position in sequence / substituted amino acid]. For example, asparagine (N) at position 36 of an amino acid sequence that has been mutated to histidine (H) can be indicated interchangeably as "N36H" or "N36 to H".

[0033] As used herein, the term "protein of interest (POI)" includes any synthetic or naturally occurring protein or peptide. Thus, the term encompasses those compounds that are traditionally considered drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, and the like. Examples of therapeutic agents are described in well-known references such as 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 that become biologically active or more active after they have been placed in a physiological environment.

[0034] As used herein, "isolated peptide" or "purified peptide" is intended to mean a peptide (or fragment thereof) that is substantially free of materials with which the peptide is normally associated in nature, or free of 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 (e.g., mammalian cells), expression of a recombinant nucleic acid encoding the peptide (e.g., in a cell or in a cell-free translation system), or chemical synthesis of the peptide. In addition, peptide fragments can be obtained by any of these methods or by cleavage of full-length proteins and / or peptides.

[0035] As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0036] As used herein, the phrase "nucleic acid" refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridizing with a complementary nucleic acid by Watson-Crick base pairing. The nucleic acids of the present invention may also include nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acids (PNA) or thiodiester linkages). In particular, nucleic acids may include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0037] As used herein, "isolated nucleic acid" or "purified nucleic acid" is intended to mean DNA that is free of genes that flank the genes in the naturally occurring genome of the organism from which the DNA of the present 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 present as a separate molecule (e.g., a cDNA or genomic or cDNA fragment produced by PCR, restriction endonuclease digestion, or chemical synthesis or in vitro synthesis). It also includes recombinant DNA that is part of a hybrid gene encoding another polypeptide sequence. The term "isolated nucleic acid" also refers to RNA, for example, an mRNA molecule encoded by an isolated DNA molecule, or chemically synthesized, or separated from or substantially free of at least some cellular components, such as other types of RNA molecules or peptide molecules.

[0038] As used herein, "extein" refers to a portion of an intein-modified protein that is not part of the intein and that can be spliced ​​or cleaved following excision of the intein.

[0039] "Intein" refers to an in-frame insertion sequence in a protein. An intein can catalyze its own excision from a protein by 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 the intein N-terminus, or the intein C-terminus, or both intein-extein ends. As used herein, "intein" encompasses mini-inteins, modified or mutated inteins, and split inteins.

[0040] As used herein, the term "split intein" refers to any intein in which one or more peptide bond breaks occur 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 recombine or reconstitute into an intein functional for a splicing or cleavage reaction. Any catalytically active intein or fragment thereof can be used to derive a split intein for use in the systems and methods disclosed herein. For example, in one aspect, the split intein can be derived from a eukaryotic intein. In another aspect, the split intein can be derived from a bacterial intein. In another aspect, the 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 a splicing reaction.

[0041] As used herein, an "N-terminal intein segment" or "N-intein" refers to any intein sequence comprising an N-terminal amino acid sequence that is functional for a splicing and / or cleavage reaction when combined with a corresponding C-terminal intein segment. Thus, an N-terminal intein segment also comprises sequences that are spliced ​​away when splicing occurs. An N-terminal intein segment may comprise a modified sequence that is a modified N-terminal portion of a naturally occurring (native) intein sequence. Non-intein residues may also be genetically fused to an intein segment to provide additional functionality, such as the ability for affinity purification or covalent immobilization.

[0042] As used herein, a "C-terminal intein segment" or "C-intein" refers to any intein sequence comprising a C-terminal amino acid sequence that 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 comprises a sequence that is spliced ​​off when splicing occurs. In another aspect, the C-terminal intein segment is cleaved from a peptide sequence fused to its C-terminus. The sequence cleaved from the C-terminus of the C-terminal intein is referred to herein as a "target protein POI", discussed in more detail below. The C-terminal intein segment may comprise a modified sequence that is a C-terminal portion of a naturally occurring (native) intein sequence. For example, the C-terminal intein segment may comprise additional amino acid residues and / or mutated residues, as long as the inclusion of such additional and / or mutated residues does not render the C-terminal intein segment non-functional for splicing or cleavage.

[0043] A consensus sequence is a DNA, RNA or protein sequence that represents an aligned sequence of interest. The consensus sequence of a sequence of interest can be defined in different ways, but is usually 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.

[0044] As used herein, the term "splice" or "splices" means the removal of 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 by the action of a cleavage intein.

[0045] As used herein, the term "cleave" or "cleaves" means splitting a single polypeptide to form two or more smaller polypeptide molecules. In some cases, cleavage is mediated by the addition of exogenous endopeptidases, 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.

[0046] The term "fused" means covalently bonded to. For example, a first peptide is fused to a second peptide when two peptides are covalently bonded to each other (e.g., via a peptide bond).

[0047] As used herein, an "isolated" or "substantially pure" material is one that has been separated from components with which it is naturally associated. Typically, a polypeptide is substantially pure when it is at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, and 99%) free by weight from other proteins and naturally occurring organic molecules with which it is naturally associated.

[0048] As used herein, "bind" or "binds" means that one molecule recognizes and adheres to another molecule in a sample, but does not substantially recognize or adhere to other molecules in the sample. 5 Up to 10 6 A molecule "specifically binds" another molecule if the binding affinity is 1.1 L / mole.

[0049] 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 these methods are well known in the art.

[0050] As used herein, the term "modified" or "mutated" as in "modified intein" or "mutated intein" refers to one or more modifications in the referenced nucleic acid sequence or amino acid sequence, e.g., intein, when compared to the native or naturally occurring structure. Such modifications may be substitutions, additions or deletions. The modification may occur in one or more amino acid residues or one or more nucleotides of the referenced structure, e.g., intein.

[0051] As used herein, the term "modified peptide", "modified protein" or "modified protein of interest" or "modified target protein" refers to a protein that has been modified.

[0052] As used herein, "operably linked" refers to the association of two or more biomolecules in such a way that their configuration relative to each other allows the normal function of the biomolecules to be performed. With respect to nucleotide sequences, "operably linked" refers to the association of two or more nucleic acid sequences by means of enzymatic ligation or other means in such a way that their configuration relative to each other allows the normal function of the sequences to be performed. For example, a nucleotide sequence of a pre-coding sequence or secretory leader is operably linked to a nucleotide sequence of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote the translation of the sequence.

[0053] "Sequence homology" can refer to a 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 of 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 term "similarity" or "identity" or "homology" with respect to nucleotide sequences is intended to indicate a quantitative measure of the homology between two sequences.

[0054] Alternatively or in addition, "similarity" about a sequence refers to the number of positions with equivalent nucleotides divided by the number of nucleotides in the shorter sequence of the two sequences, wherein the comparison of the two sequences can be determined according to Wilbur and Lipman algorithm (1983) Proc.Natl.Acad.Sci.USA 80:726.For example, a window size of 20 nucleotides, a word length of 4 nucleotides and a gap penalty of 4 can be used, and commercially available programs (e.g., Intelligenetics™ Suite, Intelligenetics Inc. CA) can be used to conveniently perform computer-assisted analysis and interpretation of sequence data including comparison.When an RNA sequence is said to be similar to a DNA sequence or to have a certain degree of sequence identity, the thymidine (T) in the DNA sequence is considered to be equal to the uracil (U) in the RNA sequence. The following references also provide algorithms for comparing the relative identity or homology or similarity of the amino acid residues of two proteins, and relative to the aforementioned additionally or alternatively, references can be used to determine homology or identity or similarity percentage. 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, e.g., Sambrook, "Molecular Cloning, A Laboratory Manual" 2nd ed., CSH Press, Cold Spring Harbor, 1989; "Nucleic Acid Hybridization, A Practical Approach", Hames and Higgins, eds., IRL Press, Oxford, 1985; see also Figure 2 and their description herein, where sequence comparisons are present.

[0055] The terms "plasmid" and "vector" and "box" refer to extrachromosomal elements, which 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, genome integration sequences, phages or nucleotide sequences derived from any source, linear or circular single-stranded or double-stranded DNA or RNA, many of which have been connected or recombined into unique constructs that can introduce promoter fragments and the DNA sequence of selected gene products into cells together with appropriate 3' non-translated sequences. Usually, a "vector" is a modified plasmid that contains additional multiple insertion sites and "expression cassettes" for cloning, which contain the DNA sequence of the selected gene product for expression in the host cell (i.e., transgenic). This "expression cassette" usually includes a 5' promoter region, a transgenic ORF and a 3' terminator region, as well as all necessary regulatory sequences required for ORF transcription and translation. Therefore, the expression cassette is integrated into the host to allow the transgenic ORF in the box to be expressed.

[0056] The term "buffer" or "buffered solution" refers to a solution that resists changes in pH through the action of its conjugate acid-base domain.

[0057] The term "loading buffer" or "equilibrating buffer" refers to a buffer containing one or more salts, which is mixed with the protein preparation for loading the protein preparation onto the column. The buffer is also used to equilibrate the column before loading and to wash the column after loading the protein.

[0058] The term "wash buffer" is used herein to refer to a buffer that is passed through, for example, a column after the loading of a protein of interest (e.g., a protein coupled to a C-terminal intein fragment) and before elution of the protein of interest. The wash buffer may act to remove one or more contaminants without significantly eluting the desired protein.

[0059] The term "elution buffer" refers to a buffer used to elute the desired protein from the column. As used herein, the term "solution" refers to a buffered solution or a non-buffered solution, including water.

[0060] The term "washing" means passing an appropriate buffer over or through a solid support, such as a chromatography resin.

[0061] The term "eluting" a molecule (eg, a desired protein or a contaminant) from a solid support means removing the molecule from such material.

[0062] The term "contaminant" or "impurity" refers to any foreign or harmful molecule present in the protein sample to be purified, in addition to the protein to be purified, especially biological macromolecules, such as DNA, RNA or protein. Contaminants include, for example, other proteins from cells expressing and / or secreting the protein to be purified.

[0063] The terms "separating" or "isolating" as used in connection with protein purification refer to separating a desired protein from a second protein or other contaminants or impurity mixture in a mixture containing both the desired protein and the second protein or other contaminants or impurity mixture, so 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 contaminants or impurity mixture.

[0064] The terms "purify" or "purifying" a desired protein from a composition or solution comprising the desired protein and one or more contaminants mean increasing the purity of the desired protein in the composition or solution by removing (completely or partially) at least one contaminant from the composition or solution.

[0065] N-intein protein variants

[0066] The present invention relates to affinity chromatography and affinity tag cleavage mechanisms in a single step using a split intein system according to the present invention that cleaves with a broad tolerance of amino acids to generate a tag-free protein of interest (POI) as a final product. The two halves of the intein are an affinity ligand (N-intein) and an affinity tag (C-intein), and they bind rapidly. Immobilization of one half (N-intein) to a chromatography resin enables the capture of the other half (C-intein) coupled to the POI from solution. In the presence of Zn 2+ In the presence of ions, the cleavage reaction is inhibited, allowing the formation of a stable complex while the impurities are washed away. After the impurities are eliminated, a chelating agent or reducing agent is added and the cleavage reaction continues, allowing the POI to be collected while the intein tag remains non-covalently bound to the cognate intein linked to the chromatography resin.

[0067] Preferably, the present invention provides a native split intein or an N-intein protein variant sequence derived from a consensus sequence of a native intein and a split intein, wherein the N-intein variant is modified to eliminate all asparagine (N) amino acid residues present in the sequence compared to the native sequence or the consensus sequence. Preferably, all such sequences do not include a cysteine ​​(C) at position 1 of the N-intein variant sequence.

[0068] Preferably, the present invention provides N-intein protein variant sequences that do not include an asparagine (N) at position 36 of the variant sequence. This position is calculated starting from the initial catalytic cysteine ​​numbered 1 according to conventional cluster alignments with natural split inteins. This position is conserved for N in the prior art and natural N-intein sequences, but the inventors have found that this position can be mutated to an amino acid that provides increased alkaline stability compared to the natural 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 alkaline stability is histidine (H or His) or glutamine (Q or Gln).

[0069] Natural inteins are known in the art. A list of inteins is found in Table 1 below. All inteins have the potential to be made into split inteins, while some inteins naturally exist in a split form. All inteins found in the table either exist as split inteins or have the potential to be made into split inteins modified according to the present invention at position 36 such that the conserved N is replaced with another amino acid such as H or Q that confers alkaline stability.

[0070] Table 1 - Naturally occurring inteins

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] The split intein of the disclosed compositions or the split intein that can be used in the disclosed methods can be a modified or mutated intein. The modified intein can include modifications to the N-terminal intein segment, the C-terminal intein segment, or both. The modification can include additional amino acids at the N-terminus, the C-terminus of either portion of the split intein, or the modification can be within either portion of the split intein. Table 2 shows a list of amino acids, their abbreviations, polarity, and charge.

[0108] Table 2 - List of amino acids

[0109]

[0110] Preferably, the present invention provides an N-intein protein variant of a native N-intein domain of Nostoc punctata (Npu), wherein the native N-intein domain has the following sequence: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRV (SEQ ID NO: 1)

[0111] wherein the protein variant comprises an amino acid substitution of asparagine (N) at position 36 of SEQ ID NO: 1 with an amino acid that increases the alkaline stability of the N-intein protein variant compared to the alkaline stability of the native N-intein of SEQ ID NO: 1.

[0112] Preferably, the present invention provides an N-intein protein variant of SEQ ID NO: 1, wherein the protein variant comprises an amino acid substitution of cysteine ​​(C) at position 1 of SEQ ID NO: 1 with any other amino acid than cysteine, plus an amino acid substitution of asparagine (N) at position 36 of SEQ ID NO: 1 with an amino acid that increases the alkaline stability of the N-intein protein variant compared to the alkaline stability of the native N-intein of SEQ ID NO: 1.

[0113] The present invention also provides N-intein protein variants of a reference protein, wherein the 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 the 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 of asparagine (N) at position 36 of the reference protein with an amino acid that increases the alkaline stability of the N-intein protein variant compared to the alkaline stability of the native N-intein of SEQ ID NO: 1.

[0114] In another embodiment, the N-intein comprises the amino acid sequence of SEQ ID NO: 2, which is a consensus derivative sequence of the N-intein. The N-intein variant sequence based on SEQ ID NO: 2 further comprises an amino acid other than N at position 36, which increases the alkaline stability of the N-intein protein variant compared to the alkaline stability of the native N-intein of SEQ ID NO: 1. Preferably, the amino acid that increases alkaline stability is an amino acid that is less sensitive to deamidation than asparagine (N). The amino acid sequence of SEQ ID NO: 2 is as follows:

[0115] ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLEDGSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV (SEQ ID NO: 2)

[0116] in

[0117] X in positions 20, 35, 70, 73 and 95 is each independently selected from K, R or A;

[0118] X in position 28 is C, A or S;

[0119] X in position 36 is N, H or Q;

[0120] X in position 25 is N or R;

[0121] X in position 59 is D or C;

[0122] X in position 80 is E or Q; and

[0123] X in position 90 is Q, R or K.

[0124] Preferred embodiments of the N-intein according to the present invention are selected from the group of N-intein variants referred to herein as A48, B22, B72 and A41, wherein:

[0125] A48 has the sequence of SEQ ID NO: 2, wherein:

[0126] X in positions 20, 35, 70, 73 and 95 is R;

[0127] The X in position 28 is A;

[0128] X in position 36 is H;

[0129] X in position 25 is N;

[0130] The X in position 59 is D;

[0131] X in position 80 is E; and

[0132] The X in position 90 is Q;

[0133] B22 has the sequence of SEQ ID NO: 2, wherein:

[0134] X in positions 20, 35, 70, 73 and 95 is A;

[0135] The X in position 28 is A;

[0136] X in position 36 is H;

[0137] X in position 25 is N;

[0138] The X in position 59 is D;

[0139] X in position 80 is E; and

[0140] The X in position 90 is Q;

[0141] B72 has the sequence of SEQ ID NO: 2, wherein:

[0142] X in positions 20, 35, 70, 73 and 95 is K;

[0143] X in position 28 is C;

[0144] X in position 36 is H;

[0145] X in position 25 is N;

[0146] The X in position 59 is D;

[0147] X in position 80 is E; and

[0148] The X in position 90 is Q

[0149] A40 has the sequence of SEQ ID NO: 2, wherein:

[0150] X in positions 20, 35, 70, 73 and 95 is R;

[0151] The X in position 28 is A;

[0152] X in position 36 is N;

[0153] X in position 25 is N;

[0154] The X in position 59 is D;

[0155] X in position 80 is E; and

[0156] The X in position 90 is Q.

[0157] A41 has the sequence of SEQ ID NO: 2, wherein:

[0158] X in positions 20, 35, 70, 73 and 95 is K;

[0159] The X in position 28 is A;

[0160] X in position 36 is N;

[0161] X in position 25 is N;

[0162] The X in position 59 is D;

[0163] X in position 80 is E; and

[0164] The X in position 90 is Q;

[0165] Comparative ligand A53 has the sequence of SEQ ID NO: 2, wherein:

[0166] X in positions 20, 35, 70, 73 and 95 is K;

[0167] X in position 28 is C;

[0168] X in position 36 is N;

[0169] X in position 25 is N;

[0170] The X in position 59 is D;

[0171] X in position 80 is E; and

[0172] The X in position 90 is Q.

[0173] The N-intein of the present invention can be coupled to a solid phase such as a membrane, fiber, particle, 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.

[0174] In this case, the solid phase can be formed by one or more polymer nanofiber substrates such as electrostatic spinning 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 nanofiber is not particularly limited. The polymer nanofiber can be a monofilament nanofiber appropriately, and can, for example, have a circular, elliptical or substantially circular / elliptical cross section. Generally, one or more polymer nanofibers are provided in the form of one or more nonwoven sheets, each of which comprises one or more polymer nanofibers. The nonwoven sheet comprising 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 so that one or more nanofibers take a specific pattern. The nonwoven sheet generally has an area density of 1 to 40 g / m2. The nonwoven sheet generally has a thickness of 5 to 120 μm. The polymer should be suitable for use as a chromatography medium, i.e., a polymer of 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.

[0175] The N-intein according to the invention can be immobilized to a solid support to a very high degree, with 0.2 - 2 µmole / ml N-intein coupled per ml of resin (swollen gel).

[0176] The N-intein according to the invention may be coupled to the solid phase via a Lys tail at the C-terminus, said Lys tail comprising one or more, for example at least two Lys. Alternatively, the N-intein is coupled to the solid phase via a Cys tail at the C-terminus.

[0177] C-intein protein variants

[0178] Preferably, the present invention also provides a C-intein comprising the following sequence SEQ ID NO 3:

[0179] VKIVSRKSLGVQNVYDIGVEKDHNFLLANGLIASN (SEQ ID NO: 3)

[0180] or a sequence that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and preferably a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0181] It will be appreciated that the selection of the N-intein and 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 intein or consensus split intein sequences, as it has been found that the affinity of the N fragment for different C fragments (e.g., Npu N fragment or variants thereof for Ssp C fragment or variants thereof) still maintains sufficient binding affinity for use in the disclosed methods.

[0182] Vectors comprising the intein variants of the present invention

[0183] In a third aspect, the present invention relates to a vector comprising a C-intein of SEQ ID NO: 3 above and a gene encoding a protein of interest (POI). Also disclosed herein are vectors comprising nucleic acids encoding a C-terminal intein segment, and cell lines comprising the vectors. As used herein, a plasmid or viral vector is an agent that transports the disclosed nucleic acids (e.g., those encoding the C-terminal intein segment and the peptide of interest) into a cell without degradation, and includes a promoter that produces gene expression in a cell 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 able to carry larger genetic payloads, i.e., transgenic or marker genes, and are therefore commonly used vectors. However, they are not as useful in non-proliferating cells. Adenoviral vectors are relatively stable and easy to handle, have high titers, and can be delivered in aerosol formulations, 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.

[0184] Split intein system

[0185] Preferably, the present invention provides a split intein system for affinity purification of a protein of interest (POI), comprising an N-intein and a C-intein as described above.

[0186] Preferably, the N-intein comprises an N36H mutation for increased alkaline stability.

[0187] Preferably, the N-intein is attached to the solid phase and the C-intein is co-expressed with the POI and used as a tag for 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.

[0188] The alkaline stability of the N-intein ligand in the split intein system according to the invention enables regeneration under alkaline conditions such as 0.05-0.5 M NaOH after cleavage of the POI from the solid phase. The solid phase can be regenerated up to 100 times.

[0189] In one embodiment, the C-intein and an additional tag are co-expressed with the POI. The additional tag may be any conventional chromatography tag, such as an IEX tag or an affinity tag.

[0190] Method for purifying target protein (POI)

[0191] 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, comprising binding a C-intein and an N-intein at a neutral pH, e.g. 6-8, and in the presence of divalent cations (which impair spontaneous cleavage); washing the solid phase in the presence of divalent cations; 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, e.g. 0.5 M NaOH.

[0192] This protocol is suitable for proteins that are not sensitive to Zn. The advantage is that it allows long contact time with the resin and the addition of large sample volumes. Sample loading can be performed for a long time, for example up to 1.5 hours.

[0193] According to the present invention, a POI yield of more than 30%, preferably 50%, most preferably more than 80% is obtained in less than 4 hours of cutting.

[0194] The present invention enables high ligand densities when the N-intein is immobilized to a solid phase. 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 C-intein bound POI / ml resin settled.

[0195] Affinity tags

[0196] The present invention also relates to a method for purifying a protein of interest (POI), comprising the steps of: 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 the POI and the C-intein; binding the C-intein to an N-intein attached to a solid phase at neutral pH and cleaving 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 double tag: increase purity (making dual affinity purification possible), solubility, detectability.

[0197] Affinity tags can be peptides or protein sequences cloned in frame with protein coding sequences that change the behavior of proteins. Affinity tags can be attached to the N or C termini of proteins, which can be used in methods for purifying proteins from cells. Cells expressing peptides containing affinity tags can be expressed in supernatants / cell culture media together with signal sequences. Cells expressing peptides containing affinity tags can also be precipitated, lysed, and cell lysates applied to columns, resins or other solid supports, which display ligands for affinity tags. Affinity tags and any fused peptides are bound to solid supports, which can also be washed several times with buffer to eliminate unbound (contaminated) proteins. If attached to affinity tags, the target protein can be eluted from the solid support via buffer, which causes the affinity tag to dissociate from the ligand, resulting in purified protein, or soluble proteases can be used to cut from the bound affinity tags. As disclosed herein, affinity tags are cut by the self-cleavage mechanism of the C-intein segment in the active intein complex.

[0198] Examples of affinity include, but are not limited to, maltose binding protein, which can bind to immobilized maltose to facilitate purification of fusion target proteins; 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 metals; FLAG octapeptide, which can bind to immobilized anti-FLAG antibodies.

[0199] Affinity tags can also be used to facilitate purification of target proteins using the disclosed modified peptides by a variety of methods including, but not limited to, selective precipitation, ion exchange chromatography, binding to precipitating ligands, dialysis (by altering the size and / or charge of the target protein), and other highly selective separation methods.

[0200] In some aspects, such affinity tags can be used, which do not actually bind to the ligand, but selectively precipitate or act as a ligand for the corresponding binding domain of the fixed. 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 the fusion peptide to be purified by centrifugation. Another example is an antibody Fc domain, which acts as a ligand for a fixed protein A or protein G binding domain.

[0201] Target protein

[0202] Target proteins for all protocols are: any recombinant protein, especially proteins requiring a native or near-native N-terminal sequence, such as therapeutic protein candidates, biologics, antibody fragments, antibody mimetics, protein scaffolds, enzymes, recombinant proteins or peptides, such as growth factors, cytokines, chemokines, hormones, antigen (viral, bacterial, yeast, mammalian) production, vaccine production, cell surface receptors, fusion proteins.

[0203] The invention will now be described more precisely with reference to some non-limiting examples and to the accompanying drawings. Example

[0204] Experiment 1: Alkaline stability of the N-intein ligands of the present invention

[0205] The N-intein ligands A40, A41 and A48 according to the present invention were immobilized to a Biacore™ CM5 sensor chip (Cytiva, Sweden) in an amount sufficient to give an immobilization level of about 450 response units (RU) or more. To track the relative binding capacity of the C-intein-tagged POI to the immobilization surface, 20 μg / ml of C-intein (SEQ ID NO: 3)-tagged green fluorescent protein (GFP) was flowed over the chip for 1 minute and the signal intensity was recorded. The surface was then cleaned in situ (CIP) by rinsing with 100 mM NaOH, 4 M guanidine-HCl for 10 minutes at room temperature 22 ± 3°C. This was repeated for 50 cycles and the alkaline stability of the immobilized ligand was tracked after each cycle as the relative loss of binding capacity (signal intensity) relative to the C-intein-tagged GFP.

[0206] Results are shown in Figure 1 In the present invention, ligand A48 (with N36H mutation) is shown to have improved alkaline stability compared to ligands A41 and A40. Alkaline stability is further improved compared to the native sequence. In addition, the N36H mutation significantly improves alkaline stability compared to the wild-type Npu N-intein sequence (A52 with C1A mutation compared to SEQ ID NO: 1).

[0207] After 50 CIP cycles, the relative remaining binding capacity (%) was 55% for A40 and A41 and 69% for A48. The alkaline stability using 0.5 M NaOH is shown in Figure 5 middle.

[0208] Figure 5 Results for A40 and A48 during 20 cycles are shown. Relative remaining binding capacity (%)

[0209] CIP: 2 min. 100 mM NaOH, 4 M Gdn-HCl, then 2 min. 0.5 M NaOH.

[0210] Experiment 2: Alkaline stability of the N-intein ligands of the present invention

[0211] Purified N-intein ligands A53, B72, B22 and A48 were immobilized to a Biacore™ CM5 sensor chip (Cytiva, Sweden) in amounts sufficient to give immobilization levels of approximately 450 response units (RU) or greater. To track the relative binding capacity of the non-cleavable C-intein-tagged POI to the immobilization surface, 20 µg / ml of non-cleavable 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) by rinsing with 100 mM NaOH, 4 M guanidine-HCl for 10 minutes at room temperature 22 ± 3°C. This was repeated for 50 cycles and the alkaline stability of the immobilized ligand was tracked after each cycle as the relative loss of non-cleavable C-intein-tagged IL-1b binding capacity (signal intensity).

[0212] Results are shown in Figure 2 In the figure, all three ligands (A48, B22 and B72) with N36H mutations have improved alkaline stability compared to ligand A53. After 50 CIP cycles, the relative remaining binding capacity (%) of A53 is only 20%, while it is 28% for B72, 30% for B22, and 35% for A48.

[0213] Experiment 3: Immobilization of N-intein ligand A48 to agarose gel resin

[0214] 5 milliliters of epoxy-activated cross-linked activated gel resins are added to a polypropylene test tube. 2.7 milliliters of N-intein ligand A48 corresponding to 135 milligrams of Lys tails at the C-terminal end are added to the tube, followed by 1.3 milliliters of phosphate buffer (pH 12.1), so that the agarose resin slurry is adjusted to about 50%, then 2 grams of sodium sulfate are added. The pH of the resulting reaction mixture is adjusted to 11.5. And the reaction mixture is heated on an oscillating table until 33 ° C, and kept vibrating for 4 hours at 33 ° C. The slurry is then transferred to a glass filter, and washed 3 times with 10 milliliters of distilled water. After washing, the gel is transferred to a three-necked round-bottom flask (RBF), and 5 milliliters of Tris buffer (pH 8.6) and 375 microliters of thioglycerol are added. The reaction mixture is placed on an oscillating table for 2 hours at 45 ° C. After the reaction, the slurry is 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 alkaline / acid wash was repeated 2 more times for a total of 18 washes in this step. The gel resin was then washed 10 times with 5 ml of distilled water. The washed and drained gel was kept in 20% ethanol in a refrigerator prior to analysis.

[0215] The dry weight of the gel resin was determined by measuring the weight of 1 ml of gel. In the sample preparation, 2 grams of drained gel resin were thoroughly mixed with 2 grams of water to give a resin slurry of about 50%, which was then added to a 1 mL Teflon cube. A vacuum was then applied to drain the gel in the cube and thus obtain 1 mL of gel. The gel was transferred to a dry weight balance. The weight was determined after 35 minutes, with the drying temperature set at 105°C.

[0216] Amino acid analysis was measured after dry weight determination. From the corresponding dry weight and information on protein size and primary amino sequence, the ligand density can be derived in mg / mL gel resin.

[0217] The result for the coupled agarose resin was 90.6 mg / ml dry weight with a ligand content of 18.4 mg / ml, which corresponds to 1.38 umole / ml.

[0218] Experiment 4: Static Binding Capacity as a Function of Ligand Density

[0219] The proposed volumetric method presented herein can measure the binding capacity of a resin in a test tube.

[0220] Reaction Settings

[0221] Briefly, prototype resin with immobilized A48 ligand at various ligand densities and dual-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 2.5% resin slurry was added to the ILLUSTRA™ micro spin column followed by 150 µL of diluted A43 (SEQ ID NO: 5). The reaction was allowed to incubate at 22°C with shaking at 1450 rpm for fixed time points of 2 hours and then centrifuged at 3000 rcf for 1 minute.

[0222] SDS-PAGE

[0223] The centrifuged samples (containing cleaved and unbound uncleaved proteins) were mixed 1:1 with 2x SDS-PAGE reducing sample buffer, boiled at 95°C for 5 minutes, and then subjected to SDS-PAGE (18µL loaded). C-intein-tagged test protein A43 (SEQ ID NO: 5) standards (typically a five-point standard of 18.75-300µg / mL) were added to enable concentration calculation based on optical density volume. The gel was Coomassie stained for 60 minutes (~100mL / gel) followed by destaining at room temperature with gentle agitation for 120-180 minutes (until the background is completely transparent). Densitometric quantification of uncleaved / unbound and cleaved test proteins was performed using IQTL software. The optical density raw data was then exported to Microsoft Excel.

[0224] SBC calculation

[0225] Since the test protein input in the reaction is known, we can indirectly calculate the static binding capacity (SBC) using the following equation:

[0226]

[0227] 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.

[0228] Experiment 5: Purification of Elongation Factor G with and without Zn Protocol

[0229] In this example, a resin prototype with immobilized ligand A48 was used to purify the thermophilic anaerobic bacillus from Tengchong ( Thermoanaerobacter tengcongensis ) was expressed in Escherichia coli strain BL21 (DE3) with a C-intein (SEQ ID NO3) tag.

[0230] The frozen cell pellet after fermentation harvest was thawed and resuspended with extraction buffer (20 mMTris-HCl, pH 8.0) by magnetic agitation. DNase I (bovine pancreas) and 1 mM MgSO4 were added, followed by lysozyme (egg). After stirring at room temperature for 30 minutes, the resuspended and lysozyme-treated cell suspension 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.

[0231] Purification using the Zn-free protocol was done on an ÄKTA™ Avant system at 2 ml / min and then 1 ml / min during sample loading and washing. A 1 ml HiTrap™ column containing immobilized A48 ligand was used. 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.

[0232] After 4 hours of incubation on the HiTrap™ column, 17.8 mg of pure, tag-free EfG eluted. According to mass spectrometry analysis, the mass difference between the eluted protein and the CIP protein was equal to the mass of the C-intein tag. The purity according to SDS-PAGE was as high as SEC analysis on Superdex™ 200 Increase. The total protein amount was calculated based on the theoretical UV absorption coefficient at 280 nm and the UV signal of the diluted elution and CIP fractions.

[0233] The scheme that comprises Zn ion in use balance buffer and clarification sample repeats purification.Final Zn concentration is 1.6mM.Flow velocity is reduced to 0.5 ml / minute during sample application, and is then increased to 1 ml / minute during washing and elution.Complete washing and elution with 50 mM Tris-HCl, 20 mM imidazole buffer pH 7.5.Only collect an elution peak in this purification, and this incubation after post washing is after 4 hours.

[0234] After 4 hours of incubation on the HiTrap™ column, 16.6 mg of pure, tag-free EfG eluted. The purity was 92% according to SEC analysis on Superdex™ 200 Increase. 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.

[0235] Experiment 6: Purification of IL-1β

[0236] A 1 ml HiTrap™ column containing an immobilized A48 ligand was used to purify the target protein IL-1β (SEQ ID NO: 5) tagged with a C-intein, which was expressed intracellularly in E. coli BL21 (DE3) and lysed by sonication. Soluble protein was harvested by centrifugation and loaded onto a 1 mL HiTrap™ column immobilized with an A48 ligand. During sample loading and washing, a Zn-free protocol (as in Experiment 4) was used on the ÄKTA™ Avant system at 4 ml / min (600 cm / hr linear flow rate). The run was then paused for 4 hours, and then the flow was restarted at 1 mL / min to elute the cleaved protein (4-hour cleavage fraction). The run was then paused again for another 12 hours, and then the flow was started at 1 mL / min to elute the protein that was not cleaved after 4 hours. The combination and balance of washing and elution were performed with a single buffer. The chromatogram from the purification is shown in Figure 4A The raw material, flow-through, wash fractions, 4-hour and 16-hour elution fractions were subjected to SDS-PAGE and Coomassie staining, and subsequent analysis using IQTL software ( Figure 4B ).

[0237] After 4 hours of incubation on the HiTrap™ column, 9.4 mg of cleaved IL-1β was eluted, followed by another 1.1 mg after 16 hours. According to SDS-PAGE analysis, the purity was 99.5 (4 hours) and 99.8% (16 hours). The total protein amount was calculated based on the theoretical UV absorption coefficient of the cleaved protein at 280 nm.

[0238] Experiment 7: Purification of the receptor binding domain of SARS-COV-2

[0239] The receptor binding domain (RBD) of SARS-COV-2 NCBI tagged with a C-intein was expressed in ExpiHEK cells and secreted into the cell culture medium. Using the ÄKTA™ Avant FPLC system, approximately 210 mL of supernatant was loaded onto a 1 mL HiTrap column with an immobilized A48 ligand, and no salt or other additives were added to the cell culture supernatant. Sample application and washing were performed at 4 mL / min (loading time ~52.5 minutes (600 cm / hour linear flow rate)), followed by 6 column volumes of washing, followed by a pause / hold step for a total of 4 hours. The elution phase was performed at 1 mL / min. The column was left to rest for another 68 hours, 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.

[0240] Theoretical absorbance 0.1% coefficient was used to determine protein concentration and yield within Unicorn™ 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 is ~25 kDa, while experimental SDS-PAGE analysis indicated a molecular weight of 33 kDa, which can be explained by two glycosylations and was also determined by mass spectrometry analysis.

[0241] The CCT-RBD protein has the following sequence:

[0242] METDTLLLWVLLLWVPGSTGVKIVSRKSLGVQNVYDIGVEKDHNFLLANGLIASNRVQPTESIVRFPN ITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEV RQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV EGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFHHHHHH (SEQ ID NO:4)

[0243] Signal sequence - bold underlined.

[0244] CCT-Tag-Dot Underline.

[0245] The RBD domain is double underlined.

[0246] His tag - dotted underline

[0247] Purity results from the cleaved proteins are found in Table 3.

[0248] Table 3

[0249] Elution Cutting time purity Target protein yield 4 hours 4 hours 96.5% 4.9 mg 72 hours 72 hours 99.4% 9.2 mg

[0250] Experiment 8: Tandem tagging and affinity purification on two columns

[0251] E. coli BL21 (DE3) was transformed with the A43 expression plasmid TwinStrep™ and IL-1b tagged with a C-intein (SEQ ID NO 3) and plated on agar plates containing 50 µg / ml kanamycin. The next day, a single colony was picked and grown to OD600 0.6 in 5 ml Luria-Bertani (LB) broth. The culture was transferred to 200 ml LB broth containing the same antibiotics and grown at 37°C until OD600 was 0.6. Protein expression was induced at 22°C for 16 hours by adding isopropyl bD-1-thiogalactopyranoside (IPTG, 0.5 mM). After expression, cells were harvested by centrifugation at 4,000 xg for 15 minutes and stored at -80°C until use.

[0252] For purification, cell pellets were resuspended in 10 ml per gram wet weight in buffer A1 (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) and disrupted by sonication (Sonics Vibracell, microtip, 30% amplitude, 2 sec on, 4 sec off, 3 min total).

[0253] After centrifugation at 40,000 xg for 20 minutes at 4°C, the supernatant containing the soluble fraction was collected and passed through a 5 ml HiTrap™ column, Streptactin™ XT (GE Healthcare, Sweden). The column was washed with the same buffer A1 until the UV absorbance at 280 nm was less than 20 mAU. The bound C-intein-tagged IL-1b was eluted in buffer B1 (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 50 mM biotin, pH 8.0) and collected.

[0254] The purified protein was immediately applied to a 1 ml HiTrap™ column packed with resin containing immobilized N-intein ligand A48 without added inhibitor ZnCl 2. The cleaved, untagged IL-1b was collected in the flow-through.

[0255] MSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK GGGSGGGS VKIVSRKSLGVQNVYDIGVEKDHNFLLAN GLIASNAFVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLS CVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDF TMQFVSS AAA (SEQ ID NO: 5)

[0256] TwinStrep - dot underline

[0257] CCT-Bold Underline

[0258] IL1b (test protein) - underlined

[0259] The patents and scientific literature mentioned herein 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 incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated by reference.

[0260] Although the present invention has been particularly shown and described with reference to its preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims. It should also be understood that the embodiments described herein are not mutually exclusive and that features from the various embodiments may be combined in whole or in part according to the present invention.

Claims

1. An N-intein variant consisting of the following sequence: ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLED GSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV,where, X in positions 20, 35, 70, 73 and 95 is R; The X in position 28 is A; X in position 36 is H; X in position 25 is N; The X in position 59 is D; X in position 80 is E; and The X in position 90 is Q; And compared with SEQ ID NO:1, the alkaline stability of the N-intein variant is increased.

2. An N-intein variant consisting of the following sequence: ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLED GSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV,where, X in positions 20, 35, 70, 73 and 95 is A; The X in position 28 is A; X in position 36 is H; X in position 25 is N; The X in position 59 is D; X in position 80 is E; and The X in position 90 is Q; And compared with SEQ ID NO:1, the alkaline stability of the N-intein variant is increased.

3. An N-intein variant consisting of the following sequence: ALSYDTEILTVEYGFLPIGXIVEEXIEXTVYSVDXXGFVYTQPIAQWHNRGEQEVFEYXLED GSIIRATXDHXFMTTDGXMLPIDEIFEXGLDLXQV,where, X in positions 20, 35, 70, 73 and 95 is K; X in position 28 is C; X in position 36 is H; X in position 25 is N; The X in position 59 is D; X in position 80 is E; and The X in position 90 is Q; And compared with SEQ ID NO:1, the alkaline stability of the N-intein variant is increased.

4. The N-intein variant according to any one of claims 1 to 3, which is coupled to a solid phase.

5. The N-intein variant according to any one of claims 1 to 3, which is coupled to a membrane, a fiber, a particle, a bead or a chip.

6. The N-intein variant according to claim 4, wherein the solid phase is a chromatography resin of natural or synthetic origin.

7. The N-intein variant of claim 4, wherein the solid phase is a chromatography resin.

8. The N-intein variant of claim 4, wherein the solid phase is a natural or synthetic chromatography resin.

9. The N-intein variant of claim 7, wherein the solid phase is a polysaccharide.

10. The N-intein variant according to claim 7, wherein the solid phase is agarose.

11. The N-intein variant according to claim 6, wherein the solid phase is provided with embedded magnetic particles.

12. The N-intein variant of claim 4, wherein the solid phase is a non-diffusion limiting resin / fiber material.

13. The N-intein variant according to claim 4, wherein the N-intein variant is coupled to a solid phase via a Lys tail comprising one or more Lys at the C-terminus.

14. The N-intein variant according to claim 4, wherein the N-intein variant is coupled to a solid phase via a Cys tail on the C-terminus.

15. The N-intein variant according to claim 4, wherein 0.2-2 μmol of the N-intein variant is coupled per ml of solid phase.

16. The N-intein variant according to claim 7, wherein 0.2-2 μmol of the N-intein variant is coupled per ml of chromatography resin.

17. The N-intein variant according to any one of claims 1-3, wherein the N-intein variant is stable under alkaline conditions corresponding to 0.05M-0.5M NaOH.

18. The N-intein variant according to any one of claims 1-3, wherein the N-intein variant is stable under alkaline conditions corresponding to 0.1-0.5 M NaOH.

19. A split intein system for affinity purification of a target protein POI, comprising the N-intein variant according to any one of claims 1 to 18; and a C-intein. 20 . The split intein system according to claim 19 , comprising the N-intein variant and the C-intein sequence shown in SEQ ID NO:

3.

21. The split intein system of claim 19 or 20, wherein the C-intein and additional tag are co-expressed with the POI.

22. The split intein system of claim 19, wherein the N-intein variant is immobilized to a solid phase.

23. Use of the split intein system according to any one of claims 19 to 22 for affinity purification of a protein of interest POI, wherein the C-intein is co-expressed with the POI, the N-intein variant is immobilized to a solid phase, and the solid phase is regenerated under alkaline conditions of 0.05-0.5 M NaOH after the POI is cleaved from the solid phase.

24. Use according to claim 23, wherein the solid phase is regenerated for up to 100 cycles.

25. The use according to claim 23, wherein the solid phase is regenerated for up to 50 cycles.

26. A chromatography column comprising a chromatography resin comprising one or more N-intein variants, wherein the N-intein variant is as described in any one of claims 1-18.

27. A method for purifying a C-intein-tagged protein POI using the split intein system according to any one of claims 19 to 22, wherein the N-intein variant is immobilized on a solid phase; the method comprises contacting the C-intein and the N-intein variant at neutral pH and in the presence of divalent cations; washing the solid phase in the presence of divalent cations; Adding a chelating agent to allow spontaneous cleavage between the C-intein and the POI; The tag-free POI is collected; and the solid phase is regenerated under alkaline conditions, wherein the neutral pH is 6-8, or the alkaline conditions are 0.05-0.5 M NaOH.

28. A method for purifying a C-intein-tagged protein POI using the split intein system according to any one of claims 19-22, wherein the N-intein variant is immobilized on a solid phase; the method comprises contacting the C-intein and the N-intein variant at a neutral pH; 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, wherein the neutral pH is 6-8, the C-intein and the N-intein variant are contacted at a high flow rate, or the alkaline conditions are 0.05-0.5M NaOH.

29. A method for purifying a protein of interest POI, which uses the split intein system according to any one of claims 19-22 and comprises the following steps: co-expressing the POI with a C-intein according to SEQ ID NO 3 and an additional tag; allowing the additional tag to bind to its binding partner on a first solid phase; cleaving the POI and the C-intein; allowing the C-intein to bind to an N-intein variant attached to a second solid phase at a neutral pH, and cleaving the bound C-intein and N-intein variant from the POI; and regenerating the second solid phase under alkaline conditions, wherein the alkaline conditions are 0.05-0.5 M NaOH.

30. The method of claim 29, wherein the additional tag is an affinity tag or ion exchange.

31. The method of any one of claims 27-30, wherein the alkaline conditions are combined with a chaotropic agent and the solid phase can be regenerated up to 100 times.

32. The method of claim 31 , wherein the basic conditions are combined with guanidine or urea.

33. The method of any one of claims 27-30, wherein the POI is: a native N-terminal sequence of a protein.

34. The method of any one of claims 27-30, wherein the N-intein variant is immobilized to a chromatography resin and has a static binding capacity of 0.2-2 μmol / ml.

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