Modified inteins and methods of use

Abstract

Disclosed herein are modified inteins and methods of using them. These modified inteins are mutants of ΔI-CM which possess desired properties such as reduced premature cleavage.

Description

MODIFIED INTEINS AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONSThis application claims benefit of U.S. Provisional Application No. 63 / 586,725, filed September 29, 2023, incorporated herein by reference in its entirety.GOVERNMENT SUPPORT CLAUSEThis invention was made with government support under grant / contract no. 1158394 awarded by The National Science Foundation and grant / contract no. W91 INF-11-1-0118 awarded by the Army Research, Development and Engineering Command. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTINGThe sequence listing submitted on September 27, 2024, as an .XML file entitled “103361- 529WO1_ST26,” created on September 25, 2024, and having a file size of 10,372 bytes, is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUNDInteins (Intervening Proteins) are a class of protein element that was discovered in the early 1990’s, and modified inteins have found many uses in biotechnology. Natural intein genes are found as DNA sequence insertions in a variety of host proteins throughout nature. Once the intein and host protein are translated into one long precursor protein, the intein removes itself in a process now known as protein splicing. The ability to rearrange peptide bonds during the splicing reaction has made inteins highly valuable in biotechnology applications that include protein engineering and protein purification.The purification application of inteins includes applications where an intein is modified to cleave off one of the flanking segments rather than splice them together, effectively enabling the development of self-removing affinity tags[1]. In practice, the intein is flanked on one side by a conventional affinity tag, while the desired target protein is placed at the other end of the intein (Figure 1). The three-part precursor protein is expressed and purified using the conventional affinity tag, at which point the intein is induced to cleave the target through some change in buffer (typically pH or thiol addition) or temperature conditions. This application of inteins wascommercialized in the late 1990’s, when it generated significant interest as an efficient and inexpensive method for generating tagless target proteins via an affinity separation. During this time, the AI-CM intein was developed, which exhibited accelerated C-terminal cleaving under mildly acidic pH. Thus, proteins could be expressed and then purified at high pH (pH 8.5), but then cleaved from their tags a lower pH (pH 6.2), providing a simple platform for purifying a variety of tagless proteins (see Figure IB).Intein-based self-cleavable tags have now been successfully utilized to purify a wide range of traceless, scarless proteins[2'5]. This purification technology can achieve high degrees of product purity, which is characteristic of affinity capture, but with the added benefit of recovering a tagless protein from a single column step (without modification to the amino acid sequence). The advantages of intein-mediated self-cleaving purification have sparked great commercial interest among many companies.The AI-CM intein with pH-inducible rapid C-terminal cleavage has shown great promise as a protein purification platform, especially for proteins expressed in microbial cells such as E. coli (U.S. Patent 6,933,362). However, a significant obstacle to the commercial deployment of this intein has been premature cleavage of the tag at moderate temperature and pH, leading to a loss of the tag before the protein can be purified. Specifically, due to the temperature sensitivity of the AI-CM intein, the cleaving activity of the intein can be easily activated during protein expression under typical culture conditions, typically 37°C, resulting in pre-cleavage of the tag during expression of the tagged target protein. As a consequence, the protein of interest cleaves from the intein and tag prior to binding to the affinity column, thereby making it impossible to achieve purification of the target[2](Figure 2).The difficulty of controlling the intein self-cleavage reaction, combined with the incompatibility of bacterial culture with the production of disulfide-bonded and glycosylated proteins, has prevented the use of intein tags for purification at scale in industrially relevant hosts. Attempts to improve / control the kinetics of contiguous intein splicing reactions have been largely successful[6'10], but engineering control over the C-terminal self-cleavage reaction has been far more problematic. Typical evolutionary methods rely on genetic selections of active inteins at given intracellular conditions[6], which cannot be modified significantly, and thus isolation of a truly pH / temperature controlled self-cleaving intein for bioseparations has remained an elusive target due to the limited condition space that may be successfully explored.Lowering the expression temperature can alleviate pre-cleavage to some extent, and therefore, the AI-CM self-cleaving purification tag system is still an extremely powerful tool for recombinant protein purification with E. coli as the expression host. However, there are somesignificant limitations to the types of proteins that can be produced in bacterial culture. In particular, protein products that contain disulfide bonds for stability or that require post- translational modifications for activity cannot be readily produced using E. coli. In these cases, recombinant protein expression must be carried out in eukaryotic host cells, such as yeast, insect, or mammalian cells. The most commonly used eukaryotic expression host in the biotechnology industry is the Chinese Hamster Ovary (CHO) cell, which is an anchorage-independent mammalian expression system.[21>22]For self-cleaving intein tag technology to become a universal platform for recombinant protein purification it must be compatible with any expression host, including CHO. The major impediment to implementation of self-cleaving intein tags in mammalian expression systems such as CHO is premature intein cleavage during protein expression. If the intein cleaves during expression (before the purification protocol), then the cleaved target protein will not be captured during the initial purification step, which leads directly to product loss and makes the process untenable. The problem of premature cleavage is illustrated in Figure 2, which is a plot of the cleaving rate of the AI-CM intein as a function of both temperature and pH. The intein system works well in E. coli because the intracellular pH is slightly above 7.0 and the expression temperature can be lowered to reduce the amount of premature cleavage. However, mammalian expression systems are not compatible with reduced expression temperature. The rate of the cleaving reaction of the AI-CM intein at the conditions of CHO cell culture (37°C, pH 7.2) is about 10-fold higher than the rate for E. coli expression (Figure 2). In addition, protein expression in mammalian cells must be carried out for much longer than in E. coli. The combination of these factors leads to a significant amount (close to 100%) of premature cleavage of the intein during mammalian cell expression, precluding the use of intein-based purifications in mammalian cells.Overall, pre-cleavage is a common issue with contiguous intein-based self-cleaving tags. Thus, scientists and engineers have been attempting to address this issue and have tried many potential solutions with varying degrees of success. There are two main approaches that have emerged:One approach to avoid pre-cleavage is to use split inteins instead of contiguous inteins. This method takes advantage of the fact that split inteins do not have cleaving activity before they are assembled. A successful example is the engineered Npu DnaE split intein purification system developed by Wood and Shi at Ohio State University, which was granted patents recently. (US Patent No. 10,066,027 and US Patent No. 10,669,351). Currently, Protein Capture Science is producing related products ( / CapTag™) for protein purification. However, the instability of splitintein segments has been identified as an issue that can cause misfolding of tagged target proteins for artificially split inteins.Another approach focuses on the development of homologous recombination mutations in the contiguous intein to improve its application performance. Many of these mutations are designed by predicting the evolution direction of mutation based on the intein structure model. Recently, Lin et al. were able to decrease the pre-cleavage of ALCM intein by mutating the amino acid residues surrounding the H429 and H439 histidine residues. According to kinetic analyses, the engineered variants demonstrated a decrease in cleavage rates while still maintaining pH dependence.

[0023] However, since the understanding of the roles played by different amino acids within the intein during C-terminal cleavage is currently not well-established, this method has only resulted in mildly improved performance, but has not yet achieved the desired results.What is needed are modified inteins with reduced premature cleavage during expression in microbial cells, but which still exhibit reasonably fast cleavage under optimized conditions. The inteins disclosed here were identified using an evolutionary method based on yeast surface display, where the entire intein sequence was mutated and selected for optimal pH and temperature performance. The resulting intein mutants exhibit significantly reduced premature cleaving, but still highly controllable cleaving in response to pH changes, making them highly attractive for self-removing tag applications.SUMMARYA polypeptide comprising a functional intein with isolated C-terminal cleaving activity, wherein said intein is a variant of SEQ ID NO: 1, wherein said variant comprises reduced premature cleavage of the intein during expression in a host cell or cell-free expression system compared to SEQ ID NO: 1.Also disclosed herein is a polypeptide comprising SEQ ID NO: 2 or 3, or a functional variant thereof, wherein said functional variant exhibits reduced premature cleavage of the intein.Further disclosed is a method of producing a purified protein of interest, the method comprising providing the polypeptide comprising a functional intein, wherein said intein is a variant of SEQ ID NO: 1, under suitable conditions for self-cleavage of the intein, thereby producing a purified protein of interest.DESCRIPTION OF DRAWINGSFIGS. 1A-1B show schematics of intein-based purification schemes. (FIG. 1 A) Schematic diagram outlining intein-mediated affinity purification for a protein of interest. (FIG. IB) Steps of intein-mediated affinity purification include protein expression, high-pH protein capture via the tag, washing / decreasing the pH, and elution after cleavage low pH.FIG. 2 shows C-terminal cleavage kinetics of the AI-CM intein. The cleavage rate is presented for the AI-CM intein as a function of temperature and pH. Cleavage at mammalian cell culture conditions (circle on top line) is greater than lOx faster than cleavage during expression in E. coli (circle on bottom line), demonstrating the current limitations of intein-based purification schemes.FIGS. 3A-3B show non-mAb protein purification schemes. (FIG. 3A) The difficulty of developing a non-mAb protein purification platform arises from the lack of conserved structural and chemical properties. (FIG. 3B) Typical process workflow for developing a process for purifying a non-mAb, where an optimal scheme must be identified / optimized for each new molecule before adoption at commercial scale.FIG. 4 shows the half-life of intein cleavage of AI-CM and the 1-12 and 1-29 mutants under different conditions.FIGS. 5A-5C show cleavage test results of the AI-CM and the 1-12 and 1-29 inteins at pH 7.3 (FIG. 5 A) Precursor percentage plot of different intein at 37°C; (FIG. 5B) Precursor percentage plot of different intein at 20°C; (FIG. 5C) SDS-PAGEs of cleavage test at pH 7.3. Lanes: L: protein ladder; CL: clarified lysate; FW: flow-through wash; Oh: start of cleavage reaction timepoint zero, 0.5h-24h: timepoints 0.5-hour, 1 hour, 2 hours, 3.5 hours, 5 hours, and 24 hours after timepoint zero.FIGS. 6A-6C show cleavage test results of the AI-CM and the 1-12 and 1-29 inteins at pH 6.2 (FIG. 6A) cleavage efficiency of different intein at 20°C; (FIG. 6B) cleavage efficiency of different intein at 37°C; (FIG. 6C) SDS-PAGEs of cleavage test at pH 6.2. Lanes: L: protein ladder; CL: clarified lysate; FW: flow-through wash; Oh: start of cleavage reaction timepoint zero, 0.5h-24h: timepoints 0.5-hour, 1 hour, 2 hours, 3.5 hours, 5 hours, and 24 hours after timepoint zero.FIGS. 7A-7B show results of cleavage efficiency of ELP-Intein-GFP at pH 8.5 and pH 6.2 with the AI-CM and the 1-12 and 1-29 inteins. (FIG. 7A) cleavage efficiency of different inteins at 37°C (note: the precursor of AI-CM intein at pH 6.2 is not enough to detect after 5 hours, so the trendline elongated with the dashed line, the final cleavage efficiency was assumed to be higher than 90%); (FIG. 7B) SDS-PAGEs of cleavage efficiency of different intein at pH 6.2 and pH 8.5;Lanes: L: protein ladder; Oh: start of cleavage reaction timepoint zero, 0.5h-5h: timepoints 0.5- hour, 1 hour, 2 hours, 3.5 hours, 5 hours after timepoint zero.FIGS. 8A-8B show a statistical optimization of a purification process based on these inteins in combination with an ELP precipitation tag. (FIG. 8A) The effects test for product recovery and product purity; (FIG. 8B) The prediction profiler of self-cleaving ELP purification.FIG. 9 shows a schematic of the purification process of self-cleaving ELP tag.FIGS. 10A-10D show results of in vivo premature cleavage with the ALCM and the 1-12 and 1-29 inteins. (FIG. 10A) Percentage of intact ELP-Intein-GFP precursor; (FIG. 10B) Percentage of intact ELP-Intein-MBP precursor of different intein; (FIG. 10C) Percentage of intact ELP-Intein-P-gal precursor different intein; (FIG. 10D) SDS-PAGEs of pre-purified samples; Lanes: L: protein ladder; Cl : condition 1 (16°C, 24 hours); C2: condition 2 (37°C, 3 hours).FIGS. 11A-11F show SDS-PAGEs of ELP-Intein-sfGFP purification with the ALCM (FIG. 11B, FIG. HE) and the 1-12 (FIG. 11C, FIG. 1 IF) and 1-29 (FIG. 11 A, FIG. 11D) inteins; Lanes: L: protein ladder; WL: whole lysate; CL: clarified lysate; SUP: supernatant of first precipitation; WS: supernatant of second precipitation; TO: start of cleavage reaction timepoint zero, T5: timepoints 5 hours after TO; T18: timepoints 18 hours after TO; EL: elution sample (purified protein in supernatant).FIGS. 12A-12F show SDS-PAGEs of ELP-Intein-P-gal purification with the ALCM (FIG. 12B, FIG. 12E) and the 1-12 (FIG. 12C, FIG. 12F) and 1-29 (FIG. 12A, FIG. 12D) inteins; Lanes: L: protein ladder; WL: whole lysate; CL: clarified lysate; SUP: supernatant of first precipitation; WS: supernatant of second precipitation; TO: start of cleavage reaction timepoint zero, T5: timepoints 5 hours after TO; T18: timepoints 18 hours after TO; EL: elution sample (purified protein in supernatant).FIGS. 13A-13F show SDS-PAGEs of ELP-Intein-MBP purification with the ALCM (FIG. 13B, FIG. 13E) and the 1-12 (FIG. 13C, FIG. 13F) and 1-29 (FIG. 13 A, FIG. 13D) inteins; Lanes: L: protein ladder; WL: whole lysate; CL: clarified lysate; SUP: supernatant of first precipitation; WS: supernatant of second precipitation; TO: start of cleavage reaction timepoint zero, T5: timepoints 5 hours after TO; T18: timepoints 18 hours after TO; EL: elution sample (purified protein in supernatant).FIG. 14 shows alignment data for ALCM intein and the 1-12 and 1-29 mutants. The “consensus” and ALCM are SEQ ID NO: 1, 1-29 is SEQ ID NO: 2, and 1-12 is SEQ ID NO: 3.DETAILED DESCRIPTIONGeneral DefinitionsUnless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed 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.The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur."Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level.Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.By “reduce,” or “abrogate,” (used interchangeably) or other forms of the word, such as “reducing” or “reduction,” or “abrogating” or “abrogation” is meant lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.By “increase” or other forms of the word, such as “increasing,” is meant raising or elevating. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, chickens, ducks, geese, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.“Control” refers to a sample or standard used for comparison with an experimental sample. In some embodiments, the control is a sample obtained from a healthy subject (or a plurality of healthy subjects), such as a subject or subjects not expected or known to have a particular polymorphism. In additional embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample or plurality of such samples), or group of samples that represent baseline or normal values. A positive control can be an established standard that is indicative of a specific methylated nucleotide. In some embodiments a control nucleic acid is one that lacks a particular methylated nucleotide, and is used in assays for comparison with a test nucleic acid, to determine if the test nucleic acid includes the methylated nucleotide.“Detecting” is used herein to identify the existence, presence, or fact of something. General methods of detecting are known to the skilled artisan and may be supplemented with the protocols and reagents disclosed herein. For example, included herein are methods of detecting cleavage of an intein. Detection can include a physical readout, such as fluorescence output.An “isolated” biological component (such as a protein) has been substantially separated, produced apart from, or purified away from other biological components. Polypeptides which have been “isolated” include molecules purified by standard purification methods, as well as thosechemically synthesized. Isolated does not require absolute purity, and can include polypeptides that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% isolated.As used herein, the term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.“Peptide” as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A peptide is comprised of consecutive amino acids. The term “peptide” encompasses naturally occurring or synthetic molecules.In addition, as used herein, the term “peptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The peptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the peptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given peptide can have many types of modifications. Modifications include, without limitation, linkage of distinct domains or motifs, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, 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 a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to 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)).As used herein, “isolated peptide” or “purified peptide” is meant to mean a peptide (or a fragment thereof) that is substantially free from the materials with which the peptide is normally associated in nature, or from the materials with which the peptide is associated in an artificialexpression or production system, including but not limited to an expression host cell lysate, growth medium components, buffer components, cell culture supernatant, or components of a synthetic in vitro translation system. The peptides disclosed herein, or fragments thereof, can be obtained, for example, by extraction from a natural source (for example, a mammalian cell), by expression of a recombinant nucleic acid encoding the peptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the peptide. In addition, peptide fragments may be obtained by any of these methods, or by cleaving full length proteins and / or peptides.The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.The phrase “nucleic acid” as used herein 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 hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.As used herein, “isolated nucleic acid” or “purified nucleic acid” is meant to mean DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is 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 which exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR, restriction endonuclease digestion, or chemical or in vitro synthesis). It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences. The term “isolated nucleic acid” also refers to RNA, e.g., an mRNA molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components, for example, other types of RNA molecules or peptide molecules.The identity / similarity between two or more nucleic acid or protein sequences is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad.Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology (NCBI, National Library of Medicine, Building 38 A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1166=1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15=20*100=75). One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above.By “contacting” is meant placement in direct physical association, for example solid, liquid or gaseous forms. Contacting includes, for example, direct physical association of fully- and partially-solvated molecules.As used herein, “kit” means a collection of at least two components constituting the kit.Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising aninstruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.As used herein, the terms “target protein”, “protein of interest” and “therapeutic agent” include any synthetic or naturally occurring protein or peptide. In the context of this invention, a “protein of interest” is a protein that is to be purified using intein purification technology by an end user in a laboratory or manufacturing setting. This definition would apply to any protein or peptide requiring purification for study or other research applications. The term additionally encompasses those compounds traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, and the like. Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14th edition), the Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (1st edition), and they include, without limitation, medicaments; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.As used herein, “variant” refers to a molecule that retains a functional activity that is the same or substantially similar to that of the original sequence. The variant may be from the same or different species or be a synthetic sequence based on a natural or prior molecule. Moreover, as used herein, “variant” refers to a molecule having a structure attained 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 that based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities compared to the parentmolecule. For example, substituting specific amino acids in a given peptide can yield a variant peptide with similar activity to the parent.“Intein” refers to an in-frame intervening sequence in a protein as described by Perler (Perler, Davis et al. 1994). An intein can catalyze its own excision from the protein through a post- translational protein splicing process to yield the free intein and a mature protein. An intein can also catalyze the cleavage of the intein-extein bond at either the intein N-terminus, or the intein C-terminus, or both of the intein-extein termini. As used herein, “intein” encompasses mini-inteins and modified or mutated inteins.The term “Sensitivity Enhancing Motif’ (SEM) refers to an amino acid sequence of three or more residues expressed in fusion with the intein, which renders the cleaving activity of an intein complex highly sensitive to extrinsic conditions as described previously in U.S. Patent 10,066,027, herein incorporated by reference in its entirety. The SEM is a constitutive element of an intein.The term “immobilization moiety” refers to one or more amino acid residues (e.g. Cys), expressed in fusion with the intein, which allows for covalent immobilization of the intein (and its fusion partners by extension).The classification “linker moiety” or “linker” refers to one or more amino acid residues expressed in fusion with the intein that confers structure, spacing, or flexibility between the intein, the immobilization moiety, and / or other fusion partners. Common examples of linker moieties include, but are not limited to: Glycine-Serine repeat ((GlynlSern2)n3), Polyproline dyad ((XaaPro)n), and a-helical (A(EAAAK)nA) linker motifs.The classification “tag moiety” or “tag” refers to a peptide, domain, or a specific amino acid motif that is expressed in fusion with a protein, and aids in purification, detection, and / or enhances soluble expression of its fusion partners. Examples of common “tag” moieties include but are not limited to: purification tags (e.g. poly-His, poly-Arg, GST, CBD, MBP, CBP, Strep- Tag, FLAG-tag, etc.), detection tags (e.g. GFP, luciferase, epitope tags (i.e. FLAG, HA, c-myc), HRP, etc.), and expression / solubility enhancing tags (e.g. T7-tag, NusA, TrxA, DsbA, DsbC, GST, MBP, etc ).As used herein, the terms "cleave", "cleaves", “cleavage” and “a cleaving event” refer to a chemical reaction in which a peptide bond within a polypeptide is broken, thereby dividing a single polypeptide to form two or more smaller polypeptide molecules. In some cases, cleavage is mediated by the addition of an extrinsic endopeptidase, which is often referred to as “proteolytic cleavage”. In other cases, cleaving can be mediated by the intrinsic activity of one or both of thecleaved peptide sequences, which is often referred to as “self-cleavage”. Cleavage can be controlled by extrinsic conditions (such as buffer pH), as described herein.By the term “fused” or “in fusion with” is meant covalently bonded to. For example, a first peptide is fused to a second peptide when the two peptides are covalently bonded to each other (e.g., via a peptide bond). Peptides and / or protein domains conjoined by peptide bonds may also be referred to as “fusion partners”.Herein, “bind”, “binds”, “binding” or “binding event” 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. The terms “bind”, “binds”, “binding” and “binding event” also imply the interaction between two molecules is non-covalent and reversible. These terms are used interchangeably with “associate with,” “associates with,” or “associating with.”As used herein, the terms “modified” or “mutated,” as in “modified intein” or “mutated intein,” refer to one or more modifications in either the nucleic acid or amino acid sequence being referred to, such as an intein, when compared to the native, or naturally occurring structure. Such modification can be a substitution, addition, or deletion. The modification can occur in one or more amino acid residues or one or more nucleotides of the structure being referred to, such as an intein.The terms “plasmid” and “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of the central metabolism of the cell and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3 ' untranslated sequence into a cell. Typically, a “vector” is a modified plasmid that contains additional multiple insertion sites for cloning and an “expression cassette” that contains a DNA sequence for a selected gene product (i.e., a transgene) for expression in the host cell. This “expression cassette” typically includes a 5 ' promoter region, the transgene ORF, and a 3 ' terminator region, with all necessary regulatory sequences required for transcription and translation of the ORF. Thus, integration of the expression cassette into the host permits expression of the transgene ORF in the cassette.The term “buffer” or “buffered solution” refers to solutions which resist changes in pH by the action of its conjugate acid-base range.The term “loading buffer” or “binding buffer” refers to the buffer containing the salt or salts which is mixed with the protein preparation for loading the protein preparation onto a column. This buffer is also used to equilibrate the column before loading, and to wash to column after loading the protein.The term “wash buffer” is used herein to refer to the buffer that is passed over a column (for example) following loading of a protein of interest and prior to elution of the protein of interest. The wash buffer may serve to remove one or more contaminants without substantial elution of the desired protein.The term “elution buffer” refers to the buffer used to elute the desired protein from the column. As used herein, the term “solution” refers to either a buffered or a non-buffered solution, including water.The term “washing” means passing an appropriate buffer through or over a solid support, such as a chromatographic resin.The term “eluting” a molecule (e.g. a desired protein or contaminant) from a solid support means removing the molecule from such material.The term “contaminant” or “impurity” refers to any foreign or objectionable molecule, particularly a biological macromolecule such as a DNA, an RNA, or a protein, other than the protein being purified, that is present in a sample of a protein being purified. Contaminants include, for example, other proteins from cells that express and / or secrete the protein being purified.The term “separate” or “isolate” as used in connection with protein purification refers to the separation of a desired protein from a second protein or other contaminant or mixture of impurities in a mixture comprising both the desired protein and a second protein or other contaminant or impurity mixture, such that at least the majority of the molecules of the desired protein are removed from that portion of the mixture that comprises at least the majority of the molecules of the second protein or other contaminant or mixture of impurities.The term “purify” or “purifying” a desired protein from a composition or solution comprising the desired protein and one or more contaminants means increasing the degree of purity of the desired protein in the composition or solution by removing (completely or partially) at least one contaminant from the composition or solution.The terms “chromatography media” or “chromatographic medium” refer to any type of stationary phase substrate (solid support), scaffold, or matrix used for chromatography or purification, in which an intein is affixed, immobilized, bonded, or grafted (covalently or otherwise), for the purpose of separating, enriching, or purifying a secondary molecule of interest.Common examples of chromatography media include but are not limited to: chromatography resins (e.g. crosslinked agarose, polymer, or silica-based particles / porous beads); functionalized membranes; micro- and nano-scale magnetic particles; and structured pore / structured channel media (e.g. monoliths and monolithic columns).General DescriptionRecent advances in cell culture technologies have dramatically increased the productivity and titers of recombinantly-expressed therapeutic proteins to the point where downstream capture and purification are routinely the bottlenecks in the manufacturing process.[11’13]Moreover, identification of a purification scheme that meets scalability and purity requirements necessitates extensive process optimization that is unique for each new molecule, depending upon the physical and chemical properties of the protein of interest (Figure 3)

[0014] . Such laboratory-scale optimizations are not only expensive to conduct, but are also time-consuming, labor-intensive, and extend the time-to-market for each new drug. As a result, there is a clear demand for the development of simple, economical, and efficient purification platform technologies

[0015] . Such platform technologies offer potentially dramatic simplifications to downstream process development by providing robust process performance that requires relatively minor optimizations from molecule-to-molecule

[0016] . In addition to simplifying facility footprints, added process flexibility decreases development costs by constricting the process condition space that must be explored, by offering a robust framework for process validation and regulatory oversight, decreasing buffer consumption, and by shortening time-to-market.The quintessential example of such a platform technology is the protein A capture platform for immunoglobulin G (IgG) monoclonal antibodies (mAbs)[16'18],havehistorically dominated product pipelines in the biopharmaceutical industry. Protein A binds polyvalently to a conserved region of the mAb (Fc domain), thus providing a broadly generalizable method for the capture of mAbs on-column via interaction with immobilized protein A. The availability of this reliable method for mAb purification has been partially responsible for the dominance of mAb- based therapies in the biopharmaceutical arena. While mAbs still represent a majority of the therapies in development, product pipelines are increasingly diversifying to include non-mAb therapeutics, such as recombinant human enzymes

[0019] . Unfortunately, no purification platform analogous to protein A has been developed for non-mAb proteins, which lack shared domains and structural features. This lack of a universal modality for isolation of non-mAbs represents a significant obstacle to the field of downstream process development. Although affinity tag technology represents a potentially transformative approach that provides a generalizable platformfor capture of non-mAbs, these approaches are currently only viable at laboratory-scale due to a variety of economic and technical considerations. Regrettably, the tag removal step severely limits the robustness of affinity-capture platforms, considering that proteases used for proteolytic tag removal are expensive, must be removed in subsequent polishing steps, and are capable of non-specific cleavage within the target protein

[0020] . Consequently, affinity tag technologies that do not rely on protease-mediated tag removal would provide many practical benefits to the field of downstream process development. The intein technology described herein can increase the usability of inteins for a number of purification and related applications in pure research and the biopharmaceutical industry.AI-CM Variants and Polypeptides Comprising InteinsThe invention disclosed herein relates to mutant variants of the previously reported AI- CM mini intein (US Patent 6,933,362, herein incorporated by reference in its entirety for its teaching concerning AI-CM and its uses). AI-CM is referred to herein as SEQ ID NO: 1. Two of these mutants are referred to herein as the 1-12 variant (SEQ ID NO: 3) and the 1-29 variant (SEQ ID NO: 2), where both have reduced premature cleavage during expression in microbial cells, but still exhibit reasonably fast cleavage under optimized conditions (see Example 1). They are useful in purifying proteins via a self-removing tag method, and for drug delivery applications amongst other things.The specific mutations (numbering relative to the AI-CM parent intein, SEQ ID NO: 1) of these inteins are:1-29: H41R, I108V, R111P, E152V.1-12: V32L, H41R, T53M, G64S, A69V, T70S.It is noted that each of these mutations can be individually present in SEQ ID NO: 1 alone, or in any combination that they are found in either or both of 1-29 and 1-12. For example, SEQ ID NO: 1 can be modified with any one of the following mutations: H41R, I108V, R111P, E152V, V32L, T53M, G64S, A69V, or T70S. In another embodiment, SEQ ID NO: 1 can be modified with any two of these mutations: H41R, I108V, R111P, E152V, V32L, T53M, G64S, A69V, or T70S. In another embodiment, SEQ ID NO: 1 can be modified with any three of these mutations: H41R, I108V, R11 IP, E152V, V32L, T53M, G64S, A69V, or T70S. In another embodiment, SEQ ID NO: 1 can be modified with any four of these mutations: H41R, I108V, R11 IP, E152V, V32L, T53M, G64S, A69V, or T70S. In another embodiment, SEQ ID NO: 1 can be modified with any five of these mutations: H41R, I108V, R111P, E152V, V32L, T53M, G64S, A69V, or T70S. In another embodiment, SEQ ID NO: 1 can be modified with any six of these mutations: H41R,I108V, R111P, E152V, V32L, T53M, G64S, A69V, or T70S. In another embodiment, SEQ ID NO: 1 can be modified with any seven of these mutations: H41R, I108V, R11 IP, E152V, V32L, T53M, G64S, A69V, or T70S. In another embodiment, SEQ ID NO: 1 can be modified with any eight of these mutations: H41R, I108V, R111P, E152V, V32L, T53M, G64S, A69V, or T70S. Finally, SEQ ID NO: 1 can be modified with all nine of these mutations: H41R, I108V, R111P, El 52V, V32L, T53M, G64S, A69V, or T70S.In addition to these mutations, SEQ ID NO: 1, 2, or 3 may be modified with any other additional mutations. These mutations can be additions, deletions, or substitutions of other amino acids. These additional mutations can confer other specific, desired properties. Therefore, disclosed herein are proteins with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional mutations to SEQ ID NOS: 1, 2, or 3. Put another way, disclosed herein are sequences with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOS: 1, 2, or 3, or any amount above, below, or in-between these percentages.The modified cleavage rate features of these modified inteins can greatly alleviate the precleavage issue in the expression of fusion proteins containing a contiguous intein, leading to significantly improved stability and yield of precursor (tagged target protein) expression under typical 37°C expression conditions (e.g., E. coh. CHO cells). Meanwhile, these modified inteins retain the feature of relatively rapid pH-controllable C-terminal cleavage. Because these inteins can substantially reduce premature cleaving, the yields of proteins purifications employing them can be greatly increased with only minimal changes in process design and cleaving time.Therefore, disclosed herein is a polypeptide comprising a functional intein, wherein said intein is a variant of SEQ ID NO: 1, wherein said variant comprises reduced premature cleavage of the intein compared to SEQ ID NO: 1. These variants are described above, and are typified by SEQ ID NOS: 2 and 3.By “reduced premature cleavage” is meant that cleavage of the intein is reduced when it is within cells, right after mRNA translation into protein. When cleavage occurs at this stage, it leads to the separation of a significant amount of target protein and tag before harvest, resulting in only a small quantity of purified products. The mutants disclosed herein can reduce premature cleavage when compared to premature cleavage of AI-CM by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%, or by 2, 3, 4, 5, 6, 7,8, 9, or 10 times, or any amount below, above, or in between these values.Described another way, the mutants disclosed herein can have a longer half-life after expression than AI-CM by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or by 2, 3, 4, 5, 6, 7,8, 9, or 10 times, or any amount below, above, or in between these values. These results are shown in Figure 4, for example.The inteins described herein are cleaved upon exposure to various controllable conditions. These conditions can be physical, chemical, or both. Examples of inteins which are controllable under various conditions are known in the art, for example, those found in U.S. Patent 10,066,027 and U.S. Patent 10,100,080, both of which are incorporated by reference in their entirety. Cleavage can be self-cleavage, for instance, triggered by a change in physical condition(s) and / or chemical condition(s) e.g., a change in one or more physical condition and / or one or more chemical condition (such that a combination of physical condition(s) and chemical condition(s) being possible), for instance, any one, or more, or a combination of any two or all, of change in pH, temperature, oxidative potential and ionic strength. The result can then be a cleavage of the product protein from the fusion product, with isolation of the purified product protein resulting therefrom (e.g., rinsing column after triggering self-cleavage or elution of product from column, to obtain purified protein).The protein of interest, which is also referred to herein as the target protein, can be any protein which requires purification or identification using an intein system. Examples include, but are not limited to, proteins and biomolecular assemblies (e.g., produced by recombinant DNA technology), such as, e.g., hormones (e.g. insulin, human growth hormone, erythropoietin, interferons, granulocyte colony stimulating factor, tissue plasminogen activator), monoclonal antibodies (mAbs) and mAb-derivatives (e.g., bi-specific mAbs, Fabs, scFvs, shark and camelid antibodies), scaffold-derived therapeutics (e.g., DARPins, Affibodies, anticalins), therapeutic enzymes (e.g., alpha galactosidase A, alpha-L-iduronidase, N-acetylgalactosamine-4-sulfatase, glucocerebrosidase), toxins (e.g. botulinum, CRM 197, ricin), recombinant vaccines (e.g., anthrax, diphtheria, tetanus, pneumonia, hepatitis B virus, human papilloma virus), virus-like particles (e.g., hepatitis B, human papilloma, influenza, parvovirus, Norwalk viruses), as well asindustrial enzymes (e.g., papain, bromelain, trypsin, proteinase K, BENZONASE™ enzyme, DENERASE™ enzyme, urease, pepsin, etc.) and diagnostic reagents (e.g., glucose and lactate dehydrogenase, DNA polymerases, alkaline phosphatase, horseradish peroxidase, restriction enzymes, hybridoma-derived antibodies, etc.). In a particular embodiment, the protein if interest is an antibody (e.g., a monoclonal antibody) to a therapeutic target.Affinity tags can be peptide or protein sequences expressed in fusion to intein, which confers specific chemical or physical properties that can aid in purifying the protein from cells. Cells expressing a peptide comprising an affinity tag can be pelleted, lysed, and the cell lysate applied to a column, resin or other solid support that displays a corresponding ligand that binds to the affinity tags. The affinity tag and any fused peptides are bound to the solid support, which can also be washed several times with buffer to eliminate unbound (contaminant) proteins. A protein of interest, if attached to an affinity tag or affinity tag / intein combination, can be eluted from the solid support via a buffer that causes the affinity tag to dissociate from the ligand resulting in a purified fusion protein, or can be cleaved from the bound affinity tag using a soluble protease, or the purified target protein can be released from the immobilized tag using an intein-mediated selfcleaving reaction.Examples of affinity tags can be found in Kimple et al. Curr Protoc Protein Sci 2004 Sep; Arnau et al. Protein Expr Purif 2006 Jul; 48(1) 1-13; Azarkan et al. J Chromatogr B Analyt Technol Biomed Life Sci 2007 Apr 15; 849(1-2) 81-90; and Waugh et al. Trends Biotechnol 2005 Jun; 23(6) 316-20, all hereby incorporated by reference in their entirety for their teaching of examples of affinity tags.Affinity tags can also be used to facilitate the purification of a protein of interest using the disclosed modified peptides through a variety of methods, including, but not limited to, selective precipitation, ion exchange chromatography, binding to precipitation-capable ligands, dialysis (by changing the size and / or charge of the target protein) and other highly selective separation methods. In each case, the purified protein of interest is subsequently separated from the tag via self-cleaving of the intein moiety.Nucleic Acids, Vectors, and CellsAlso disclosed herein are nucleic acids which encode the polypeptides disclosed herein. For example, SEQ ID NO: 4 is a nucleic acid which encodes ALCM, SEQ ID NO: 5 is a nucleic acid which encodes the 1-29 mutant, and SEQ ID NO: 6 is a nucleic acid which encodes the 112 mutant. Disclosed herein are nucleic acids which are 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 4-6, or any amount in between, above, or below these percentages.Polynucleotides that encode intein fusions are also described herein. The polynucleotides can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. In certain embodiments, the polynucleotides are isolated. In certain embodiments, the polynucleotides are substantially pure.Such polynucleotides can, for example, be incorporated in an expression vector for producing intein fusion proteins as discussed herein. An expression vector can contain expression control elements including the promoter. The fusion protein-coding gene is operatively linked to the expression vector to permit the promoter sequence to direct RNA polymerase binding and expression of the fusion protein-encoding gene. Useful in expressing the polypeptide coding gene are promoters that are inducible, viral, synthetic, constitutive as described by Paszkowski et al., 1989 EMBO J., 3:2719 and Odell et al., 1985 Nature, 313:810, as well as temporally regulated, spatially regulated, and spatiotemporally regulated as given in Chua et al., 1989 Science, 244: 174- 181.Expression vectors compatible with eukaryotic cells, such as those compatible with cells of prokaryotes (E. coif), mammals, algae or insects and the like, are contemplated herein. Such expression vectors can also be used to form the recombinant DNA molecules of the present invention. Prokaryotic and eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Normally, such vectors contain one or more convenient restriction sites for insertion of the desired DNA segment and promoter sequences. Optionally, such vectors contain a selectable marker specific for use in prokaryotic or eukaryotic cells.The choice of which expression vector and ultimately to which promoter a fusion proteinencoding gene is operatively linked depends directly on the functional properties desired, e.g., the location and timing of protein expression, and the host cell to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention can direct the replication, and preferably also the expression (for an expression vector) of the fusion protein gene included in the DNA segment to which it is operatively linked.The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host / vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expressioncontrol sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Esherichia coli. including pCR 1, pBR322, pMB9, pET vectors and their derivatives, and wider host range plasmids, such as Ml 3 and filamentous single-stranded DNA phages.In some embodiments, a vector comprising a polynucleotide that encodes an intein further comprises a multiple cloning site. A multiple cloning site is a polynucleotide sequence comprising one or more unique restriction sites. Non-limiting examples of the restriction sites include EcoRI, SacI, Kpnl, Smal, Xmal, BamHI, Xbal, Hindi, PstI, SphI, Hindlll, Aval, or any combination thereof.Multiple cloning sites can be used in vectors comprising a polynucleotide encoding an intein to simplify the insertion of a polynucleotide encoding a heterologous polypeptide into the vector such that the vector can be used to express a fusion protein comprising the intein and the heterologous polypeptide.The vectors can comprise at least one promoter. The promoter can be any sequence that is suitable for driving expression of an intein domain or intein fusion.Different hosts often have preferences for a particular codon to be used for encoding a particular amino acid residue. Such codon preferences are well known and a DNA sequence encoding a desired fusion protein sequence can be altered, using in vitro mutagenesis for example, so that host-preferred codons are utilized for a particular host in which the fusion protein is to be expressed.A recombinant nucleic acid molecule such as a DNA molecule, comprising a gene vector or construct containing one or more regulatory sequences (control elements) such as a promoter suitable for driving the expression of the gene in a compatible bacterial or eukaryotic host cell organism operatively linked to an exogenous nucleic acid segment (e.g., a DNA segment or sequence) that defines a gene that encodes a contemplated fusion protein, as discussed above, is also contemplated. More particularly, also contemplated is a recombinant DNA molecule that comprises a gene vector comprising a promoter for driving the expression of the fusion protein in host organism cells operatively linked to a DNA segment that defines a gene encodes an intein domain linked to a heterologous polypeptide. That recombinant DNA molecule, upon suitable transfection and expression in a host cell, provides a contemplated fusion protein.As is well known in the art, so long as the required nucleic acid, illustratively DNA sequence, is present, (including start and stop signals), additional base pairs can usually be present at either end of the DNA segment, and that segment can still be utilized to express the protein. This, of course, presumes the absence in the segment of an operatively linked DNA sequence thatrepresses expression, expresses a further product that consumes the fusion protein desired to be expressed, expresses a product that consumes a wanted reaction product produced by that desired fusion protein, or otherwise interferes with expression of the gene of the DNA segment.Thus, so long as the DNA segment is free of such interfering DNA sequences, a DNA segment of the invention can be about 500 to about 15,000 base pairs in length. The maximum size of a recombinant DNA molecule, particularly an expression vector, is governed mostly by convenience and the vector size that can be accommodated by a host cell, once all of the minimal DNA sequences required for replication and expression, when desired, are present. Minimal vector sizes are well known.A DNA segment that encodes a fusion protein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., 1981 J. Am. Chem. Soc., 103:3185. Of course, by chemically synthesizing the coding sequence, any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence.DNA segments containing a gene encoding the fusion protein can also be obtained from recombinant DNA molecules (plasmid vectors) containing that gene.MethodsDisclosed herein are methods of producing a purified protein of interest, the method comprising providing the polypeptide comprising an intein, as discussed above, purifying the protein of interest by use of an affinity tag fused to the intein, and subsequently providing suitable conditions for self-cleavage of the intein, thereby producing a purified protein of interest. The polypeptides comprising the intein of the present invention can be used in any methods in which conditional self-cleaving of an intein are useful. These methods can occur in the absence of any other enzymes, chemical additives, or treatments, or can include other elements as needed.In some embodiments, a protein of interest can be cleaved from an intein domain. This method is discussed above. For example, a heterologous polypeptide (protein of interest) can be cleaved from the intein after immobilization on a support. In some embodiments, this reaction occurs in the presence of a denaturing agent, e.g., to increase protein solubility. In some embodiments, the reaction occurs in the presence of urea. In some embodiments, the reaction occurs in a presence of no more than about 6.5M, about 6M, about 5M, about 4.5M, about 4M, about 3.5M, about 3M, about 2.5M, about 2M, about 1.5M, about IM, or about 0.5M urea. In some embodiments, the reaction occurs in the presence of about 0.5 to about 6M, about 0.5 to about 4M, about 1 to about 4M, about 2 to about 4M, or about 3 to about 4M urea. In someembodiments, the reaction occurs in the presence of about 0.5 to about 2M, or about 0.5 to IM urea.In some embodiments, the reaction results in a yield at least about 50%, at least about 55%, at least about 60%, at least about 65%, of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% purified protein of interest. In some embodiments, an intein self-cleavage reaction can be initiated by shifting the conditions, e.g., the temperature or pH, in which the polypeptide comprising the intein is incubated. In some embodiments, self-cleavage is initiated by a pH or temperature shift.In some embodiments, the fusion protein can comprise a binding component. This component can be used to attach the fusion protein to a resin, e.g., for the purpose of protein separation or purification such as those provided, for example, in Lu et al., Journal of Chromatography A 1218: 2553-2560 (2011) and Elleuche and Poggeler, Appl. Microbiol. Biotechnol 87:479-489 (2010), which are herein incorporated by reference. In addition, a polypeptide comprising an intein can be in solution, bound to affinity beads or a column, anchored to a cell membrane, or on a phage surface. Affinity binders (binding components) can include His- tags, chitin binding domains, a maltose binding protein, or a glutathione-S-transferase, for example. The polypeptide comprising the intein can be inside or outside a cell.In some embodiments, intein reactions can be used in protein purification, (e.g., using chromatographic tags or non-chromatographic tags and / or in large-scale processes), in protein circularization, in protein polymerization, and in selenoprotein production, for example as described in Elleuche and Poggeler, Appl. Microbiol. Biotechnol 87:479-489 (2010), and Evans T. et al., Biopolymers 51 :333-342 (1999), which are herein incorporated by reference in their entireties. The high efficiency of the inteins provided herein makes them particular amenable to large-scale industrial applications.In some embodiments, an intein reaction can be used to produce a protein of interest. The protein of interest can also be a polypeptide that is cleaved from a sequence it was previously linked to, as described above.EXAMPLESExample 1: Characterization of two mutants of AI-CM inteinAn intein screening system was developed where the intein library was inserted as a protein of interest (POI) between the HA (hemagglutinin) and c-myc epitope tags of a yeast surface displayed fusion protein. Due to the C-terminal cleavage properties of the intein, the c- myc tag can be released. By labeling The HA and c-myc tags with two different fluorescentantibodies, the amount of both HA and c-myc tags on the surface of a given yeast cell can be tracked and analyzed by flow cytometry. This allows the characterization of intein cleavage under different conditions using fluorescent signals, facilitating a corresponding selection process for inteins that cleave or don’t cleave under specific conditions of pH and temperature. Simultaneously, the signal of the POI displayed on the yeast surface is amplified due to the presence of thousands of intein fusion copies on each cell. This enables the application of flow cytometry and fluorescence-activated cell sorting (FACS) to screen for stringent selection conditions at high throughput. Two variants of AI-CM inteins with slower cleaving rates under 37°C were successfully isolated using this system, and they are herein referred to as the 1-29 and 1-12 intein mutants.In this study, the on-column cleaving kinetic test method was used to comprehensively characterize the two variants by replacing the C-extein with streptokinase (SK) as a model protein of interest. Based on the characterization results, we further explored combining the two intein mutants with different purification tags to create novel self-cleaving tags, which were applied to protein purification using both chromatographic and non-chromatographic methods. Prior to the on-column cleaving test, purification was performed using chitin resin, thereby demonstrating the feasibility of using the two mutant variants combined with the CBD tag as self-cleaving tags for affinity chromatography purification on a conventional affinity resin column. The purification method conditions varied depending on the specific intein mutant. In this work, we have also used our novel intein mutants (SEQ ID NOS: 2 and 3) in conjunction with an ELP (elastin-like- polypeptide) aggregating tag to develop an easy to use and cheap purification method which results in >90% pure bioactive proteins with high yields.Materials and MethodsChemical and reagentsAmmonium Sulfate, 2-amino-2-methyl-l,3-propanediol (AMPD), PIPES, Yeast Extract and Tryptone were purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium Chloride was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All restriction enzymes - EcoRI, BsrGI, Hindlll and Xbal were purchased from New England Biolabs (Ipswich, MA, USA). The cloning primers were synthesized by Sigma Aldrich (St. Louis, MO, USA). Chitin resin was purchased from New England Biolabs (Ipswich, MA, USA)CloningUsing one of the previously constructed pET-CBD-ALCM-SK, a cut and paste cloning strategy was employed to replace the AI-CM intein with the Al- 12 intein mutant and AL29 inteinmutant, resulting in the construction of CBD-AI-12-SK and CBD-AI-29-SK plasmids, respectively.Using one of the previously reported pET-ELP plasmid backbones, pET-ELP-AI-CM- sfGFP, a cut and paste cloning strategy was employed to replace the AI-CM intein with the Al- 12 intein mutant and AI-29 intein mutant, resulting in the construction of ELP-AI-12-sfGFP and ELP- AI-29-sfGFP plasmids, respectively. Subsequently, based on these three plasmids, a cut and paste cloning approach was utilized to replace the target protein (sfGFP) with P-gal and MBP individually, thereby completing the construction of other plasmids.Protein expressionThe aforementioned plasmids, upon completion of construction, were transformed into E. coli BLR (DE3) strain for protein expression in this study. The transformed cells were grown on LB plates supplemented with 100 pg / ml ampicillin at 37°C. Single colonies were selected and inoculated into 5 mL LB broth containing 100 pg / mL ampicillin and cultured at 37°C for 16-18 hours. Subsequently, the culture was diluted 1 : 100 (v / v) into Terrific Broth (TB) medium containing 100 pg / mL ampicillin and incubated in Ultra Yield™ flasks at 37°C for 3-4 hours until reaching an optical density at 600 nm (OD600) of 0.6-0.8. Following that, protein expression was induced by adding 0.5 mM IPTG (final concentration) to the culture, and the cultivation was continued at 16°C for 24 hours or at 37°C for 3 hours.Cell lysis and recoveryFollowing completion of the expression, cells were collected by centrifugation at 8000 rpm for 10 minutes at 4°C. The resulting pellet containing the precipitated cells was stored at - 20°C for subsequent use. Prior to cell lysis, the stored cell pellet was resuspended in a pH 8.5 buffer (20 mM AMPD / PIPES) at 10% volume of the original culture. The suspension was subjected to 10 rounds of sonication at 6-7 watts on ice, with each round consisting of 30 seconds of sonication followed by a 30 second pause. Finally, the suspension was centrifuged at 12000 rpm for 10 minutes at 4°C, and the pellet containing cell debris and other insoluble materials was discarded. The resulting clarified supernatant was used for the subsequent purification.Protein purification on chitin resinFirst, 300 pl chitin binding resins were packed in each 10 ml gravity-flow column at room temperature. The storage buffer was allowed to drain from the resin by gravity flow, and 10 CVs loading buffers were used to equilibrate resins. After the equilibration buffer was drained from the column, the prepared samples were added to the resin. The flowthroughs were collected and re-applied once to maximize binding. Then resins were washed by 10 CVs of pH 8.5 wash buffer(20 mM AMPD, 20 mM PIPES, 500 mM NaCl) and 10 CVs of cleaving buffer at different pH (20mM AMPD, 20 mM PIPES) sequentially.Protein purification by ELP tagThe elastin-like polypeptide tag is a biopolymer comprising of variable number of repeats of the pentapeptide (Val-Pro-Gly-X-Gly) where X is any guest amino acid residue. ELP tags undergo a phenomenon known as inverse transition cycling, wherein they are soluble in aqueous solutions below their inverse transition temperature and reversibly precipitate at temperatures greater than their transition temperature. ELP tags also selectively precipitate at room temperature in relatively low concentrations of ammonium sulfate. The reversible phase transition feature of ELP tags allows for multiple product wash steps to substantially increase product purity, making these tags quite attractive for protein purification applications. The precipitation characteristics of ELP tags depend on many factors, which include the number of repeats, the properties of the guest residue, molecular weight and salt concentration. ELP tags have also been known to improve the overall solubility of the fused target protein, making it possible to purify difficult-to-express and insoluble proteins such as nanobodies and Human Interleukin-3. ELP tags have also been used in conjunction with split intein tags for purifying proteins from E. coli cell lysates and mammalian cell culture supernatants.Two novel mutants of the Mtu RecA ALCM mini-intein were tested, which were obtained through yeast surface display, for protein purification applications. These mutants exhibit remarkably reduced premature cleavage activity during expression in E. coli compared to the original Mtu RecA ALCM mini-intein, thereby increasing yields of the purified target proteins. Fusion of these novel intein mutants to the ELP tag results in an easy to use purification method that generally produces bioactive proteins of modest purity (>80 %) in a single non- chromatographic step.During the ELP purification process, 2 M ammonium sulfate was added to the 1 ml clarified lysate obtained from the last section, reaching a final concentration of either 0.4 M or 0.8 M. The mixture was then incubated at room temperature for 15 minutes, followed by centrifugation at 15,000 rpm for 5 minutes. After discarding the supernatant, a second round of precipitation was performed to enhance purity. The precipitate was resuspended in 1ml high-salt wash buffer (20 mM AMPD, 20 mM PIPES, 200 mM NaCl, pH 8.5), and 2 M ammonium sulfate was added again to achieve a final concentration of 0.8 M. The mixture was incubated at room temperature for 15 minutes, and then centrifuged at 15,000 rpm for 5 minutes. Following the removal of the supernatant, the precipitate was resuspended in 0.5 ml elution buffer (20 mM AMPD, 20 mM PIPES, 200 mM NaCl, pH 6.0), and incubated at room temperature or 37°C for aperiod of time (5 hours or 18 hours). Finally, 2 M ammonium sulfate was added to the suspension to reach a final concentration of 0.4 M or 0.8 M, followed by incubation at room temperature for 15 minutes, and centrifugation at 15,000 rpm for 5 minutes. The ELP tag was precipitated, while the purified tag less target protein was harvested in the supernatant.Cleavage testsFor the ELP tag, during the final incubation step of the purification process, samples were taken at different time points for cleavage analysis. Specifically, after suspending the precipitate in 0.5 ml elution buffer (20 mM AMPD, 20 mM PIPES, 200 mM NaCl, pH 6.0), an immediate 20 pl sample was taken from the suspension. This sample was mixed with 20 pl of 2x SDS loading dye and heated at 98°C for 5 minutes to serve as the 0-hour time point. Sampling was continued in subsequent time intervals using the same methodology. Finally, the intensity of the precursor and target protein bands at each time point on the SDS-PAGE was measured using ImageJ (NIH). This measurement was used to calculate the cleavage percentage at different time points.For the CBD tag, after the environment of the column was changed to acidic conditions (pH 6.2) during the purification process, the column was immediately sealed, and 1 CV of cleaving buffer was added to the column and the resin resuspended into a slurry by vortexing. A sample of the resin slurry was immediately collected following a standard procedure, and this sample was designated as the starting time point (TO). Subsequent samples were taken at 30 minutes, 1 hour, 2 hours, 3.5 hours, 5 hours, and 24 hours after TO using the same procedure. For each time point, the resin slurry was thoroughly mixed, and 20 pl of the mixture was immediately taken and mixed with 20 pl of 2X sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading dye. The mixture was then heated at 98°C for 10 minutes to stop the reaction. SDS-PAGE was performed to analyze the samples at each time point.Activity assay for green fluorescent proteinDiluted samples of 100 pL each for clarified lysates and elutions were read in a BioTek Synergy 2 plate reader with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. All assays were performed in triplicate. Given the pH dependent fluorescence of green fluorescence protein, all the elution samples were titrated to pH 8.5 using 0.2 M NaOH.Activity assay for beta-galactosidaseA micro assay 96-well plate format was used, and the change in A420 per minute was measured. One unit was defined as 1 nmol of ortho-Nitrophenyl-P-galactosidase (ONPG) solution from Sigma Aldrich (St. Louis, MO, USA) hydrolyzed per minute. The absorbance at 420 nm divided by 0.0045 corresponds to the number of nmol hydrolyzed per mL reaction volume at 37°C.The A420 was measured in a 96-well plate format using a BioTek Synergy 2 spectrophotometer. 5 pL of clarified lysate and 10 pL of elution samples were diluted to 100 pL to perform the assay. All assays were performed in triplicate.Purity assessment using SDS-PAGEA 20 pl sample was taken from the purified product obtained in the final step of the purification process. The sample was mixed with 20 pl of 2x SDS loading dye and then heated at 98°C for 5 minutes. The intensity of the target protein band and other impurity bands on the SDS- PAGE were measured using ImageJ (NIH) to calculate the proportion of the target protein in the purified product. This calculation reflects the purity of the product.Yield assessment using A280 measurementsProtein yields were assessed by measuring the absorbance of the elution samples at 280 nm. The theoretical extinction coefficients for sfGFP, beta-galactosidase, and MBP were estimated to be 21890, 260,690 and 66350 M-lcm-1, respectively. All measurements were performed in triplicate.Experimental design and statistical analysisAll statistical analysis was performed using JMP 16 software (SAS Institute). The significance level value (a) was set to 0.05 for all the analyses. All experimental runs were randomized to minimize random errors. Further, duplicates were performed for all runs to achieve more reliable results.ResultsCharacterization of AI-12 and AI-29 inteinOne of the major limitations in the application of AI-CM intein has been its premature cleavage as a contiguous intein within cells, occurring right after mRNA translation into protein. This leads to the separation of a significant amount of target protein and tag before harvest, resulting in only a small quantity of purified products. In order to address this issue, two mutants were selected using a developed yeast surface display system. The cleaving rates of these mutants were systematically characterized at different temperatures and pH values using the on-column cleaving test method. To ensure sufficient precursor availability for testing, samples were expressed at a low temperature of 16°C for 6 hours. The original AI-CM intein was used as a control, the cleavage reaction was assumed to be a first-order reaction, so the half-life was estimated using the calculation formula for first-order reactions.Compared to the other two inteins, AI-29 exhibits reduced differences in the half-life of the cleavage reaction across different temperatures, indicating a reduced temperature sensitivity (Figure 4). At pH 7.3, the cleavage half-life of AI-29 is significantly longer than that of the other two inteins, demonstrating its ability to solve the premature cleavage issue. Especially under conditions that simulate the majority of cell cultures, such as 37°C, the cleavage half-life of AI- 29 reaches almost three times that of AI-CM intein. This implies AI-29 can potentially make a significant improvement in cases where AI-CM intein alone could only produce a small amount or even no precursor. Moreover, under pH 6.0 conditions, the cleavage half-life of AI-29 is only slightly longer than the other two inteins. At room temperature and pH 6.0, the cleavage half-life is merely one hour longer than AI-CM intein, and significantly shorter than its own half-life under room temperature and pH 7.3 conditions. This indicates a significant pH sensitivity of the cleavage reaction, allowing the mutant to employ a pH control mechanism similar to AI-CM intein. At pH 6.0, the cleavage of AI-12 exhibits a relatively short half-life, particularly at room temperature under pH 6.0 conditions, where its half-life is the shortest among three inteins. This indicates an enhanced pH sensitivity. At pH 7.3, the cleavage half-life of AI-12 falls between that of AI-29 and AI-CM intein.The overall results of these half-life characterizations are important, as they demonstrate that the three inteins, including the AI-CM intein, form a combination with different cleavage rates. Since the cleavage rate of AI-CM intein can be affected by the C-extein in practical applications, this implies that regardless of the specific impact of the C-extein, an ideal cleavage rate can always be achieved by pairing it with AI-CM or one of the 1-12 or 1-29 mutant inteins.Application of AI-12 / AI-29 intein with CBD tag as a self-cleaving tag for affinity chromatography purificationBy genetically fusing a CBD tag to the N-terminus of AI-CM / Al- 12 / AI-29 inteins, a selfcleaving tag can be constructed for affinity chromatography purification of recombinant proteins. The underlying principle involves forming a fusion protein by connecting the C-terminus of the self-cleaving tag to the target protein. Upon expression, the precursor and other impurities are separated from the fusion protein using the affinity between the CBD tag and the chitin column. Subsequently, activation of the intein's C-terminal cleavage is achieved by lowering the pH to 6.2, resulting in the release of the purified tagless target protein. Due to the distinct cleavage rates of the three AI-CM inteins, a series of experiments was conducted to investigate the performance of this set of self-cleaving tags with regard to premature cleaving and convenient cleavage rate during the purification process. SK protein was chosen as the test C-extein. After purifying the precursoron a chitin column, columns were incubated under specific conditions to measure their cleave rates under relevant conditions for expression and purification of the target protein. Resin samples were collected at different time points by mixing with the same volume of 2X SDS loading dye and heated at 95°C to stop the cleavage reaction and release the cleaved and uncleaved protein from the resin, as well as the cleaved tag. The proportions of the various cleavage products or precursors were then analyzed using SDS-PAGE. Subsequently, curves were plotted to illustrate the cleavage rate properties of inteins.Assessment of precursor lost by premature cleavageInitially, when utilizing the CBD tag with AI-CM intein, the loss of precursors during the expression process under neutral conditions at 37°C and 20°C was investigated by simulating the expression conditions (the intracellular pH is typically considered to be about pH 7.3, with typical expression temperatures ranging from about 20°C to about 37°C) on a chitin column (Figure 5).Due to the loss during the expression process, each group started with approximately 80% of precursors. Under pH 7.3 conditions, designed to mimic thew intracellular pH conditions of the expressing cells, all three inteins exhibit slower cleavage at 20°C. However, AI-29 intein shows only a 9% difference between different 20°C and 37°C after 5 hours, indicating a weakened temperature sensitivity and substantially slower cleaving at 37°C (Figure 5a vs 5b). Even at 37°C, AI-29 intein still retains close to 60% of precursors after 5 hours, which is twice as much as the other two inteins (Figure 5a). This indicates that its performance in preventing premature cleavage at 37°C will be significantly better than the other two inteins. At 20°C, the results suggest that AI- 12 performs better than AI-CM, but AI-29 still offers the highest amount of precursors. After 24 hours at 20°C, approximately 50% of precursors remain (The 24 hours data are from the same SDS-PAGE and not plotted in the graph).Assessment of cleavage efficiency at pH 6.2Next, the cleavage efficiency for the different inteins at pH 6.2 was characterized at 20°C and 37°C to determine the duration of chitin column incubation required after activating cleavage before elution when using the CBD tag for purification (Figure 6). Since the time required for other steps remains constant, this result could demonstrate the time required for purification.Based on the results shown in Figure 6, under low pH conditions (pH 6.2), all three inteins exhibit faster cleavage rates at 37°C compared to room temperature. However, the temperature sensitivity is not significant for AI-12 and AI-29 inteins. On the other hand, the AI-CM intein shows a significant difference, with a cleavage efficiency of over 60% within the first hour at 37°C, while at 20°C it is less than 30% within the first hour, reaching 60% only after 3 hours. Both AI-CM and AI-12 exhibit faster cleavage rates under low pH, with over 80% cleavageefficiency after 3 hours at 37°C and over 85% cleavage efficiency after 5 hours at 20°C. The difference lies in the fact that Al- 12 exhibits a faster rate at room temperature, while AI-CM shows a faster rate at 37°C. And AI-29 has a significantly slower cleavage rate than the other two inteins; after five hours, the cleavage rate of AI-29 reaches only 60% at 37°C, which is even lower at room temperature.Based on these data, the selection of inteins can be based on the cleavage rate when paired with the target protein, as well as the specific requirements of the application. For target proteins with faster cleavage rates, selecting the AI-29 intein would prevent premature cleavage and allow for obtaining a greater number of precursors and achieving higher recovery rates during the purification process. On the other hand, for target proteins with slower cleavage rates, choosing the AI-CM intein ensures completed cleavage within a reasonable timeframe for purification. The Al- 12 intein can be considered as a moderate option, providing a balanced cleavage rate. In general, this chromatography purification method offers high-purity products, and the selfcleavage ability of inteins allows for the convenient removal of the tag after purification. It is a classic application of self-cleaving tags in purification, and our lab previously developed the CBD- AI-CM tag based on the Original AI-CM intein. However, when using the original AI-CM intein, the issue of premature cleavage due to its fast cleavage rate resulted in limitations in the expression level of the precursor. By introducing two mutant variants with different cleavage rates under various conditions, these limitations have been effectively improved. The combination of the three inteins expands the application range of this self-cleaving tag to a broader set of target proteins.Application of AI-12 / AI-29 intein with the ELP tag as a self-cleaving tag for non- chromatographic purificationAssessment of in vivo behavior for intein mutantsThroughout the conventional expression of fusion proteins containing AI-CM intein in E. coli cells, a high cleavage activity within the cellular environment led to premature cleavage easily. To mitigate this issue, it is typically necessary to lower the cultivation temperature during expression, albeit at the expense of extending the expression duration to ensure adequate levels of expression. Recently identified variants of the AI-CM intein exhibit a slower cleaving rate at 37°C compared to the original AI-CM intein. Therefore, the behavior of fusion proteins containing these two intein mutants, in conjunction with an ELP tag, was primarily investigated within the cellular environment.Specifically, E. coli BLR cells carrying different plasmids were separately cultured to an appropriate cell density and induced with IPTG. Subsequently, expression was carried out at 37°Cfor 3 hours and at 16°C for 24 hours, respectively. To clearly compare the expression levels of the precursor, clarified lysates were initially subjected to aggregation purification after harvest, removing a significant portion of impurities present in the clarified lysate (Figure 10(d)). Samples were taken right prior to activating intein cleavage by reducing the pH value, and the expression levels of the precursor were analyzed using SDS-PAGEs and ImageJ software. The results of analyzing the proportion of precursors obtained from different samples on SDS-PAGE are presented graphically in Figure 10(a-c) and in Table 1.Table 1: Percentage of precursor in clarified lysate sample of different intein and target proteinIt is evident that, regardless of the conditions, both mutants provide a higher proportion of precursors compared to the original AI-CM intein. Moreover, the AI-29 intein exhibits the highest capability in providing precursors. When applied to the three target proteins, it consistently yields a proportion of precursors exceeding 70%. When the target protein is MBP, the proportion of precursor can even reach close to 90%. The Al- 12 intein generally provides at least around 50% of precursors. Another characteristic of the two mutants is that there is no significant increase in premature cleavage due to higher expression temperatures. While the proportion of precursors obtained with the AI-CM intein significantly increases when expressed at 16°C, the improvement with the two mutants under low-temperature expression conditions is minimal, especially for the AI-29 intein. When the target protein is MBP, the proportion of precursors is the same for both expression conditions. This observation is consistent with their low temperature sensitivity.In summary, utilizing the two mutants for aggregation purification benefits from a significant improvement in premature cleavage. It enables the expression of a higher proportion of precursors than the original AI-CM intein, thus facilitating purification yields. The choice ofexpression conditions regarding temperature becomes more flexible, allowing the use of 37°C for shorter expression durations.Assessment of pH sensitivity of intein mutantsAs a self-cleaving tag, the cleavage controllability of the intein composing the tag directly affects the yield and recovery of the final product. The C-terminal cleavage of the AI-CM intein is pH-inducible, which gives it an advantage over other thiol-induced inteins. To confirm the controllability of the AI-CM intein and its mutants when combined with the ELP tag, sfGFP was used as a model protein, and the cleavage behavior of the AI-CM intein and its two mutants at different pH conditions was observed (Figure 7). Initially, the clarified lysate was subjected to preliminary aggregation purification. The purified precursor was then cultivated for five hours in buffer solutions with pH 8.5 and pH 6.2, respectively. Samples were taken at different time points during the cultivation process, and the proportions of precursor and cleaved protein were analyzed using SDS-PAGE to observe the cleavage process of the intein.It can be observed that intein mutants maintain pH sensitivity when combined with an ELP tag to form a self-cleaving tag. Overall, the AL29 intein possesses the slowest cleavage rate. Under pH 6.2 and 37°C conditions, the final cleavage efficiency of AI-CM and AL12 can reach 95%, and it is nearly complete after approximately 2.5 hours. Although the cleavage efficiency of AL 29 is slightly lower, it can also reach almost 80% after 5 hours. Both AI-12 and AL29 cleavage reactions demonstrate excellent pH controllability. Under pH 8.5 and 37°C conditions, both mutants exhibit almost zero cleavage efficiency in the first two hours and remain below 20% after 5 hours. Throughout the entire process, the cleavage efficiency remains below one-fourth of that under pH 6.2 conditions. The AI-CM intein also exhibits a certain degree of pH controllability, with approximately 20% cleavage efficiency after 2 hours and reaching around 50% after 5 hours. This excellent pH controllability can effectively inhibit the cleavage reaction that may occur during precipitation and washing processes, thereby reducing precursor loss before the final stage of purification. Additionally, performing the cleavage reaction at room temperature could result in lower cleavage activity and further minimize precursor loss. However, once the pH is lowered and the temperature is increased to 37°C, the cleavage of both mutants can be rapidly activated. Particularly, in the case of AI-12, almost complete cleavage can be achieved within two and a half hours. Since the precipitation and washing process just takes 1-2 hours, utilizing AI-12 intein could allow the entire purification process to be completed in approximately 3-4 hours. Despite the AL29 intein requiring more than five hours for cleavage, its slower cleavage rate typically provides a higher amount of precursor during the expression. This trade-off is acceptable considering that the increased precursor yield compensates for the longer cleavage time.Optimization of ammonium sulfate concentration for purificationAs part of this study, we first optimized the concentration of ammonium sulfate required to effectively precipitate the aggregating tag target protein fusion. Next, we wanted to identify the optimal amount of salt in the wash step, to achieve high purity without sacrificing recovery significantly. These twin objectives were achieved by employing the response surface methodology (Figure 8). The independent variables were ammonium sulfate concentration (varied between 0 M and 1 M) and salt concentration in the wash buffer (varied between 0 M and 0.5 M), while the dependent response variables were product purity and recovery. Interestingly, it was observed after performing an effects test that both product recovery and purity were strong functions of ammonium sulfate concentration and did not depend on the salt concentration used in the wash step. Both product purity and product recovery were found to have quadratic dependence on the concentration of ammonium sulfate. Figure 8 (a) shows the effect test results for both product recovery and product purity. Figure 8 (b) shows the prediction profiler. Based on the results of the model, we decided to use 0.8 M Ammonium sulfate to precipitate the aggregating tag to maximize product recovery. Further, given the statistically insignificant effect of wash salt concentration on product yield and purity, we decided to use a buffer with 200 mM salt in it to perform the wash stepExample of protein purification using self-cleaving ELP tagThe ELP tag used in this study is derived from the previous ELP-ALCM tag developed by our laboratory. To obtain three similar ELP-intein tags, ALCM intein was replaced with two new variants. This ELP tag consists of 110 repeated VPGXG units, where X represents Vai, Ala, or Gly in a ratio of 5:2:3.Figure 9 illustrates the purification process of self-cleaving ELP tags based on ALCM and its variants. After the harvest of the cell culture, an appropriate amount of ammonium sulfate was added to the lysate to precipitate the ELP -tagged precursor, separating it from other impurities. The precipitate was washed once with a wash buffer to improve the purity and then precipitated again. The solutions were maintained at pH 8.5 throughout this process to suppress intein cleavage activity. The washed precipitate was resuspended in a pH 6.2 buffer to activate cleavage and incubated at room temperature or 37 °C for a certain period to allow most fusion proteins to be cleaved. Finally, ammonium sulfate was added to precipitate the cleaved ELP tag, obtaining a supernatant containing high-purity tagless protein. To demonstrate the generality of this method and observe the performance of three inteins, purification was performed on three target proteins: sfGFP (25.1 kDa - Figure 11), P-gal (116.5 kDa - Figure 12), and MBP (40.3 kDa - Figure 13), the purification results are shown in Table 2.Table 2: Summary of ELP purification results with different inteins and target proteins.All three target proteins could be successfully purified using the ELP intein tag, and the purity could reach above 90%. The performance of the three inteins also aligns with the expected characteristics. AI-29 provides the highest amount of expressed precursors, while AI-CM offers more efficient cleavage, and Al- 12 exhibits a balanced performance between the other two.Interestingly, the optimal yields for each protein are obtained when paired with different inteins, indicating that the concept of interchanging the three intein systems is rational.DiscussionIn this example, two mutants of AI-CM intein obtained through yeast surface display system were characterized. The performance of the mutants was consistent with the expectations of the screening, demonstrating the reliability of the developed yeast surface display screening system. This is of great significance in the optimization of intein activity, as the system offers high throughput, flexible condition settings, and strong applicability. It is a valuable and beneficial to apply this system to optimize other inteins.Since its development, the AI-CM intein has been widely used for its pH-inducible fast C- terminal cleavage advantages. However, the persistent issue of excessive premature cleavage during expression at 37°C has limited its application range, making it challenge to be used in mammalian cell expression systems. Although attempts have been made to utilize artificial split AI-CM inteins, their effectiveness has been underwhelming, leading to their replacement by natural split inteins. While split inteins have proven satisfactory in purification applications, contiguous inteins still remain irreplaceable in certain applications. The significant improvement in premature cleavage demonstrated by the two mutant variants of AI-CM intein described herein offers a novel solution. Remarkably, the AI-29 intein exhibits a remarkable slowing of cleavage at 37 °C while maintaining pH-inducible fast cleavage. 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Nature Biotechnology 15(6): 553-557SEQUENCESSEQ ID NO: 1 : AI-CMALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLHARPVVSWFDQGTRDVIGL RIAGGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPIPARVQALADA LDDKFLHDMLAEELRYSVIREVLPTRRARTFGLEVEELHTLVAEGVVVHNSEQ ID NO: 2: AI-CM with 1-29 VariantsALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLRARPVVSWFDQGTRDVIGL RIAGGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPVPAPVQALAD ALDDKFLHDMLAEELRYSVIREVLPTRRARTFGLVVEELHTLVAEGVVVHNSEQ ID NO: 3: AI-CM with 112 VariantsALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVLAAAKDGTLRARPVVSWFDQGMRDVIG LRIAGSAILWVSPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPIPARVQALAD ALDDKFLHDMLAEELRYSVIREVLPTRRARTFGLEVEELHTLVAEGVVVHNSEQ ID NO: 4: AI-CM nucleic acid gccctcgcagagggcactcggatcttcgatccggtcaccggtacaacgcatcgcatcgaggatgttgtcggtgggcgcaagcctattcatg tcgtggctgctgccaaggacggaacgctgcatgcgcggcccgtggtgtcctggttcgaccagggaacgcgggatgtgatcgggttgcgg atcgccggtggcgccatcctgtgggcgacacccgatcacaaggtgctgacagagtacggctggcgtgccgccggggaactccgcaag ggagacagggtggcgcaaccgcgacgcttcgatggattcggtgacagtgcgccgattccggcgcgcgtgcaggcgctcgcggatgcc ctggatgacaaattcctgcacgacatgctggcggaagaactccgctattccgtgatccgagaagtgctgccaacgcggcgggcacggac gttcggcctcgaggtcgaggaactgcacaccctcgtcgccgaaggggttgttgtacacaacSEQ ID NO: 5 Al- 12 nucleic acid gccctcgcagagggcactcggatcttcgatccggtcaccggtacaacgcatcgcatcgaggatgttgtcggtgggcgcaagcctattcatg tcttggctgctgccaaggacggaacgctgcgtgcgcggcccgtggtgtcctggttcgaccagggaatgcgggatgtgatcgggttgcgg atcgccggtagcgccatcctgtgggtgtcacccgatcacaaggtgctgacagagtacggctggcgtgcggccggggaactccgcaagg gagacagggtggcgcaaccgcgacgcttcgatggattcggtgacagtgcgccgattccggcgcgcgtgcaggcgctcgcggatgccct ggatgacaaattcctgcacgacatgctggcggaagaactccgctattccgtgatccgagaagtgctgccaacgcggcgggcacggacgt tcggcctcgaggtcgaggaactgcacaccctcgtcgccgaaggggttgttgtacacaacSEQ ID NO: 6 AI-29 nucleic acidGccctcgcagagggcactcggatcttcgatccggtcaccggtacaacgcatcgcatcgaggatgttgtcggtgggcgcaagcctattcat gtcgtggctgctgccaaggacggaacgctgcgtgcgcggcccgtggtgtcctggttcgaccagggaacgcgggatgtgatcgggttgc ggatcgccggtggcgccatcctgtgggcgacacccgatcacaaggtgctgacagagtacggctggcgtgccgccggggaactccgca agggagacagggtggcgcaaccgcgacgcttcgatggattcggtgacagtgcgccggttccggcgcccgtgcaggcgctcgcggatg ccctggatgacaaattcctgcacgacatgctggcggaagaactccgctattccgtgatccgagaagtgctgccaacgcggcgggcacgg acgttcggcctcgtggtcgaggaactgcacaccctcgtcgccgaaggggttgttgtacacaac

Claims

WHAT IS CLAIMED IS:

1. A polypeptide comprising a functional intein with isolated C-terminal cleaving activity, wherein said intein is a variant of SEQ ID NO: 1, wherein said variant comprises reduced premature cleavage of the intein during expression in a host cell or cell-free expression system compared to SEQ ID NO: 1.

2. The polypeptide of claim 1, wherein said variant of SEQ ID NO: 1 comprises one or more mutations to SEQ ID NO: 1.

3. The polypeptide of claim 2, wherein at least one of the one or more mutations occur at position 32, 41, 53, 64, 69, 70, 108, 111, and / or 152 of SEQ ID NO: 1.

4. The polypeptide of claim 3, wherein at least one of the mutations comprises H41R, I108V, R111P, E152V, V32L, H41R, T53M, G64S, A69V, or T70S.

5. The polypeptide of claim 2, wherein the variant comprises mutations at positions H41R, I108V, R111P, and E152V of SEQ ID NO: 1, which is represented by SEQ ID NO: 2.

6. The polypeptide of claim 5, wherein the variant comprises at least one additional mutation which exhibits reduced premature cleavage compared to SEQ ID NO: 1.

7. The polypeptide of claim 2, wherein the variant comprises mutations at positions V32L, H41R, T53M, G64S, A69V, and T70S, which is represented by SEQ ID NO: 3.

8. The polypeptide of claim 7, wherein the variant comprises at least one additional mutation which exhibits reduced premature cleavage compared to SEQ ID NO: 1.

9. The polypeptide of any one of claims 1-8, wherein cleavage activity of the intein is controllable by varying at least one physical condition, one chemical condition, or both.

10. The polypeptide of claim 9, wherein the cleavage activity is controlled by varying pH.

11. The polypeptide of claim 9, wherein the cleavage activity is controlled by varying temperature.

12. The polypeptide of any one of claims 1-11, wherein the polypeptide further comprises a protein of interest.

13. The polypeptide of claim 12, wherein the polypeptide further comprises a binding component.

14. A nucleic acid encoding any one of the polypeptides of claims 1-13.

15. A vector comprising the nucleic acid of claim 14.

16. A cell comprising the vector of claim 15.

17. A method of producing a purified protein of interest, the method comprising providing the polypeptide of claim 13 under suitable conditions for self-cleavage of the intein, thereby producing a purified protein of interest.

18. The method of claim 17, wherein, prior to self-cleavage of the intein, a fused affinity tag component immobilizes the polypeptide to a solid support.

19. The method of claim 18, wherein the solid support comprises a chromatographic media, including a porous resin, a membrane, a monolith or a magnetic bead.

20. The method of claim any one of claims 17-19, wherein the intein cleaves the protein of interest from both itself and the binding component, thereby releasing the purified protein of interest while the binding component and tag remain immobilized on the solid support.

21. The method of any one of claims 17-20, wherein conditions comprise a change in pH and / or temperature.

22. The method of any one of claims 17-21, wherein the polypeptide is produced by a cell.

23. The method of claim 22, wherein the cell is a prokaryotic or eukaryotic cell.

24. The method of any one of claims 17-21, wherein the polypeptide is produced by a cell-free expression system.

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