Patent Publication Number: US-10323235-B2

Title: Reversible regulation of intein activity through engineered new zinc binding domain

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. patent application Ser. No. 14/418,858, filed Jan. 30, 2015, now U.S. Pat. No. 9,796,967, which is a 371 of PCT/US2013/053195, filed Aug. 1, 2013, which claims benefit of U.S. Provisional Application No. 61/678,303, filed Aug. 1, 2012, and U.S. Provisional Application No. 61/777,068, filed Mar. 12, 2013, each of which are hereby incorporated herein by reference in their entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under contract HHSN261200800001E awarded by the National Cancer Institute and the National Institutes of Health. The government has certain rights in the invention. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to the field of molecular and protein biology including polypeptides and modified polypeptides. This application also relates to the field of protein purification. The present invention also relates generally to the field of protein preparation, protein recovery and protein purification. 
     BACKGROUND OF THE INVENTION 
     Inteins are naturally occurring, self-splicing protein subdomains that are capable of excising out their own protein subdomain from a larger protein structure while simultaneously joining the two formerly flanking peptide regions (“exteins”) together to form a mature host protein. 
     The interesting behavior of inteins has led to an emerging area of research regarding the structural, mechanistic, and biological features of inteins. For instance, a variety of x-ray crystal structures, computational models, and mutational studies have led to a general consensus that inteins are distinct from common proteolytic enzymes in that they do not degrade peptide bonds other than the two bonds linking the intein to the flanking “exteins.” This is highly useful because unwanted proteolytic damage to other proteins is mitigated when inteins are used as transient coupling elements within fusion proteins. 
     What is needed is a method for selectively and reversibly inactivating an intein. Such an approach could allow for affinity based purification of a wide variety of proteins. The availability of such a generic protein recovery and purification system would have significant applications in the areas of rapid anti-infectious disease vaccine manufacture, bioterrorism defense, and personalized anti-cancer antigen generation, as well as contributing to the acceleration of new drug evaluation and optimization. 
     SUMMARY 
     Disclosed herein are polypeptides, compositions, and methods of making and use of said polypeptides and compositions. 
     A critical application of inteins has been the development of self-cleaving affinity tags for protein purification. In these applications, the inteins are modified to exhibit isolated cleaving of the intein-extein bonds at either their N- or C-terminus, or both. By replacing one extein with an affinity tag, and the other extein with a desired target protein, the target protein can be easily purified by the tag, where tag removal is subsequently facilitated by the intein self-cleaving activity. Critical to this application, however, is the ability to suppress cleaving during expression of the tagged target protein, but induce rapid self-cleaving of the intein from the purified target protein. This ability had proven elusive, and is critical to the application of inteins to general protein purification methods of using affinity tags for protein purification. In this case critical application of inteins has been the development of self-cleaving activity. 
     Disclosed herein are modified peptides comprising a controllable intervening protein sequence (CIPS) wherein the modified peptide comprises the structure: X1-CIPS, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible zinc-binding motif and an intein. 
     Also disclosed herein are modified peptides comprising a CIPS wherein the modified peptides comprise the structure: X1-CIPS-X2, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible zinc-binding motif and an intein, and wherein X2 comprises a protein of interest. 
     Disclosed herein are controllable intervening protein sequences (CIPSs) comprising an intein and a reversible zinc-binding motif wherein the reversible zinc-binding motif comprises the structure: aa1-aa2-aa3-aa4, wherein aa1 is a non-polar amino acid, aa2 is a negatively-charged amino acid, aa3 is a non-polar amino acid and aa4 is a positively charged amino acid. 
     Disclosed herein are CIPSs comprising an intein and a reversible zinc-binding motif wherein the reversible zinc-binding motif comprises the structure: aa1-aa2-aa3-aa4-aa5, wherein aa1 is a non-polar amino acid, aa2 is a negatively-charged amino acid, aa3 is a non-polar amino acid, aa4 is a positively charged amino acid and aa5 is a non-polar amino acid. 
     Disclosed herein are isolated nucleic acids capable of encoding the peptides or CIPSs disclosed herein as well as plasmids, vectors, and cell lines comprising nucleic acids capable of encoding the peptides or CIPSs disclosed herein. 
     Disclosed herein are methods of producing a modified peptide comprising a CIPS, the method comprising: preparing a nucleic acid that encodes one or more peptides comprising a CIPS; transforming a host cell with the nucleic acid; and culturing the transformed host cell under conditions suitable for the expression of the modified peptide comprising a CIPS encoded by the nucleic acid. Such methods can further comprise isolating the modified peptide comprising a CIPS. 
     Disclosed herein are methods of producing a modified peptide comprising a CIPS, the method comprising: preparing a nucleic acid that encodes one or more peptides comprising a CIPS; transforming a host cell with the nucleic acid; and culturing the transformed host cell under conditions suitable for the expression of the modified peptide comprising a CIPS encoded by the nucleic acid and further comprising exposing the modified peptide comprising the CIPS to a chemical reagent which inhibits splicing or cleavage. 
     Disclosed herein are methods of producing a modified peptide comprising a CIPS, the method comprising: preparing a nucleic acid that encodes one or more peptides comprising a CIPS; transforming a host cell with the nucleic acid; and culturing the transformed host cell under conditions suitable for the expression of the modified peptide comprising a CIPS encoded by the nucleic acid and further comprising exposing the modified peptide to a chelating agent, a change in pH, a change in temperature, dialysis, or dilution. 
     Disclosed herein are methods of producing a protein of interest comprising: (a) preparing a nucleic acid that encodes a modified peptide comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS-X2, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible zinc-binding motif and an intein, and wherein X2 comprises the protein of interest; (b) transforming a host cell with the nucleic acid; (c) culturing the transformed host cell under conditions suitable for the expression of the modified peptide; (d) exposing the modified peptide to a concentration of zinc which inhibits splicing or cleavage by the CIPS; (e) isolating the modified peptide; and (f) removing the zinc, thereby allowing for splicing or cleavage of the protein of interest from the modified peptide, thereby producing the protein of interest. 
     Disclosed herein are methods for binding and eluting a phage-displayed polypeptide from a protein of interest comprising: producing a modified peptide comprising the structure X1-CIPS-X2 wherein X1 is an affinity tag, and wherein X2 is a protein of interest; binding the modified peptide to a solid support; contacting a phage-displayed polypeptide with the support-bound modified peptide, thereby permitting binding of the phage-displayed polypeptide with the modified peptide comprising the CIPS; removing unbound phage-displayed polypeptides; and eluting the bound phage-displayed polypeptide by inducing cleavage of the protein of interest by the CIPS. 
     Disclosed are kits comprising the peptides, vectors, nucleic acids or cells as disclosed herein. The disclosed kits can further comprise zinc. 
     While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
       The accompanying figures and drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention. These are non-limiting examples. 
         FIG. 1  shows natural intein function. Native inteins remove themselves from within host proteins in a self-splicing reaction. 
         FIG. 2  shows a self-cleaving affinity tag method. The inteins or CIPSs disclosed herein can allow for efficient, single-column purification of target proteins by providing an effective means for tag self-cleaving. 
         FIG. 3  shows that inteins isolated from different sources share certain common features. Specifically, almost all inteins end in a histidine-asparagine dipeptide, which can be used to assist in binding metal ions. SEQ ID NOs:51-64 are present in  FIG. 3 . The sequences read from left to right and top to bottom. For example, SEQ ID NO:51 is AILYVGCFAKGT and SEQ ID NO:52 is NQVVVHNCGERGN. 
         FIG. 4  shows the chemical control of intein cleavage. The zinc concentration required to decrease the cleavage rate by 50% decreased from 100 μM in the ΔI-CM intein to 17 μM in the LN-004b intein is shown. Thus, the concentration of zinc required to fully suppress intein cleaving also decreased by a factor of approximately 10 (from about 600 μM to less than 100 μM). This change allows the safe use of zinc to suppress intein cleaving in CHO cell culture because CHO cells can easily tolerate 100 μM zinc but suffer at 600 μM zinc. 
         FIGS. 5A-F  show Schrodinger Q-SITE and Impact structures of exemplary ZBMs that included the zinc metal ion and the amino acid side chain, separated from the molecular modeling region using hydrogen caps between the alpha and beta carbons. The simulations were run with and without a metal center to look for structures that are able to tightly bind to zinc ion in a way that the bound zinc will sequester the critical conserved histidine residue of the active site. The structures represented a range of conformations, all of which had potential to inhibit the intein active site upon zinc-binding. A) The top two sequences are SEQ ID NO:45. The bottom sequence is SEQ ID NO:46. B) The top sequence is SEQ ID NO:47. The bottom sequence is SEQ ID NO:46. C) The top two sequences are SEQ ID NO:45. The bottom sequence is SEQ ID NO:46. D) The top two sequences are SEQ ID NO:48. The bottom sequence is SEQ ID NO:46. E) The top sequence is SEQ ID NO:49. The bottom sequence is SEQ ID NO:46. F) The top two sequences are SEQ ID NO:50. The bottom sequence is SEQ ID NO:46. 
         FIGS. 6A and 6B  show the Affinity Fusion Technologies. (A) Conventional method (left) requires protease treatment to remove the affinity tag from the purified fusion protein. Subsequent purification is required to remove the protease and cleaved tag. (B) Inteins (right) allow self-cleaving of the affinity tag, providing a pure product in a single column step. 
         FIG. 7  shows the comparison of conditions experienced by proteins expressed in  E. coli  and CHO relative to the expected cleaving rate of the intein module. Rapid cleaving under CHO cell culture conditions leads to unacceptable levels of premature cleaving during expression in vivo. 
         FIGS. 8A, 8B, and 8C  show evidence that the penultimate histidine residue of the ΔI-CM intein plays a critical role in regulating the C-terminal cleaving reaction. (A) Protein sequence alignment of seven canonical inteins, showing the high degree of conservation of the penultimate histidine residue. SEQ ID NOs:51-64 are present in  FIG. 8A . The sequences read from left to right and top to bottom. (B) The cleavage rate versus pH curve for the C-terminal cleaving reactions of several representative inteins, showing similarity to the pKa curve for the histidine side chain. (C) Extended position of the penultimate histidine (shown in ball-and-stick) in the ΔICM crystal structure. 
         FIG. 9  shows the strategy for grafting a metal-binding motif (shown schematically in yellow) to the N-terminus of the mini-intein. The site is designed to sequester the catalytic H439 side chain. 
         FIGS. 10A and 10B  show the design and zinc binding characterization of the of the LN-004b intein mutant. A) Active site configuration showing bound zinc ion. B) Repression of cleaving versus zinc concentration of several constructed mutants. 
         FIG. 11  is a representative single column purification of maltose binding protein (target protein) using a self-cleaving choline binding protein on Q-sepharose. 
         FIG. 12  shows protein purification by self-cleaving ELP precipitation tag. 
         FIG. 13  is a western blot result showing the effect of zinc on premature cleavage in a cell-free expression system. The antibody in the blot is directed to the protein of interest (Lac, or beta-lactamase in this case). CM refers to the parent ΔI-CM intein, while 4b refers to the LN-004b intein and 02 refers to the LN-002 intein. In each case, the ‘+’ symbol indicates expression of the tagged protein in the presence of 300 μM zinc ion, while the ‘−’ symbol indicates the absence of zinc. As can be observed, the CM intein shows very little sensitivity to zinc, and exhibits significant cleavage of the CI-Lac precursor protein to produce cleaved Lac regardless of zinc presence. The 4b and 02 inteins cleave more quickly than the CM intein, and are almost completely cleaved in the absence of zinc, but their cleaving is significantly reduced in the presence of zinc ion. 
         FIG. 14  is a western blot result showing the effect of zinc on premature cleavage in a cell-free expression system. The antibody in the blot is directed to the protein of interest (Seap; Secreted Alkaline Phosphatase). CM refers to the parent ΔI-CM intein, while 4b refers to the LN-004b intein and 02 refers to the LN-002 intein. In each case, the ‘+’ symbol indicates expression of the tagged protein in the presence of 600 μM zinc ion, while the ‘−’ symbol indicates the absence of zinc. As can be observed, the CM intein shows very little sensitivity to zinc, and exhibits significant cleavage of the CI-Seap precursor protein to produce cleaved Seap regardless of zinc presence. The designed inteins are more sensitive to zinc ion, and the 4b intein in particular is almost completely inactivated in the presence of zinc at this concentration. The controllability of the intein is expected to increase with optimized pH and temperature of expression. 
         FIG. 15  is a western blot result showing the effect of different zinc concentrations on premature cleavage of the LN-004b intein in a cell-free expression system. The antibody in the blot is directed to the protein of interest (Seap; Secreted Alkaline Phosphatase). In each case, the zinc concentration is noted above each lane. As can be observed, the amount of premature cleaving is reduced as the zinc concentration increases. It is anticipated that a higher degree of control will be attained through optimization of pH and temperature of expression. 
         FIG. 16  shows results of CHO expression of Fc-Intein-SEAP at 37° C., 5% CO2 incubation, and harvested 72 h after transfection. Lane 1: The secreted form of Fc-I-SEAP precursor in the presence of 200 μM of Zinc. Lane 2: The intracellular Fc-I-SEAP precursor and cleaved SEAP in the presence of 200 μM of Zinc. Lane 3: The secreted form of Fc-I-SEAP precursor in the absence of 200 μM of Zinc. Lane 4: The intracellular Fc-I-SEAP precursor and cleaved SEAP in the absence of 200 μM of Zinc. Numbers to the left of the gel indicate molecular weights of the predicted species. Note the presence of significant cleaved SEAP in the absence of zinc (lane 3), while no cleaved SEAP is observed in the secreted products in the presence of zinc (lane 1). 
         FIG. 17  is a table showing the Zinc Binding Motifs (ZBMs), Intein Sequences, and ZBM plus Intein Sequences. 
         FIG. 18  is a ribbon diagram of ΔI-CM mini intein. 
         FIG. 19  is a ribbon diagram of ΔI-CM mini intein with the conserved His residue shown in ball-and-stick. 
         FIG. 20  is a ribbon diagram of ΔI-CM mini intein with the conserved His residue shown in ball-and-stick along with zinc. 
         FIG. 21  is a protein gel showing the zinc control of parent ΔI-CM Intein cleaving. 
         FIGS. 22A and 22B  are ribbon diagrams of ΔI-CM mini intein with the conserved His residue shown in ball-and-stick. A) Shows the zinc binding motif added to the N-terminus of the intein. B) Shows zinc interacting with zinc binding motif and His residue. 
         FIGS. 23A and 23B  are schematic diagrams of a conventional (A) and a self-cleaving tag-based (B) purification method for comparison. 
     
    
    
     Additional advantages of the disclosed compositions and methods will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. 
     DETAILED DESCRIPTION 
     All patents, patent applications, and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. 
     It is to be understood that this invention is not limited to specific synthetic methods, or to specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, to specific pharmaceutical carriers, or to particular pharmaceutical formulations or administration regimens, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. 
     DEFINITIONS AND NOMENCLATURE 
     The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art. 
     As used in the specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of compounds, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like. 
     Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. 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. 
     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 phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, 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. 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. 
     As used herein, “extein” refers to the portion of an intein-modified protein that is not part of the intein and which can be spliced or cleaved upon excision of the intein. 
     “Intein” refers to an in-frame intervening sequence in a protein. 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, modified or mutated inteins, and split inteins. 
     As used herein, “protein of interest” or “peptide of interest” is a protein or peptide sought to be produced, made or purified. A protein or peptide of interest can be attached to an intein in one or more of the peptides disclosed herein comprising a CIPS. A “peptide of interest” can be a protein, such as an enzyme. The terms “protein of interest” and “peptide of interest” can be used interchangeably throughout the specification. 
     As used herein, “zinc-sensitive” means that a given peptide is responsive to or influenced by the presence of zinc. For example, a “zinc-sensitive” intein responds differently in the presence of zinc than in the absence of zinc. 
     As used herein, “variant” refers to a molecule that retains a biological 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 parent molecule. For example, substituting specific amino acids in a given peptide can yield a variant peptide with similar activity to the parent. 
     Nucleic acids, nucleotide sequences, proteins or amino acid sequences referred to herein can be isolated, purified, synthesized chemically, or produced through recombinant DNA technology. All of these methods are well known in the art. 
     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. 
     As used herein, the term “modified peptide”, “modified protein” or “modified protein of interest” or “modified target protein” refers to a protein which has been modified. For example a modified peptide can be a peptide modified by the insertion of a CIPS. A modified peptide can also comprise a CIPS as well as a protein of interest and/or an affinity tag. 
     As used herein, “operably linked” refers to the association of two or more biomolecules in a configuration relative to one another such that the normal function of the biomolecules can be performed. In relation to nucleotide sequences, “operably linked” refers to the association of two or more nucleic acid sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed. For example, the nucleotide sequence encoding a pre-sequence or secretory leader is operably linked to a nucleotide sequence for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation of the sequence. 
     “Sequence homology” can refer to the situation where nucleic acid or protein sequences are similar because they have a common evolutionary origin. “Sequence homology” can indicate that sequences are very similar. Sequence similarity is observable; homology can be based on the observation. “Very similar” can mean at least 70% identity, homology or similarity; at least 75% identity, homology or similarity; at least 80% identity, homology or similarity; at least 85% identity, homology or similarity; at least 90% identity, homology or similarity; such as at least 93% or at least 95% or even at least 97% identity, homology or similarity. The nucleotide sequence similarity or homology or identity can be determined using the “Align” program of Myers et al. (1988) CABIOS 4:11-17 and available at NCBI. Additionally or alternatively, amino acid sequence similarity or identity or homology can be determined using the BlastP program (Altschul et al. Nucl. Acids Res. 25:3389-3402), and available at NCBI. Alternatively or additionally, the terms “similarity” or “identity” or “homology,” for instance, with respect to a nucleotide sequence, are intended to indicate a quantitative measure of homology between two sequences. 
     Alternatively or additionally, “similarity” with respect to sequences refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm. (1983) Proc. Natl. Acad. Sci. USA 80:726. For example, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics™ Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar, or have a degree of sequence identity with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. The following references also provide algorithms for comparing the relative identity or homology or similarity of amino acid residues of two proteins, and additionally or alternatively with respect to the foregoing, the references can be used for determining percent homology or identity or similarity. Needleman et al. (1970) J. Mol. Biol. 48:444-453; Smith et al. (1983) Advances App. Math. 2:482-489; Smith et al. (1981) Nuc. Acids Res. 11:2205-2220; Feng et al. (1987) J. Molec. Evol. 25:351-360; Higgins et al. (1989) CABIOS 5:151-153; Thompson et al. (1994) Nuc. Acids Res. 22:4673-480; and Devereux et al. (1984) 12:387-395. “Stringent hybridization conditions” is a term which is well known in the art; see, for example, Sambrook, “Molecular Cloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor, 1989; “Nucleic Acid Hybridization, A Practical Approach”, Hames and Higgins eds., IRL Press, Oxford, 1985; see also  FIG. 2  and description thereof herein wherein there is a sequence comparison. 
     The terms “plasmid” and “vector” and “cassette” refer to an extra chromosomal 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. 
     Controllable Intervening Protein Sequences 
     A variety of intein-mediated, affinity tagged intein systems are known in the art but critically suffer from a lack of a sufficient or industrially convenient control approach for the self-release step. Previous approaches have relied on pH or temperature as a triggering mechanism for the activation of intein self-cleavage, but these schemes require expression conditions that are not suitable for commercially relevant mammalian cell culture expression systems. Furthermore, these triggering mechanisms have historically suffered from premature intein activation, lengthened process time due to low temperature growth, incompatibility with certain naturally occurring protein bonds, and suboptimal target protein yields. These deficiencies have limited the practical application of inteins and as a consequence, the promise of the technology has not been fully realized. 
     Current self-cleaving intein systems fall into two categories: N-terminal cleaving systems, which require high concentrations of thiol compounds to induce cleaving, and C-terminal cleaving inteins, which are controlled by pH and temperature. N-terminal cleaving inteins cannot be used with target proteins that contain disulfide bonds due to the tendency of thiol compounds to break these bonds and destroy the target. C-terminal cleaving inteins are plagued with premature cleaving problems, where the tag self-cleaves before the target can actually be purified. These two limitations have effectively prevented intein technology from being applied to proteins expressed in mammalian cells, and these proteins comprise a large and growing majority of biopharmaceuticals. 
     Previously, inteins were engineered through the application of directed evolution methods. One of these inteins, derived from the Mtu-RecA intein, commonly referred to as the ΔI-CM mini intein, was optimized to obtain 1) a small physical size, 2) significant cleaving activity, and 3) selective activation under modulated solution temperature and pH conditions. The ΔI-CM intein ( FIG. 17 , in the Examples section) has been successfully applied to the expression of intact tag-intein-payload fusion proteins in  E. coli . However, premature intein activation is a significant drawback, and low temperature protein expression (below 25° C.) is used to maximize the proportion of intact fusion protein available for affinity capture. However, this condition substantially lengthens process time. Further, and most critically, this need for low-temperature expression conditions prevents the use of the ΔI-CM intein in the most commercially relevant mammalian cell culture based expression systems, which require growth and expression at temperatures above 32° C. and neutral pH. Clearly, a method for selectively and reversibly inactivating an intein is needed. Such methods and compositions are provided herein. 
     Disclosed herein are controllable intervening protein sequences (CIPS), which comprise an intein engineered to possess an appended (or internal) zinc-binding control sub-domain (also referred to herein as a “zinc-binding motif,” or “ZBM”). These CIPS can be used to modify or in modified peptides and are useful for a variety of methods including, but not limited to, protein production and purification. For example, disclosed herein are modified peptides comprising a CIPS. Also disclosed herein are modified peptides comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible zinc-binding motif and an intein. 
     Disclosed herein are modified peptides comprising a CIPS. Also disclosed herein are modified peptides comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible zinc-binding motif and an intein, wherein the intein is a naturally occurring intein, a modified intein, Mtu-RecA or one of the inteins listed in Table 2. 
     Disclosed herein are modified peptides comprising a CIPS. 
     Disclosed herein are modified peptides comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible ZBM and an intein, wherein the intein comprises additional amino acids at the N-terminus of a naturally occurring intein. For example, the modified intein can comprise four or more additional amino acids at the N-terminus of the naturally occurring intein. In some aspects, the modified intein can comprise four or more additional amino acids at the N-terminus of the naturally occurring intein, wherein the first amino acid at the N-terminal end of the intein is alanine, histidine, cysteine or glycine 
     Also disclosed herein are modified peptides comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS-X2, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible ZBM and an intein, and wherein X2 comprises a protein of interest. 
     Additional examples of CIPS can be further found in  FIG. 17 , as well as in the Examples section below. For example, the CIPS can comprise SEQ ID NOS: 11, 15, 19, 23, 27, or 31. A nucleic acid encoding a CIPS can comprise SEQ ID NOS: 12, 16, 20, 24, 28, or 32. 
     Also disclosed herein are splicing-intein variants of the described system that can be used to alter the tropism of viral, bacterial, or yeast vectors through the introduction of interrupting switched inteins into key vector surfaces or targeting proteins, thereby making the vectors inhibited by the presence of zinc and activated by chelating agents which bind zinc. 
     As discussed herein, the CIPSs disclosed herein comprise a reversible ZBM linked to an intein. The ZBM can be attached to the N-terminus or the C-terminus of the intein, or can be within the intein. 
     The ZBM can allow for the pulling away of the active site histidine into a metal-nitrogen chelation arrangement such that it is no longer available to activate the labile peptide bond for cission. 
     Disclosed herein are modified peptides comprising a ZBM which allows for enhanced binding of zinc when compared to an intein without a ZBM in which enhanced binding of zinc allows the bound zinc to interact with one or the other of two critical active site histidine residues. 
     The ZBM can be reversible. By “reversible” it is meant that the motif binds zinc when it is present, which can alter the behavior of the intein to which the ZBM is attached. This behavior of the intein, however, is reversible when zinc is removed. For example, an intein may cleave or splice when zinc is not present. However, this action can be inhibited in the presence of zinc. When the zinc is removed, however, because the action is reversible, the intein will continue to perform the activity which it would have done, had zinc not been present, such as cleaving or splicing. In some aspects, other divalent metals within the fourth row transition element series, other than zinc, can be used in the disclosed methods. Examples of other divalent metals within the fourth row transition element include, but are not limited to, titanium, vanadium, chromium, nickel, copper, manganese, iron, and cobalt. 
     The ZBMs disclosed herein can be designed such that when appended to, for example, the N-terminal portion of an intein, such as the Mtu-RecA intein, they introduce a zinc-binding, active-site modulating subdomain capable of reversibly switching the intein off. This sensitivity to zinc has enabled the successful use of the inteins under conditions that are compatible with commercially relevant mammalian cell culture expression platforms. For example, a reversible ZBM can be fused directly to the N-terminal end of the intein, with no linkers or additional amino acids separating the intein from the ZBM. 
     Disclosed herein are modified peptides comprising a CIPS. Also disclosed herein are peptides comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible zinc-binding motif and an intein. 
     Disclosed herein are CIPSs comprising an intein and a reversible ZBM, wherein the reversible ZBM comprises the structure: aa1-aa2-aa3-aa4, wherein aa1 is a non-polar amino acid, aa2 is a negatively-charged amino acid, aa3 is a non-polar amino acid and aa4 is a positively charged amino acid. For example, aa1 can be glycine, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, or valine; aa2 can be aspartic acid or glutamic acid; aa3 can be glycine, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, or valine; and aa4 can be arginine or lysine. 
     Disclosed herein are CIPSs comprising an intein and a reversible ZBM, wherein the reversible ZBM comprises the sequence: G-E-G-H (SEQ ID NO: 1) or G-D-G-H (SEQ ID NO: 2). 
     Disclosed herein are CIPSs comprising an intein and a reversible ZBM, wherein the reversible ZBM comprises the structure: aa1-aa2-aa3-aa4-aa5, wherein aa1 is a non-polar amino acid, aa2 is a negatively-charged amino acid, aa3 is a non-polar amino acid, aa4 is a positively charged amino acid and aa5 is a non-polar amino acid. For example, aa1 can be glycine, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, or valine; aa2 can be aspartic acid or glutamic acid or cysteine; aa3 can be glycine, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, or valine; aa4 can be arginine or lysine; and aa5 can be glycine, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, or valine. 
     Disclosed herein are CIPSs comprising an intein and a reversible ZBM, wherein the reversible ZBM comprises the sequence G-E-G-H-H (SEQ ID NO: 3), G-E-G-H-G (SEQ ID NO: 4), G-D-G-H-H (SEQ ID NO: 5), or G-D-G-H-G (SEQ ID NO: 6). 
     The corresponding nucleic acid sequences for the above ZBMs follow. Note that the underlined portion of the nucleic acid encodes the reversible ZBM, as well as the nucleic acid which codes for the first amino acid of the intein. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 GEGH 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 33) 
               
               
                   
                   GGAGAGGGACATCAC CTCGCAGAGGGCACTCGGAT 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 GEGH (alternate sequence encoding first amino 
               
               
                   
                 acid of intein)  
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 34) 
               
               
                   
                   GGAGAGGGACATTGC CTCGCAGAGGGCACTCGGAT 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 GEGHH 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 35) 
               
               
                   
                   GGAGAGGGACATCATGCC CTCGCAGAGGGCACTCG 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
                 GEGHG 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 36) 
               
               
                   
                   GGAGAGGGACATGGATGC CTCGCAGAGGGCACTCGG 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 GDGHH 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 37) 
               
               
                   
                   GGAGATGGACATCATGCC CTCGCAGAGGGCACTCGGA 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 GDGHG 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 38) 
               
               
                   
                   GGAGATGGACATGGATGC CTCGCAGAGGGCACTCGGA 
               
            
           
         
       
     
       FIG. 17  shows examples of various reversible ZBMs, as well as these ZBMs linked to an intein. 
     The CIPSs described herein can be engineered to possess an intein wherein the appended zinc-binding motif is placed (1) at a specific location near the intein&#39;s enzyme active site or (2) at other similar sites that either can directly or allosterically control the intein&#39;s active site dynamics and conformations. In some aspects, the CIPS can be engineered in a way that when the CIPS is exposed to zinc, the splicing rate can be decreased by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% when compared to an intein which has not been modified to comprise a zinc-binding domain. In the case where a Divalent Cation Binding Motif (DCBM) is used rather than a ZBM, the CIPS can be engineered in a way that when the CIPS is exposed to a divalent metal within the fourth row transition element series, the splicing rate can be decreased by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% when compared to an intein which has not been modified to comprise a DCBM. 
     Because the ZBM can be reversible, when the peptide comprising the CIPS is exposed to a chelating agent, a change in pH, dialysis, or dilution, thereby removing zinc, the CIPS can excisise the intein portion out by protein splicing, or cleavage in the absence of splicing. In some aspects, the modified peptide (comprising a CIPS, an affinity tag, and/or a protein of interest) can be subjected to these conditions. The CIPS may also be inserted into a region that substantially inhibits the activity of the protein of interest. 
     In some aspects, the zinc ion can be removed by a shift to a pH below 7.0, which can cause zinc loss via protonation of the chelating histidine residues. In some aspects, the zinc ion can be removed, for example, by addition of EDTA (Ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) to directly chelate the zinc ion. In some aspects, the zinc ion can be removed by dilution to a concentration below that required for intein inhibition, or by dialysis to remove the zinc ion from the intein. In some aspects, the zinc ion can be removed by buffer wash with an immobilized CIPS in a column chromatography format. 
     In some aspects, a CIPS can be engineered such that when zinc is present at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 750, 800, 850, 900, 1000, 1100, 1200, 1300, 1400, 1500, or more μM, the CIPS splicing activity can be prevented or reduced. One of skill in the art can readily ascertain the appropriate amount of zinc required to prevent specific intein portions of the CIPS from splicing or cleaving. 
     In some aspects, the CIPSs disclosed herein comprise a reversible DCBM linked to an intein. The DCBM can be attached to the N-terminus or the C-terminus of the intein, or can be within the intein. For example, disclosed herein are modified peptides comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible DCBM and an intein, wherein the intein comprises additional amino acids at the N-terminus of a naturally occurring intein. As used herein, a DCBM can be used instead of a ZBM, thereby allowing for other divalent metal ions to control the intein function. 
     As discussed herein, the CIPSs disclosed herein can comprise a reversible DCBM linked to an intein. The DCBM can be attached to the N-terminus or the C-terminus of the intein, or can be within the intein. 
     The DCBM can allow for the pulling away of the active site histidine into a metal-nitrogen chelation arrangement such that it is no longer available to activate the labile peptide bond for cission. 
     The DCBM can allow for enhanced binding of divalent metals within the fourth row transition element series when compared to an intein without a DCBM in which enhanced binding of a divalent metal within the fourth row transition element series allows the bound divalent metals within the fourth row transition element series to interact with one or the other of two critical active site histidine residues. 
     As described throughout, the DCBM and the corresponding divalent metal within the fourth row transition element series can be used as described herein for the ZBM and zinc so far as they relate to the methods and compositions described herein. 
     In Table 1, below, is a list of the various amino acids, their abbreviations, as well as their polarity and charge. Using this table, one of skill in the art can readily ascertain which amino acids can be used in the peptide compositions disclosed herein. 
     As discussed herein, the CIPSs disclosed herein comprise an intein. One way to control protein activity is through the use of inteins which can allow expression of an intein modified protein with a predefined activity level. As discussed herein, inteins are self-cleaving and self-ligating peptides. The collective attributes of being both self-cleaving and self-ligating are referred to as “self-splicing” or “splicing.” An intein cleaves from the protein and mediates ligation of the protein sequences (exteins) from which it cleaves to splice the protein. An intein may be inserted within the protein sequence or fused terminally to the protein. An intein insertion in a protein may allow control of a protein by yielding a modified protein that has one activity when the intein is present and another activity after intein cleavage or splicing. In some cases, the intein splicing reaction can be controlled by one or more of a variety of induction conditions, such as zinc, when the proper modification has been made to the intein. When an activity normally detrimental to the host is reduced, the intein protects the expression host from detrimental growth, physiological, or yield effects of the protein. After expression of the protein, the activity can be changed by exposing the modified protein to reaction conditions that induce intein splicing (such as removal of zinc by the methods disclosed herein). In one aspect, the modified protein that results after splicing has increased activity. Inteins can also be used in protein purification systems, described below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Amino Acids 
               
            
           
           
               
               
               
               
               
            
               
                   
                 3-Letter 
                 1-Letter 
                   
                   
               
               
                 Amino Acid 
                 Code 
                 Code 
                 Polarity 
                 Charge 
               
               
                   
               
               
                 Alanine 
                 Ala 
                 A 
                 nonpolar 
                 neutral 
               
               
                 Arginine 
                 Arg 
                 R 
                 Basic 
                 positive 
               
               
                   
                   
                   
                 polar 
               
               
                 Asparagine 
                 Asn 
                 N 
                 polar 
                 neutral 
               
               
                 Aspartic acid 
                 Asp 
                 D 
                 acidic 
                 negative 
               
               
                   
                   
                   
                 polar 
               
               
                 Cysteine 
                 Cys 
                 C 
                 nonpolar 
                 neutral 
               
               
                 Glutamic acid 
                 Glu 
                 E 
                 acidic 
                 negative 
               
               
                   
                   
                   
                 polar 
               
               
                 Glutamine 
                 Gln 
                 Q 
                 polar 
                 neutral 
               
               
                 Glycine 
                 Gly 
                 G 
                 nonpolar 
                 neutral 
               
               
                 Histidine 
                 His 
                 H 
                 Basic 
                 Positive (10%) 
               
               
                   
                   
                   
                 polar 
                 Neutral (90%) 
               
               
                 Isoleucine 
                 Ile 
                 I 
                 nonpolar 
                 neutral 
               
               
                 Leucine 
                 Leu 
                 L 
                 nonpolar 
                 neutral 
               
               
                 Lysine 
                 Lys 
                 K 
                 Basic 
                 positive 
               
               
                   
                   
                   
                 polar 
               
               
                 Methionine 
                 Met 
                 M 
                 nonpolar 
                 neutral 
               
               
                 Phenylalanine 
                 Phe 
                 F 
                 nonpolar 
                 neutral 
               
               
                 Proline 
                 Pro 
                 P 
                 nonpolar 
                 neutral 
               
               
                 Serine 
                 Ser 
                 S 
                 polar 
                 neutral 
               
               
                 Threonine 
                 Thr 
                 T 
                 polar 
                 neutral 
               
               
                 Tryptophan 
                 Trp 
                 W 
                 nonpolar 
                 neutral 
               
               
                 Tyrosine 
                 Tyr 
                 Y 
                 polar 
                 neutral 
               
               
                 Valine 
                 Val 
                 V 
                 nonpolar 
                 neutral 
               
               
                   
               
            
           
         
       
     
     Any intein can be used with the compositions and methods disclosed herein. For example, an intein can be used in the context of a CIPS. 
     Examples of inteins can be found in Perler, F. B. (2002) InBase, the Intein Database. Nucleic Acids Res. 30, 383-384, hereby incorporated by reference in its entirety for teaching examples of inteins that can be used with the compositions and methods disclosed herein. Additional examples of naturally occurring inteins include, but are not limited to, those found in Table 2. In one example, the intein can be Mtu-RecA. In some aspects, the inteins consist of two functionally and structurally distinct domains, a protein-splicing domain and an endonuclease domain. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Naturally Occurring Inteins 
               
            
           
           
               
               
               
            
               
                 Intein Name 
                 Organism Name 
                 Organism Description 
               
               
                   
               
            
           
           
               
            
               
                 Eucarya 
               
            
           
           
               
               
               
            
               
                 APMV Pol 
                   Acanthomoeba polyphaga  Mimivirus 
                 isolate = “Rowbotham- 
               
               
                   
                   
                 Bradford”, Virus, infects 
               
               
                   
                   
                 Amoebae, taxon: 212035 
               
               
                 Abr PRP8 
                   Aspergillus brevipes  FRR2439 
                 Fungi, ATCC 16899, 
               
               
                   
                   
                 taxon: 75551 
               
               
                 Aca-G186AR PRP8 
                   Ajellomyces capsulatus  G186AR 
                 Taxon: 447093, strain 
               
               
                   
                   
                 G186AR 
               
               
                 Aca-H143 PRP8 
                   Ajellomyces capsulatus  H143 
                 Taxon: 544712 
               
               
                 Aca-JER2004 PRP8 
                   Ajellomyces capsulatus  (anamorph: 
                 strain = JER2004, taxon: 5037, 
               
               
                   
                   Histoplasma capsulatum ) 
                 Fungi 
               
               
                 Aca-NAm1 PRP8 
                   Ajellomyces capsulatus  NAm1 
                 strain = “NAm1”, taxon: 339724 
               
               
                 Ade-ER3 PRP8 
                   Ajellomyces dermatitidis  ER-3 
                 Human fungal 
               
               
                   
                   
                 pathogen. taxon: 559297 
               
               
                 Ade-SLH14081 PRP8 
                   Ajellomyces dermatitidis  SLH14081, 
                 Human fungal pathogen 
               
               
                 Afu-Af293 PRP8 
                   Aspergillus fumigatus  var.  ellipticus , 
                 Human pathogenic fungus, 
               
               
                   
                 strain Af293 
                 taxon: 330879 
               
               
                 Afu-FRR0163 PRP8 
                   Aspergillus fumigatus  strain 
                 Human pathogenic fungus, 
               
               
                   
                 FRR0163 
                 taxon: 5085 
               
               
                 Afu-NRRL5109 PRP8 
                   Aspergillus fumigatus  var.  ellipticus , 
                 Human pathogenic fungus, 
               
               
                   
                 strain NRRL 5109 
                 taxon: 41121 
               
               
                 Agi-NRRL6136 PRP8 
                   Aspergillus giganteus  Strain NRRL 
                 Fungus, taxon: 5060 
               
               
                   
                 6136 
               
               
                 Ani-FGSCA4 PRP8 
                   Aspergillus nidulans  FGSC A 
                 Filamentous fungus, 
               
               
                   
                   
                 taxon: 227321 
               
               
                 Avi PRP8 
                   Aspergillus viridinutans  strain 
                 Fungi, ATCC 16902, 
               
               
                   
                 FRR0577 
                 taxon: 75553 
               
               
                 Bci PRP8 
                   Botrytis cinerea  (teleomorph of 
                 Plant fungal pathogen 
               
               
                   
                   Botryotinia fuckeliana  B05.10) 
               
               
                 Bde-JEL197 RPB2 
                 
                   Batrachochytrium dendrobatidis 
                 
                 Chytrid fungus, 
               
               
                   
                 JEL197 
                 isolate = “AFTOL-ID 21”, 
               
               
                   
                   
                 taxon: 109871 
               
               
                 Bde-JEL423 PRP8-1 
                 
                   Batrachochytrium dendrobatidis 
                 
                 Chytrid fungus, isolate 
               
               
                   
                 JEL423 
                 JEL423, taxon 403673 
               
               
                 Bde-JEL423 PRP8-2 
                 
                   Batrachochytrium dendrobatidis 
                 
                 Chytrid fungus, isolate 
               
               
                   
                 JEL423 
                 JEL423, taxon 403673 
               
               
                 Bde-JEL423 RPC2 
                 
                   Batrachochytrium dendrobatidis 
                 
                 Chytrid fungus, isolate 
               
               
                   
                 JEL423 
                 JEL423, taxon 403673 
               
               
                 Bde-JEL423 eIF-5B 
                 
                   Batrachochytrium dendrobatidis 
                 
                 Chytrid fungus, isolate 
               
               
                   
                 JEL423 
                 JEL423, taxon 403673 
               
               
                 Bfu-B05 PRP8 
                   Botryotinia fuckeliana  B05.10 
                 Taxon: 332648 
               
               
                 CIV RIR1 
                 Chilo iridescent virus 
                 dsDNA eucaryotic virus, 
               
               
                   
                   
                 taxon: 10488 
               
               
                 CV-NY2A ORF212392 
                   Chlorella  virus NY2A infects 
                 dsDNA eucaryotic 
               
               
                   
                   Chlorella  NC64A, which infects 
                 virus, taxon: 46021, Family 
               
               
                   
                 
                   Paramecium bursaria 
                 
                 Phycodnaviridae 
               
               
                 CV-NY2A RIR1 
                   Chlorella  virus NY2A infects 
                 dsDNA eucaryotic 
               
               
                   
                   Chlorella  NC64A, which infects 
                 virus, taxon: 46021, Family 
               
               
                   
                 
                   Paramecium bursaria 
                 
                 Phycodnaviridae 
               
               
                 CZIV RIR1 
                   Costelytra zealandica  iridescent virus 
                 dsDNA eucaryotic virus, 
               
               
                   
                   
                 Taxon: 68348 
               
               
                 Cba-WM02.98 PRP8 
                   Cryptococcus bacillisporus  strain 
                 Yeast, human pathogen, 
               
               
                   
                 WM02.98 (aka  Cryptococcus   
                 taxon: 37769 
               
               
                   
                   neoformans gattii ) 
               
               
                 Cba-WM728 PRP8 
                   Cryptococcus bacillisporus  strain 
                 Yeast, human pathogen, 
               
               
                   
                 WM728 
                 taxon: 37769 
               
               
                 Ceu ClpP 
                 
                   Chlamydomonas eugametos 
                 
                 Green alga, taxon: 3053 
               
               
                   
                 (chloroplast) 
               
               
                 Cga PRP8 
                   Cryptococcus gattii  (aka 
                 Yeast, human pathogen 
               
               
                   
                   Cryptococcus bacillisporus ) 
               
               
                 Cgl VMA 
                 
                   Candida glabrata 
                 
                 Yeast, taxon: 5478 
               
               
                 Cla PRP8 
                   Cryptococcus laurentii  strain 
                 Fungi, Basidiomycete yeast, 
               
               
                   
                 CBS139 
                 taxon: 5418 
               
               
                 Cmo ClpP 
                   Chlamydomonas moewusii , strain 
                 Green alga, chloroplast gene, 
               
               
                   
                 UTEX 97 
                 taxon: 3054 
               
               
                 Cmo RPB2 (RpoBb) 
                   Chlamydomonas moewusii , strain 
                 Green alga, chloroplast gene, 
               
               
                   
                 UTEX 97 
                 taxon: 3054 
               
               
                 Cne-A PRP8 (Fne-A 
                 
                   Filobasidiella neoformans 
                 
                 Yeast, human pathogen 
               
               
                 PRP8) 
                 ( Cryptococcus neoformans ) Serotype 
               
               
                   
                 A, PHLS_8104 
               
               
                 Cne-AD PRP8 (Fne- 
                 
                   Cryptococcus neoformans 
                 
                 Yeast, human pathogen, 
               
               
                 AD PRP8) 
                 ( Filobasidiella neoformans ), 
                 ATCC32045, taxon: 5207 
               
               
                   
                 Serotype AD, CBS132). 
               
               
                 Cne-JEC21 PRP8 
                   Cryptococcus neoformans  var. 
                 Yeast, human pathogen, 
               
               
                   
                   neoformans  JEC21 
                 serotype = “D” taxon: 214684 
               
               
                 Cpa ThrRS 
                   Candida parapsilosis , strain CLIB214 
                 Yeast, Fungus, taxon: 5480 
               
               
                 Cre RPB2 
                 
                   Chlamydomonas reinhardtii 
                 
                 Green algae, taxon: 3055 
               
               
                   
                 (nucleus) 
               
               
                 CroV Pol 
                   Cafeteria roenbergensis  virus BV- 
                 taxon: 693272, Giant virus 
               
               
                   
                 PW1 
                 infecting marine heterotrophic 
               
               
                   
                   
                 nanoflagellate 
               
               
                 CroV RIR1 
                   Cafeteria roenbergensis  virus BV- 
                 taxon: 693272, Giant virus 
               
               
                   
                 PW1 
                 infecting marine heterotrophic 
               
               
                   
                   
                 nanoflagellate 
               
               
                 CroV RPB2 
                   Cafeteria roenbergensis  virus BV- 
                 taxon: 693272, Giant virus 
               
               
                   
                 PW1 
                 infecting marine heterotrophic 
               
               
                   
                   
                 nanoflagellate 
               
               
                 CroV Top2 
                   Cafeteria roenbergensis  virus BV- 
                 taxon: 693272, Giant virus 
               
               
                   
                 PW1 
                 infecting marine heterotrophic 
               
               
                   
                   
                 nanoflagellate 
               
               
                 Cst RPB2 
                 
                   Coelomomyces stegomyiae 
                 
                 Chytrid fungus, 
               
               
                   
                   
                 isolate = “AFTOL-ID 18”, 
               
               
                   
                   
                 taxon: 143960 
               
               
                 Ctr ThrRS 
                   Candida tropicalis  ATCC750 
                 Yeast 
               
               
                 Ctr VMA 
                   Candida tropicalis  (nucleus) 
                 Yeast 
               
               
                 Ctr-MYA3404 VMA 
                   Candida tropicalis  MYA-3404 
                 Taxon: 294747 
               
               
                 Ddi RPC2 
                   Dictyostelium discoideum  strain 
                 Mycetozoa (a social amoeba) 
               
               
                   
                 AX4 (nucleus) 
               
               
                 Dhan GLT1 
                   Debaryomyces hansenii  CBS767 
                 Fungi, Anamorph:  Candida   
               
               
                   
                   
                   famata , taxon: 4959 
               
               
                 Dhan VMA 
                   Debaryomyces hansenii  CBS767 
                 Fungi, taxon: 284592 
               
               
                 Eni PRP8 
                   Emericella nidulans  R20 (anamorph: 
                 taxon: 162425 
               
               
                   
                   Aspergillus nidulans ) 
               
               
                 Eni-FGSCA4 PRP8 
                   Emericella nidulans  (anamorph: 
                 Filamentous fungus, 
               
               
                   
                   Aspergillus nidulans ) FGSC A4 
                 taxon: 162425 
               
               
                 Fte RPB2 (RpoB) 
                   Floydiella terrestris , strain UTEX 
                 Green alga, chloroplast gene, 
               
               
                   
                 1709 
                 taxon: 51328 
               
               
                 Gth DnaB 
                   Guillardia theta  (plastid) 
                 Cryptophyte Algae 
               
               
                 HaV01 Pol 
                   Heterosigma akashiwo  virus 01 
                 Algal virus, taxon: 97195, 
               
               
                   
                   
                 strain HaV01 
               
               
                 Hca PRP8 
                   Histoplasma capsulatum  (anamorph: 
                 Fungi, human pathogen 
               
               
                   
                   Ajellomyces capsulatus ) 
               
               
                 IIV6 RIR1 
                 Invertebrate iridescent virus 6 
                 dsDNA eucaryotic 
               
               
                   
                   
                 virus, taxon: 176652 
               
               
                 Kex-CBS379 VMA 
                   Kazachstania exigua , formerly 
                 Yeast, taxon: 34358 
               
               
                   
                   Saccharomyces exiguus , strain 
               
               
                   
                 CBS379 
               
               
                 Kla-CBS683 VMA 
                   Kluyveromyces lactis , strain CBS683 
                 Yeast, taxon: 28985 
               
               
                 Kla-IFO1267 VMA 
                   Kluyveromyces lactis  IFO1267 
                 Fungi, taxon: 28985 
               
               
                 Kla-NRRLY1140 
                   Kluyveromyces lactis  NRRL Y-1140 
                 Fungi, taxon: 284590 
               
               
                 VMA 
               
               
                 Lel VMA 
                 
                   Lodderomyces elongisporus 
                 
                 Yeast 
               
               
                 Mca-CBS113480 PRP8 
                   Microsporum canis  CBS 113480 
                 Taxon: 554155 
               
               
                 Nau PRP8 
                   Neosartorya aurata  NRRL 4378 
                 Fungus, taxon: 41051 
               
               
                 Nfe-NRRL5534 PRP8 
                   Neosartorya fennelliae  NRRL 5534 
                 Fungus, taxon: 41048 
               
               
                 Nfi PRP8 
                 
                   Neosartorya fischeri 
                 
                 Fungi 
               
               
                 Ngl-FR2163 PRP8 
                   Neosartorya glabra  FRR2163 
                 Fungi, ATCC 16909, 
               
               
                   
                   
                 taxon: 41049 
               
               
                 Ngl-FRR1833 PRP8 
                   Neosartorya glabra  FRR1833 
                 Fungi, taxon: 41049, 
               
               
                   
                   
                 (preliminary identification) 
               
               
                 Nqu PRP8 
                   Neosartorya quadricincta , strain 
                 taxon: 41053 
               
               
                   
                 NRRL 4175 
               
               
                 Nspi PRP8 
                   Neosartorya spinosa  FRR4595 
                 Fungi, taxon: 36631 
               
               
                 Pabr-Pb01 PRP8 
                   Paracoccidioides brasiliensis  Pb01 
                 Taxon: 502779 
               
               
                 Pabr-Pb03 PRP8 
                   Paracoccidioides brasiliensis  Pb03 
                 Taxon: 482561 
               
               
                 Pan CHS2 
                 
                   Podospora anserina 
                 
                 Fungi, Taxon 5145 
               
               
                 Pan GLT1 
                 
                   Podospora anserina 
                 
                 Fungi, Taxon 5145 
               
               
                 Pbl PRP8-a 
                 
                   Phycomyces blakesleeanus 
                 
                 Zygomycete fungus, strain 
               
               
                   
                   
                 NRRL155 
               
               
                 Pbl PRP8-b 
                 
                   Phycomyces blakesleeanus 
                 
                 Zygomycete fungus, strain 
               
               
                   
                   
                 NRRL155 
               
               
                 Pbr-Pb18 PRP8 
                   Paracoccidioides brasiliensis  Pb18 
                 Fungi, taxon: 121759 
               
               
                 Pch PRP8 
                 
                   Penicillium chrysogenum 
                 
                 Fungus, taxon: 5076 
               
               
                 Pex PRP8 
                 
                   Penicillium expansum 
                 
                 Fungus, taxon27334 
               
               
                 Pgu GLT1 
                   Pichia  ( Candida )  guilliermondii   
                 Fungi, Taxon 294746 
               
               
                 Pgu-alt GLT1 
                   Pichia  ( Candida )  guilliermondii   
                 Fungi 
               
               
                 Pno GLT1 
                   Phaeosphaeria nodorum  SN15 
                 Fungi, taxon: 321614 
               
               
                 Pno RPA2 
                   Phaeosphaeria nodorum  SN15 
                 Fungi, taxon: 321614 
               
               
                 Ppu DnaB 
                   Porphyra purpurea  (chloroplast) 
                 Red Alga 
               
               
                 Pst VMA 
                   Pichia stipitis  CBS 6054, 
                 Yeast 
               
               
                   
                 taxon: 322104 
               
               
                 Ptr PRP8 
                   Pyrenophora tritici - repentis  Pt-1C- 
                 Ascomycete 
               
               
                   
                 BF 
                 fungus, taxon: 426418 
               
               
                 Pvu PRP8 
                   Penicillium vulpinum  (formerly 
                 Fungus 
               
               
                   
                   P. claviforme ) 
               
               
                 Pye DnaB 
                   Porphyra yezoensis  chloroplast, 
                 Red alga, 
               
               
                   
                 cultivar U-51 
                 organelle = “plastid: chloroplast”, 
               
               
                   
                   
                 “taxon: 2788 
               
               
                 Sas RPB2 
                   Spiromyces aspiralis  NRRL 22631 
                 Zygomycete fungus, 
               
               
                   
                   
                 isolate = “AFTOL-ID 
               
               
                   
                   
                 185”, taxon: 68401 
               
               
                 Sca-CBS4309 VMA 
                   Saccharomyces castellii , strain 
                 Yeast, taxon: 27288 
               
               
                   
                 CBS4309 
               
               
                 Sca-IFO1992 VMA 
                   Saccharomyces castellii , strain 
                 Yeast, taxon: 27288 
               
               
                   
                 IFO1992 
               
               
                 Scar VMA 
                   Saccharomyces cariocanus , 
                 Yeast, taxon: 114526 
               
               
                   
                 strain = “UFRJ 50791 
               
               
                 Sce VMA 
                   Saccharomyces cerevisiae  (nucleus) 
                 Yeast, also in Sce strains 
               
               
                   
                   
                 OUT7163, OUT7045, 
               
               
                   
                   
                 OUT7163, IFO1992 
               
               
                 Sce-DH1-1A VMA 
                   Saccharomyces cerevisiae  strain 
                 Yeast, taxon: 173900, also in 
               
               
                   
                 DH1-1A 
                 Sce strains 
               
               
                   
                   
                 OUT7900, OUT7903, OUT7112 
               
               
                 Sce-JAY291 VMA 
                   Saccharomyces cerevisiae  JAY291 
                 Taxon: 574961 
               
               
                 Sce-OUT7091 VMA 
                   Saccharomyces cerevisiae  OUT7091 
                 Yeast, taxon: 4932, also in Sce 
               
               
                   
                   
                 strains OUT7043, OUT7064 
               
               
                 Sce-OUT7112 VMA 
                   Saccharomyces cerevisiae  OUT7112 
                 Yeast, taxon: 4932, also in Sce 
               
               
                   
                   
                 strains OUT7900, OUT7903 
               
               
                 Sce-YJM789 VMA 
                   Saccharomyces cerevisiae  strain 
                 Yeast, taxon: 307796 
               
               
                   
                 YJM789 
               
               
                 Sda VMA 
                   Saccharomyces dairenensis , strain 
                 Yeast, taxon: 27289, Also in 
               
               
                   
                 CBS 421 
                 Sda strain IFO0211 
               
               
                 Sex-IFO1128 VMA 
                   Saccharomyces exiguus , 
                 Yeast, taxon: 34358 
               
               
                   
                 strain = “IFO1128” 
               
               
                 She RPB2 (RpoB) 
                   Stigeoclonium helveticum , strain 
                 Green alga, chloroplast gene, 
               
               
                   
                 UTEX 441 
                 taxon: 55999 
               
               
                 Sja VMA 
                 
                   Schizosaccharomyces japonicus 
                 
                 Ascomycete fungus, 
               
               
                   
                 yFS275 
                 taxon: 402676 
               
               
                 Spa VMA 
                 
                   Saccharomyces pastorianus 
                 
                 Yeast, taxon: 27292 
               
               
                   
                 IFO11023 
               
               
                 Spu PRP8 
                 
                   Spizellomyces punctatus 
                 
                 Chytrid fungus, 
               
               
                 Sun VMA 
                   Saccharomyces unisporus , strain 
                 Yeast, taxon: 27294 
               
               
                   
                 CBS 398 
               
               
                 Tgl VMA 
                   Torulaspora globosa , strain CBS 764 
                 Yeast, taxon: 48254 
               
               
                 Tpr VMA 
                   Torulaspora pretoriensis , strain CBS 
                 Yeast, taxon: 35629 
               
               
                   
                 5080 
               
               
                 Ure-1704 PRP8 
                 
                   Uncinocarpus reesii 
                 
                 Filamentous fungus 
               
               
                 Vpo VMA 
                   Vanderwaltozyma polyspora , 
                 Yeast, taxon: 36033 
               
               
                   
                 formerly  Kluyveromyces polysporus , 
               
               
                   
                 strain CBS 2163 
               
               
                 WIV RIR1 
                   Wiseana  iridescent virus 
                 dsDNA eucaryotic 
               
               
                   
                   
                 virus, taxon: 68347 
               
               
                 Zba VMA 
                   Zygosaccharomyces bailii , strain 
                 Yeast, taxon: 4954 
               
               
                   
                 CBS 685 
               
               
                 Zbi VMA 
                   Zygosaccharomyces bisporus , strain 
                 Yeast, taxon: 4957 
               
               
                   
                 CBS 702 
               
               
                 Zro VMA 
                   Zygosaccharomyces rouxii , strain 
                 Yeast, taxon: 4956 
               
               
                   
                 CBS 688 
               
            
           
           
               
            
               
                 Eubacteria 
               
            
           
           
               
               
               
            
               
                 AP-APSE1 dpol 
                   Acyrthosiphon pisum  secondary 
                 Bacteriophage, taxon: 67571 
               
               
                   
                 endosymbiot phage 1 
               
               
                 AP-APSE2 dpol 
                 Bacteriophage APSE-2, isolate = T5A 
                 Bacteriophage of  Candidatus   
               
               
                   
                   
                   Hamiltonella defensa , 
               
               
                   
                   
                 endosymbiot of 
               
               
                   
                   
                   Acyrthosiphon pisum , 
               
               
                   
                   
                 taxon: 340054 
               
               
                 AP-APSE4 dpol 
                 Bacteriophage of  Candidatus   
                 Bacteriophage, taxon: 568990 
               
               
                   
                   Hamiltonella defensa  strain 5ATac, 
               
               
                   
                 endosymbiot of  Acyrthosiphon   
               
               
                   
                 
                   pisum 
                 
               
               
                 AP-APSE5 dpol 
                 Bacteriophage APSE-5 
                 Bacteriophage of  Candidatus   
               
               
                   
                   
                   Hamiltonella defensa , 
               
               
                   
                   
                 endosymbiot of  Uroleucon   
               
               
                   
                   
                   rudbeckiae , taxon: 568991 
               
               
                 AP-Aaphi23 MupF 
                 Bacteriophage Aaphi23, 
                 
                   Actinobacillus 
                 
               
               
                   
                   Haemophilus  phage Aaphi23 
                 
                   actinomycetemcomitans 
                 
               
               
                   
                   
                 Bacteriophage, taxon: 230158 
               
               
                 Aae RIR2 
                   Aquifex aeolicus  strain VF5 
                 Thermophilic 
               
               
                   
                   
                 chemolithoautotroph, 
               
               
                   
                   
                 taxon: 63363 
               
               
                 Aave-AAC001 
                   Acidovorax avenae  subsp.  citrulli   
                 taxon: 397945 
               
               
                 Aave1721 
                 AAC00-1 
               
               
                 Aave-AAC001 RIR1 
                   Acidovorax avenae  subsp.  citrulli   
                 taxon: 397945 
               
               
                   
                 AAC00-1 
               
               
                 Aave-ATCC19860 
                   Acidovorax avenae  subsp.  avenae   
                 Taxon: 643561 
               
               
                 RIR1 
                 ATCC 19860 
               
               
                 Aba Hyp-02185 
                   Acinetobacter baumannii  ACICU 
                 taxon: 405416 
               
               
                 Ace RIR1 
                   Acidothermus cellulolyticus  11B 
                 taxon: 351607 
               
               
                 Aeh DnaB-1 
                   Alkalilimnicola ehrlichei  MLHE-1 
                 taxon: 187272 
               
               
                 Aeh DnaB-2 
                   Alkalilimnicola ehrlichei  MLHE-1 
                 taxon: 187272 
               
               
                 Aeh RIR1 
                   Alkalilimnicola ehrlichei  MLHE-1 
                 taxon: 187272 
               
               
                 AgP-S1249 MupF 
                   Aggregatibacter  phage S1249 
                 Taxon: 683735 
               
               
                 Aha DnaE-c 
                 
                   Aphanothece halophytica 
                 
                 Cyanobacterium, taxon: 72020 
               
               
                 Aha DnaE-n 
                 
                   Aphanothece halophytica 
                 
                 Cyanobacterium, taxon: 72020 
               
               
                 Alvi-DSM180 GyrA 
                   Allochromatium vinosum  DSM 180 
                 Taxon: 572477 
               
               
                 Ama MADE823 
                 phage uncharacterized protein 
                 Probably prophage gene, 
               
               
                   
                 [ Alteromonas macleodii  ‘Deep 
                 taxon: 314275 
               
               
                   
                 ecotype’] 
               
               
                 Amax-CS328 DnaX 
                   Arthrospira maxima  CS-328 
                 Taxon: 513049 
               
               
                 Aov DnaE-c 
                 
                   Aphanizomenon ovalisporum 
                 
                 Cyanobacterium, taxon: 75695 
               
               
                 Aov DnaE-n 
                 
                   Aphanizomenon ovalisporum 
                 
                 Cyanobacterium, taxon: 75695 
               
               
                 Apl-C1 DnaX 
                 
                   Arthrospira platensis 
                 
                 Taxon: 118562, strain C1 
               
               
                 Arsp-FB24 DnaB 
                   Arthrobacter  species FB24 
                 taxon: 290399 
               
               
                 Asp DnaE-c 
                   Anabaena  species PCC7120, ( Nostoc   
                 Cyanobacterium, Nitrogen- 
               
               
                   
                 sp. PCC7120) 
                 fixing, taxon: 103690 
               
               
                 Asp DnaE-n 
                   Anabaena  species PCC7120, ( Nostoc   
                 Cyanobacterium, Nitrogen- 
               
               
                   
                 sp. PCC7120) 
                 fixing, taxon: 103690 
               
               
                 Ava DnaE-c 
                   Anabaena variabilis  ATCC29413 
                 Cyanobacterium, taxon: 240292 
               
               
                 Ava DnaE-n 
                   Anabaena variabilis  ATCC29413 
                 Cyanobacterium, taxon: 240292 
               
               
                 Avin RIR1 BIL 
                 
                   Azotobacter vinelandii 
                 
                 taxon: 354 
               
               
                 Bce-MCO3 DnaB 
                   Burkholderia cenocepacia  MC0-3 
                 taxon: 406425 
               
               
                 Bce-PC184 DnaB 
                   Burkholderia cenocepacia  PC184 
                 taxon: 350702 
               
               
                 Bse-MLS10 TerA 
                   Bacillus selenitireducens  MLS10 
                 Probably prophage gene, 
               
               
                   
                   
                 Taxon: 439292 
               
               
                 BsuP-M1918 RIR1 
                   B. subtilis  M1918 (prophage) 
                 Prophage in  B. subtilis  M1918. 
               
               
                   
                   
                 taxon: 157928 
               
               
                 BsuP-SPBc2 RIR1 
                   B. subtilis  strain 168 Sp beta c2 
                   B. subtilis  taxon 1423. SPbeta 
               
               
                   
                 prophage 
                 c2 phage, taxon: 66797 
               
               
                 Bvi IcmO 
                   Burkholderia vietnamiensis  G4 
                 plasmid = “pBVIE03”. 
               
               
                   
                   
                 taxon: 269482 
               
               
                 CP-P1201 Thy1 
                   Corynebacterium  phage P1201 
                 lytic bacteriophage P1201 
               
               
                   
                   
                 from  Corynebacterium   
               
               
                   
                   
                   glutamicum  NCHU 
               
               
                   
                   
                 87078. Viruses; dsDNA 
               
               
                   
                   
                 viruses, taxon: 384848 
               
               
                 Cag RIR1 
                 
                   Chlorochromatium aggregatum 
                 
                 Motile, phototrophic consortia 
               
               
                 Cau SpoVR 
                   Chloroflexus aurantiacus  J-10-fl 
                 Anoxygenic 
               
               
                   
                   
                 phototroph, taxon: 324602 
               
               
                 CbP-C-St RNR 
                   Clostridium botulinum  phage C-St 
                 Phage, specific_host = “ Clostridium   
               
               
                   
                   
                   botulinum  type C strain 
               
               
                   
                   
                 C-Stockholm, taxon: 12336 
               
               
                 CbP-D1873 RNR 
                   Clostridium botulinum  phage D 
                 Ssp. phage from  Clostridium   
               
               
                   
                   
                   botulinum  type D strain, 1873, 
               
               
                   
                   
                 taxon: 29342 
               
               
                 Cbu-Dugway DnaB 
                   Coxiella burnetii  Dugway 5J108-111 
                 Proteobacteria; Legionellales; 
               
               
                   
                   
                 taxon: 434922 
               
               
                 Cbu-Goat DnaB 
                   Coxiella burnetii  ‘MSU Goat Q177’ 
                 Proteobacteria; Legionellales; 
               
               
                   
                   
                 taxon: 360116 
               
               
                 Cbu-RSA334 DnaB 
                   Coxiella burnetii  RSA 334 
                 Proteobacteria; Legionellales; 
               
               
                   
                   
                 taxon: 360117 
               
               
                 Cbu-RSA493 DnaB 
                   Coxiella burnetii  RSA 493 
                 Proteobacteria; Legionellales; 
               
               
                   
                   
                 taxon: 227377 
               
               
                 Cce Hyp1-Csp-2 
                   Cyanothece  sp. ATCC 51142 
                 Marine unicellular 
               
               
                   
                   
                 diazotrophic cyanobacterium, 
               
               
                   
                   
                 taxon: 43989 
               
               
                 Cch RIR1 
                   Chlorobium chlorochromatii  CaD3 
                 taxon: 340177 
               
               
                 Ccy Hyp1-Csp-1 
                   Cyanothece  sp. CCY0110 
                 Cyanobacterium, 
               
               
                   
                   
                 taxon: 391612 
               
               
                 Ccy Hyp1-Csp-2 
                   Cyanothece  sp. CCY0110 
                 Cyanobacterium, 
               
               
                   
                   
                 taxon: 391612 
               
               
                 Cfl-DSM20109 DnaB 
                   Cellulomonas flavigena  DSM 20109 
                 Taxon: 446466 
               
               
                 Chy RIR1 
                 
                   Carboxydothermus 
                 
                 Thermophile, taxon = 246194 
               
               
                   
                   hydrogenoformans  Z-2901 
               
               
                 Ckl PTerm 
                   Clostridium kluyveri  DSM 555 
                 plasmid = “pCKL555A”, 
               
               
                   
                   
                 taxon: 431943 
               
               
                 Cra-CS505 DnaE-c 
                   Cylindrospermopsis raciborskii  CS-505 
                 Taxon: 533240 
               
               
                 Cra-CS505 DnaE-n 
                   Cylindrospermopsis raciborskii  CS-505 
                 Taxon: 533240 
               
               
                 Cra-CS505 GyrB 
                   Cylindrospermopsis raciborskii  CS-505 
                 Taxon: 533240 
               
               
                 Csp-CCY0110 DnaE-c 
                   Cyanothece  sp. CCY0110 
                 Taxon: 391612 
               
               
                 Csp-CCY0110 DnaE-n 
                   Cyanothece  sp. CCY0110 
                 Taxon: 391612 
               
               
                 Csp-PCC7424 DnaE-c 
                   Cyanothece  sp. PCC 7424 
                 Cyanobacterium, taxon: 65393 
               
               
                 Csp-PCC7424 DnaE-n 
                   Cyanothece  sp. PCC7424 
                 Cyanobacterium, taxon: 65393 
               
               
                 Csp-PCC7425 DnaB 
                   Cyanothece  sp. PCC 7425 
                 Taxon: 395961 
               
               
                 Csp-PCC7822 DnaE-n 
                   Cyanothece  sp. PCC 7822 
                 Taxon: 497965 
               
               
                 Csp-PCC8801 DnaE-c 
                   Cyanothece  sp. PCC 8801 
                 Taxon: 41431 
               
               
                 Csp-PCC8801 DnaE-n 
                   Cyanothece  sp. PCC 8801 
                 Taxon: 41431 
               
               
                 Cth ATPase BIL 
                 
                   Clostridium thermocellum 
                 
                 ATCC27405, taxon: 203119 
               
               
                 Cth-ATCC27405 TerA 
                 
                   Clostridium thermocellum 
                 
                 Probable prophage, 
               
               
                   
                 ATCC27405 
                 ATCC27405, taxon: 203119 
               
               
                 Cth-DSM2360 TerA 
                   Clostridium thermocellum  DSM 
                 Probably prophage 
               
               
                   
                 2360 
                 gene, Taxon: 572545 
               
               
                 Cwa DnaB 
                   Crocosphaera watsonii  WH 8501 
                 taxon: 165597 
               
               
                   
                 ( Synechocystis  sp. WH 8501) 
               
               
                 Cwa DnaE-c 
                   Crocosphaera watsonii  WH 8501 
                 Cyanobacterium, 
               
               
                   
                 ( Synechocystis  sp. WH 8501) 
                 taxon: 165597 
               
               
                 Cwa DnaE-n 
                   Crocosphaera watsonii  WH 8501 
                 Cyanobacterium, 
               
               
                   
                 ( Synechocystis  sp. WH 8501) 
                 taxon: 165597 
               
               
                 Cwa PEP 
                   Crocosphaera watsonii  WH 8501 
                 taxon: 165597 
               
               
                   
                 ( Synechocystis  sp. WH 8501) 
               
               
                 Cwa RIR1 
                   Crocosphaera watsonii  WH 8501 
                 taxon: 165597 
               
               
                   
                 ( Synechocystis  sp. WH 8501) 
               
               
                 Daud RIR1 
                 
                   Candidatus Desulforudis audaxviator 
                 
                 taxon: 477974 
               
               
                   
                 MP104C 
               
               
                 Dge DnaB 
                 
                   Deinococcus geothermalis 
                 
                 Thermophilic, radiation 
               
               
                   
                 DSM11300 
                 resistant 
               
               
                 Dha-DCB2 RIR1 
                   Desulfitobacterium hafniense  DCB-2 
                 Anaerobic dehalogenating 
               
               
                   
                   
                 bacteria, taxon: 49338 
               
               
                 Dha-Y51 RIR1 
                   Desulfitobacterium hafniense  Y51 
                 Anaerobic dehalogenating 
               
               
                   
                   
                 bacteria, taxon: 138119 
               
               
                 Dpr-MLMSl RIR1 
                 delta proteobacterium MLMS-1 
                 Taxon: 262489 
               
               
                 Dra RIR1 
                   Deinococcus radiodurans  R1, TIGR 
                 Radiation resistant, 
               
               
                   
                 strain 
                 taxon: 1299 
               
               
                 Dra Snf2-c 
                   Deinococcus radiodurans  R1, TIGR 
                 Radiation and DNA damage 
               
               
                   
                 strain 
                 resistent, taxon: 1299 
               
               
                 Dra Snf2-n 
                   Deinococcus radiodurans  R1, TIGR 
                 Radiation and DNA damage 
               
               
                   
                 strain 
                 resistent, taxon: 1299 
               
               
                 Dra-ATCC13939 Snf2 
                   Deinococcus radiodurans  R1, 
                 Radiation and DNA damage 
               
               
                   
                 ATCC13939/Brooks &amp; Murray strain 
                 resistent, taxon: 1299 
               
               
                 Dth UDP GD 
                   Dictyoglomus thermophilum  H-6-12 
                 strain = “H-6-12; ATCC 35947, 
               
               
                   
                   
                 taxon: 309799 
               
               
                 Dvul ParB 
                   Desulfovibrio vulgaris  subsp. 
                 taxon: 391774 
               
               
                   
                   vulgaris  DP4 
               
               
                 EP-Min27 Primase 
                 Enterobacteria phage Min27 
                 bacteriphage of 
               
               
                   
                   
                 host = “ Escherichia coli   
               
               
                   
                   
                 O157: H7 str. Min27” 
               
               
                 Fal DnaB 
                   Frankia alni  ACN14a 
                 Plant symbiot, taxon: 326424 
               
               
                 Fsp-CcI3 RIR1 
                   Frankia  species CcI3 
                 taxon: 106370 
               
               
                 Gob DnaE 
                   Gemmata obscuriglobus  UQM2246 
                 Taxon 114, TIGR genome 
               
               
                   
                   
                 strain, budding bacteria 
               
               
                 Gob Hyp 
                   Gemmata obscuriglobus  UQM2246 
                 Taxon 114, TIGR genome 
               
               
                   
                   
                 strain, budding bacteria 
               
               
                 Gvi DnaB 
                   Gloeobacter violaceus , PCC 7421 
                 taxon: 33072 
               
               
                 Gvi RIR1-1 
                   Gloeobacter violaceus , PCC 7421 
                 taxon: 33072 
               
               
                 Gvi RIR1-2 
                   Gloeobacter violaceus , PCC 7421 
                 taxon: 33072 
               
               
                 Hhal DnaB 
                   Halorhodospira halophila  SL1 
                 taxon: 349124 
               
               
                 Kfl-DSM17836 DnaB 
                   Kribbella flavida  DSM 17836 
                 Taxon: 479435 
               
               
                 Kra DnaB 
                 
                   Kineococcus radiotolerans 
                 
                 Radiation resistant 
               
               
                   
                 SRS30216 
               
               
                 LLP-KSY1 PolA 
                   Lactococcus  phage KSY1 
                 Bacteriophage, taxon: 388452 
               
               
                 LP-phiHSIC Helicase 
                   Listonella pelagia  phage phiHSIC 
                 taxon: 310539, a 
               
               
                   
                   
                 pseudotemperate marine 
               
               
                   
                   
                 phage of  Listonella pelagia   
               
               
                 Lsp-PCC8106 GyrB 
                   Lyngbya  sp. PCC 8106 
                 Taxon: 313612 
               
               
                 MP-Be DnaB 
                 Mycobacteriophage Bethlehem 
                 Bacteriophage, taxon: 260121 
               
               
                 MP-Be gp51 
                 Mycobacteriophage Bethlehem 
                 Bacteriophage, taxon: 260121 
               
               
                 MP-Catera gp206 
                 Mycobacteriophage Catera 
                 Mycobacteriophage, 
               
               
                   
                   
                 taxon: 373404 
               
               
                 MP-KBG gp53 
                   Mycobacterium  phage KBG 
                 Taxon: 540066 
               
               
                 MP-Mcjw1 DnaB 
                 Mycobacteriophage CJW1 
                 Bacteriophage, taxon: 205869 
               
               
                 MP-Omega DnaB 
                 Mycobacteriophage Omega 
                 Bacteriophage, taxon: 205879 
               
               
                 MP-U2 gp50 
                 Mycobacteriophage U2 
                 Bacteriophage, taxon: 260120 
               
               
                 Maer-NIES843 DnaB 
                   Microcystis aeruginosa  NIES-843 
                 Bloom-forming toxic 
               
               
                   
                   
                 cyanobacterium, taxon: 449447 
               
               
                 Maer-NIES843 DnaE-c 
                   Microcystis aeruginosa  NIES-843 
                 Bloom-forming toxic 
               
               
                   
                   
                 cyanobacterium, taxon: 449447 
               
               
                 Maer-NIES843 DnaE-n 
                   Microcystis aeruginosa  NIES-843 
                 Bloom-forming toxic 
               
               
                   
                   
                 cyanobacterium, taxon: 449447 
               
               
                 Mau-ATCC27029 
                   Micromonospora aurantiaca  ATCC 
                 Taxon: 644283 
               
               
                 GyrA 
                 27029 
               
               
                 Mav-104 DnaB 
                   Mycobacterium avium  104 
                 taxon: 243243 
               
               
                 Mav-ATCC25291 
                   Mycobacterium avium  subsp.  avium   
                 Taxon: 553481 
               
               
                 DnaB 
                 ATCC 25291 
               
               
                 Mav-ATCC35712 
                 
                   Mycobacterium avium 
                 
                 ATCC35712, taxon 1764 
               
               
                 DnaB 
               
               
                 Mav-PT DnaB 
                   Mycobacterium avium  subsp. 
                 taxon: 262316 
               
               
                   
                   paratuberculosis  str. k10 
               
               
                 Mbo Pps1 
                   Mycobacterium bovis  subsp.  bovis   
                 strain = “AF2122/97”, 
               
               
                   
                 AF2122/97 
                 taxon: 233413 
               
               
                 Mbo RecA 
                   Mycobacterium bovis  subsp. bovis 
                 taxon: 233413 
               
               
                   
                 AF2122/97 
               
               
                 Mbo SufB (Mbo Pps1) 
                   Mycobacterium bovis  subsp.  bovis   
                 taxon: 233413 
               
               
                   
                 AF2122/97 
               
               
                 Mbo-1173P DnaB 
                   Mycobacterium bovis  BCG Pasteur 
                 strain = BCG Pasteur 
               
               
                   
                 1173P 
                 1173P2,, taxon: 410289 
               
               
                 Mbo-AF2122 DnaB 
                   Mycobacterium bovis  subsp.  bovis   
                 strain = “AF2122/97”, 
               
               
                   
                 AF2122/97 
                 taxon: 233413 
               
               
                 Mca MupF 
                   Methylococcus capsulatus  Bath, 
                 prophage MuMc02, 
               
               
                   
                 prophage MuMc02 
                 taxon: 243233 
               
               
                 Mca RIR1 
                   Methylococcus capsulatus  Bath 
                 taxon: 243233 
               
               
                 Mch RecA 
                 
                   Mycobacterium chitae 
                 
                 IP14116003, taxon: 1792 
               
               
                 Mcht-PCC7420 DnaE-1 
                 
                   Microcoleus chthonoplastes 
                 
                 Cyanobacterium, 
               
               
                   
                 PCC7420 
                 taxon: 118168 
               
               
                 Mcht-PCC7420 DnaE-2c 
                 
                   Microcoleus chthonoplastes 
                 
                 Cyanobacterium, 
               
               
                   
                 PCC7420 
                 taxon: 118168 
               
               
                 Mcht-PCC7420 DnaE-2n 
                 
                   Microcoleus chthonoplastes 
                 
                 Cyanobacterium, 
               
               
                   
                 PCC7420 
                 taxon: 118168 
               
               
                 Mcht-PCC7420 GyrB 
                   Microcoleus chthonoplastes  PCC 
                 Taxon: 118168 
               
               
                   
                 7420 
               
               
                 Mcht-PCC7420 RIR1-1 
                   Microcoleus chthonoplastes  PCC 
                 Taxon: 118168 
               
               
                   
                 7420 
               
               
                 Mcht-PCC7420 RIR1-2 
                   Microcoleus chthonoplastes  PCC 
                 Taxon: 118168 
               
               
                   
                 7420 
               
               
                 Mex Helicase 
                   Methylobacterium extorquens  AM1 
                 Alphaproteobacteria 
               
               
                 Mex TrbC 
                   Methylobacterium extorquens  AM1 
                 Alphaproteobacteria 
               
               
                 Mfa RecA 
                 
                   Mycobacterium fallax 
                 
                 CITP8139, taxon: 1793 
               
               
                 Mfl GyrA 
                   Mycobacterium flavescens  Fla0 
                 taxon: 1776, reference 
               
               
                   
                   
                 #930991 
               
               
                 Mfl RecA 
                   Mycobacterium flavescens  Fla0 
                 strain = Fla0, taxon: 1776, ref. 
               
               
                   
                   
                 #930991 
               
               
                 Mfl-ATCC14474 RecA 
                   Mycobacterium flavescens , 
                 strain = ATCC14474, taxon: 1776, 
               
               
                   
                 ATCC14474 
                 ref #930991 
               
               
                 Mfl-PYR-GCK DnaB 
                   Mycobacterium flavescens  PYR- 
                 taxon: 350054 
               
               
                   
                 GCK 
               
               
                 Mga GyrA 
                 
                   Mycobacterium gastri 
                 
                 HP4389, taxon: 1777 
               
               
                 Mga RecA 
                 
                   Mycobacterium gastri 
                 
                 HP4389, taxon: 1777 
               
               
                 Mga SufB (Mga Pps1) 
                 
                   Mycobacterium gastri 
                 
                 HP4389, taxon: 1777 
               
               
                 Mgi-PYR-GCK DnaB 
                   Mycobacterium gilvum  PYR-GCK 
                 taxon: 350054 
               
               
                 Mgi-PYR-GCK GyrA 
                   Mycobacterium gilvum  PYR-GCK 
                 taxon: 350054 
               
               
                 Mgo GyrA 
                 
                   Mycobacterium gordonae 
                 
                 taxon: 1778, reference number 
               
               
                   
                   
                 930835 
               
               
                 Min-1442 DnaB 
                 
                   Mycobacterium intracellulare 
                 
                 strain 1442, taxon: 1767 
               
               
                 Min-ATCC13950 
                   Mycobacterium intracellulare  ATCC 
                 Taxon: 487521 
               
               
                 GyrA 
                 13950 
               
               
                 Mkas GyrA 
                 
                   Mycobacterium kansasii 
                 
                 taxon: 1768 
               
               
                 Mkas-ATCC12478 
                   Mycobacterium kansasii  ATCC 
                 Taxon: 557599 
               
               
                 GyrA 
                 12478 
               
               
                 Mle-Br4923 GyrA 
                   Mycobacterium leprae  Br4923 
                 Taxon: 561304 
               
               
                 Mle-TN DnaB 
                   Mycobacterium leprae , strain TN 
                 Human pathogen, taxon: 1769 
               
               
                 Mle-TN GyrA 
                   Mycobacterium leprae  TN 
                 Human pathogen, 
               
               
                   
                   
                 STRAIN = TN, taxon: 1769 
               
               
                 Mle-TN RecA 
                   Mycobacterium leprae , strain TN 
                 Human pathogen, taxon: 1769 
               
               
                 Mle-TN SufB (Mle 
                 
                   Mycobacterium leprae 
                 
                 Human pathogen, taxon: 1769 
               
               
                 Pps1) 
               
               
                 Mma GyrA 
                 
                   Mycobacterium malmoense 
                 
                 taxon: 1780 
               
               
                 Mmag Magn8951 BIL 
                 
                   Magnetospirillum magnetotacticum 
                 
                 Gram negative, taxon: 272627 
               
               
                   
                 MS-1 
               
               
                 Msh RecA 
                 
                   Mycobacterium shimodei 
                 
                 ATCC27962, taxon: 29313 
               
               
                 Msm DnaB-1 
                   Mycobacterium smegmatis  MC2 155 
                 MC2 155, taxon: 246196 
               
               
                 Msm DnaB-2 
                   Mycobacterium smegmatis  MC2 155 
                 MC2 155, taxon: 246196 
               
               
                 Msp-KMS DnaB 
                   Mycobacterium  species KMS 
                 taxon: 189918 
               
               
                 Msp-KMS GyrA 
                   Mycobacterium  species KMS 
                 taxon: 189918 
               
               
                 Msp-MCS DnaB 
                   Mycobacterium  species MCS 
                 taxon: 164756 
               
               
                 Msp-MCS GyrA 
                   Mycobacterium  species MCS 
                 taxon: 164756 
               
               
                 Mthe RecA 
                 
                   Mycobacterium thermoresistibile 
                 
                 ATCC19527, taxon: 1797 
               
               
                 Mtu SufB (Mtu Pps1) 
                   Mycobacterium tuberculosis  strains 
                 Human pathogen, taxon: 83332 
               
               
                   
                 H37Rv &amp; CDC1551 
               
               
                 Mtu-C RecA 
                   Mycobacterium tuberculosis  C 
                 Taxon: 348776 
               
               
                 Mtu-CDC1551 DnaB 
                   Mycobacterium tuberculosis , 
                 Human pathogen, taxon: 83332 
               
               
                   
                 CDC1551 
               
               
                 Mtu-CPHL RecA 
                 
                   Mycobacterium tuberculosis 
                 
                 Taxon: 611303 
               
               
                   
                 CPHL_A 
               
               
                 Mtu-Canetti RecA 
                   Mycobacterium tuberculosis / 
                 Taxon: 1773 
               
               
                   
                 strain = “ Canetti ” 
               
               
                 Mtu-EAS054 RecA 
                   Mycobacterium tuberculosis  EAS054 
                 Taxon: 520140 
               
               
                 Mtu-F11 DnaB 
                   Mycobacterium tuberculosis , strain 
                 taxon: 336982 
               
               
                   
                 F11 
               
               
                 Mtu-H37Ra DnaB 
                   Mycobacterium tuberculosis  H37Ra 
                 ATCC 25177, taxon: 419947 
               
               
                 Mtu-H37Rv DnaB 
                   Mycobacterium tuberculosis  H37Rv 
                 Human pathogen, taxon: 83332 
               
               
                 Mtu-H37Rv RecA 
                 
                   Mycobacterium tuberculosis 
                 
                 Human pathogen, taxon: 83332 
               
               
                   
                 H37Rv, Also CDC1551 
               
               
                 Mtu-Haarlem DnaB 
                   Mycobacterium tuberculosis  str. 
                 Taxon: 395095 
               
               
                   
                 Haarlem 
               
               
                 Mtu-K85 RecA 
                   Mycobacterium tuberculosis  K85 
                 Taxon: 611304 
               
               
                 Mtu-R604 RecA-n 
                   Mycobacterium tuberculosis  ‘98- 
                 Taxon: 555461 
               
               
                   
                 R604 INH-RIF-EM’ 
               
               
                 Mtu-So93 RecA 
                 
                   Mycobacterium tuberculosis 
                 
                 Human pathogen, taxon: 1773 
               
               
                   
                 So93/sub_species = “ Canetti ” 
               
               
                 Mtu-T17 RecA-c 
                   Mycobacterium tuberculosis  T17 
                 Taxon: 537210 
               
               
                 Mtu-T17 RecA-n 
                   Mycobacterium tuberculosis  T17 
                 Taxon: 537210 
               
               
                 Mtu-T46 RecA 
                   Mycobacterium tuberculosis  T46 
                 Taxon: 611302 
               
               
                 Mtu-T85 RecA 
                   Mycobacterium tuberculosis  T85 
                 Taxon: 520141 
               
               
                 Mtu-T92 RecA 
                   Mycobacterium tuberculosis  T92 
                 Taxon: 515617 
               
               
                 Mvan DnaB 
                   Mycobacterium vanbaalenii  PYR-1 
                 taxon: 350058 
               
               
                 Mvan GyrA 
                   Mycobacterium vanbaalenii  PYR-1 
                 taxon: 350058 
               
               
                 Mxa RAD25 
                   Myxococcus xanthus  DK1622 
                 Deltaproteobacteria 
               
               
                 Mxe GyrA 
                   Mycobacterium xenopi  strain 
                 taxon: 1789 
               
               
                   
                 IMM5024 
               
               
                 Naz-0708 RIR1-1 
                   Nostoc azollae  0708 
                 Taxon: 551115 
               
               
                 Naz-0708 RIR1-2 
                   Nostoc azollae  0708 
                 Taxon: 551115 
               
               
                 Nfa DnaB 
                   Nocardia farcinica  IFM 10152 
                 taxon: 247156 
               
               
                 Nfa Nfa15250 
                   Nocardia farcinica  IFM 10152 
                 taxon: 247156 
               
               
                 Nfa RIR1 
                   Nocardia farcinica  IFM 10152 
                 taxon: 247156 
               
               
                 Nosp-CCY9414 DnaE-n 
                   Nodularia spumigena  CCY9414 
                 Taxon: 313624 
               
               
                 Npu DnaB 
                 
                   Nostoc punctiforme 
                 
                 Cyanobacterium, taxon: 63737 
               
               
                 Npu GyrB 
                 
                   Nostoc punctiforme 
                 
                 Cyanobacterium, taxon: 63737 
               
               
                 Npu-PCC73102 DnaE-c 
                   Nostoc punctiforme  PCC73102 
                 Cyanobacterium, taxon: 63737, 
               
               
                   
                   
                 ATCC29133 
               
               
                 Npu-PCC73102 DnaE-n 
                   Nostoc punctiforme  PCC73102 
                 Cyanobacterium, taxon: 63737, 
               
               
                   
                   
                 ATCC29133 
               
               
                 Nsp-JS614 DnaB 
                   Nocardioides  species JS614 
                 taxon: 196162 
               
               
                 Nsp-JS614 TOPRIM 
                   Nocardioides  species JS614 
                 taxon: 196162 
               
               
                 Nsp-PCC7120 DnaB 
                   Nostoc  species PCC7120, ( Anabaena   
                 Cyanobacterium, Nitrogen- 
               
               
                   
                 sp. PCC7120) 
                 fixing, taxon: 103690 
               
               
                 Nsp-PCC7120 DnaE-c 
                   Nostoc  species PCC7120, ( Anabaena   
                 Cyanobacterium, Nitrogen- 
               
               
                   
                 sp. PCC7120) 
                 fixing, taxon: 103690 
               
               
                 Nsp-PCC7120 DnaE-n 
                   Nostoc  species PCC7120, ( Anabaena   
                 Cyanobacterium, Nitrogen- 
               
               
                   
                 sp. PCC7120) 
                 fixing, taxon: 103690 
               
               
                 Nsp-PCC7120 RIR1 
                   Nostoc  species PCC7120, ( Anabaena   
                 Cyanobacterium, Nitrogen- 
               
               
                   
                 sp. PCC7120) 
                 fixing, taxon: 103690 
               
               
                 Oli DnaE-c 
                   Oscillatoria limnetica  str. ‘Solar Lake’ 
                 Cyanobacterium, taxon: 262926 
               
               
                 Oli DnaE-n 
                   Oscillatoria limnetica  str. ‘Solar Lake’ 
                 Cyanobacterium, taxon: 262926 
               
               
                 PP-PhiEL Helicase 
                   Pseudomonas aeruginosa  phage 
                 Phage infects  Pseudomonas   
               
               
                   
                 phiEL 
                   aeruginosa , taxon: 273133 
               
               
                 PP-PhiEL ORF11 
                   Pseudomonas aeruginosa  phage 
                 phage infects  Pseudomonas   
               
               
                   
                 phiEL 
                   aeruginosa , taxon: 273133 
               
               
                 PP-PhiEL ORF39 
                   Pseudomonas aeruginosa  phage 
                 Phage infects  Pseudomonas   
               
               
                   
                 phiEL 
                   aeruginosa , taxon: 273133 
               
               
                 PP-PhiEL ORF40 
                   Pseudomonas aeruginosa  phage 
                 phage infects  Pseudomonas   
               
               
                   
                 phiEL 
                   aeruginosa , taxon: 273133 
               
               
                 Pfl Fha BIL 
                   Pseudomonas fluorescens  Pf-5 
                 Plant commensal organism, 
               
               
                   
                   
                 taxon: 220664 
               
               
                 Plut RIR1 
                   Pelodictyon luteolum  DSM 273 
                 Green sulfur bacteria, Taxon 
               
               
                   
                   
                 319225 
               
               
                 Pma-EXH1 GyrA 
                   Persephonella marina  EX-H1 
                 Taxon: 123214 
               
               
                 Pma-ExH1 DnaE 
                   Persephonella marina  EX-H1 
                 Taxon: 123214 
               
               
                 Pna RIR1 
                   Polaromonas naphthalenivorans  CJ2 
                 taxon: 365044 
               
               
                 Pnuc DnaB 
                   Polynucleobacter  sp. QLW- 
                 taxon: 312153 
               
               
                   
                 P1DMWA-1 
               
               
                 Posp-JS666 DnaB 
                   Polaromonas  species JS666 
                 taxon: 296591 
               
               
                 Posp-JS666 RIR1 
                   Polaromonas  species JS666 
                 taxon: 296591 
               
               
                 Pssp-A1-1 Fha 
                   Pseudomonas  species A1-1 
               
               
                 Psy Fha 
                   Pseudomonas syringae  pv. tomato 
                 Plant (tomato) pathogen, 
               
               
                   
                 str. DC3000 
                 taxon: 223283 
               
               
                 Rbr-D9 GyrB 
                   Raphidiopsis brookii  D9 
                 Taxon: 533247 
               
               
                 Rce RIR1 
                   Rhodospirillum centenum  SW 
                 taxon: 414684, ATCC 51521 
               
               
                 Rer-SK121 DnaB 
                   Rhodococcus erythropolis  SK121 
                 Taxon: 596309 
               
               
                 Rma DnaB 
                 
                   Rhodothermus marinus 
                 
                 Thermophile, taxon: 29549 
               
               
                 Rma-DSM4252 DnaB 
                   Rhodothermus marinus  DSM 4252 
                 Taxon: 518766 
               
               
                 Rma-DSM4252 DnaE 
                   Rhodothermus marinus  DSM 4252 
                 Thermophile, taxon: 518766 
               
               
                 Rsp RIR1 
                   Roseovarius  species 217 
                 taxon: 314264 
               
               
                 SaP-SETP12 dpol 
                   Salmonella  phage SETP12 
                 Phage, taxon: 424946 
               
               
                 SaP-SETP3 Helicase 
                   Salmonella  phage SETP3 
                 Phage, taxon: 424944 
               
               
                 SaP-SETP3 dpol 
                   Salmonella  phage SETP3 
                 Phage, taxon: 424944 
               
               
                 SaP-SETP5 dpol 
                   Salmonella  phage SETP5 
                 Phage, taxon: 424945 
               
               
                 Sare DnaB 
                   Salinispora arenicola  CNS-205 
                 taxon: 391037 
               
               
                 Sav RecG Helicase 
                   Streptomyces avermitilis  MA-4680 
                 taxon: 227882, ATCC 31267 
               
               
                 Sel-PC6301 RIR1 
                   Synechococcus elongatus  PCC 6301 
                 taxon: 269084 Berkely strain 
               
               
                   
                   
                 6301~equivalent name: Ssp 
               
               
                   
                   
                 PCC 6301~synonym: 
               
               
                   
                   
                 
                   Anacystis nudulans 
                 
               
               
                 Sel-PC7942 DnaE-c 
                   Synechococcus elongatus  PC7942 
                 taxon: 1140 
               
               
                 Sel-PC7942 DnaE-n 
                   Synechococcus elongatus  PC7942 
                 taxon: 1140 
               
               
                 Sel-PC7942 RIR1 
                   Synechococcus elongatus  PC7942 
                 taxon: 1140 
               
               
                 Sel-PCC6301 DnaE-c 
                   Synechococcus elongatus  PCC 6301 
                 Cyanobacterium, 
               
               
                   
                 and PCC7942 
                 taxon: 269084, “Berkely strain 
               
               
                   
                   
                 6301~equivalent name: 
               
               
                   
                   
                   Synechococcus  sp. PCC 
               
               
                   
                   
                 6301~synonym:  Anacystis   
               
               
                   
                   
                   nudulans ” 
               
               
                 Sel-PCC6301 DnaE-n 
                   Synechococcus elongatus  PCC 6301 
                 Cyanobacterium, 
               
               
                   
                   
                 taxon: 269084 “Berkely strain 
               
               
                   
                   
                 6301~equivalent name: 
               
               
                   
                   
                   Synechococcus  sp. PCC 
               
               
                   
                   
                 6301~synonym:  Anacystis   
               
               
                   
                   
                   nudulans ” 
               
               
                 Sep RIR1 
                   Staphylococcus epidermidis  RP62A 
                 taxon: 176279 
               
               
                 ShP-Sfv-2a-2457T-n 
                   Shigella flexneri  2a str. 2457T 
                 Putative bacteriphage 
               
               
                 Primase 
               
               
                 ShP-Sfv-2a-301-n 
                   Shigella flexneri  2a str. 301 
                 Putative bacteriphage 
               
               
                 Primase 
               
               
                 ShP-Sfv-5 Primase 
                   Shigella flexneri  5 str. 8401 
                 Bacteriphage, isolation_source —   
               
               
                   
                   
                 epidemic, taxon: 373384 
               
               
                 SoP-SO1 dpol 
                   Sodalis  phage SO-1 
                 Phage/isolation_source = “ Sodalis   
               
               
                   
                   
                   glossinidius  strain GA-SG, 
               
               
                   
                   
                 secondary symbiont of 
               
               
                   
                   
                   Glossina austeni  (Newstead)” 
               
               
                 Spl DnaX 
                   Spirulina platensis , strain C1 
                 Cyanobacterium, taxon: 1156 
               
               
                 Sru DnaB 
                   Salinibacter ruber  DSM 13855 
                 taxon: 309807, strain = “DSM 
               
               
                   
                   
                 13855; M31” 
               
               
                 Sru PolBc 
                   Salinibacter ruber  DSM 13855 
                 taxon: 309807, strain = “DSM 
               
               
                   
                   
                 13855; M31” 
               
               
                 Sru RIR1 
                   Salinibacter ruber  DSM 13855 
                 taxon: 309807, strain = “DSM 
               
               
                   
                   
                 13855; M31” 
               
               
                 Ssp DnaB 
                   Synechocystis  species, strain 
                 Cyanobacterium, taxon: 1148 
               
               
                   
                 PCC6803 
               
               
                 Ssp DnaE-c 
                   Synechocystis  species, strain 
                 Cyanobacterium, taxon: 1148 
               
               
                   
                 PCC6803 
               
               
                 Ssp DnaE-n 
                   Synechocystis  species, strain 
                 Cyanobacterium, taxon: 1148 
               
               
                   
                 PCC6803 
               
               
                 Ssp DnaX 
                   Synechocystis  species, strain 
                 Cyanobacterium, taxon: 1148 
               
               
                   
                 PCC6803 
               
               
                 Ssp GyrB 
                   Synechocystis  species, strain 
                 Cyanobacterium, taxon: 1148 
               
               
                   
                 PCC6803 
               
               
                 Ssp-JA2 DnaB 
                   Synechococcus  species JA-2-3B′a(2-13) 
                 Cyanobacterium, Taxon: 321332 
               
               
                 Ssp-JA2 RIR1 
                   Synechococcus  species JA-2-3B′a(2-13) 
                 Cyanobacterium, Taxon: 321332 
               
               
                 Ssp-JA3 DnaB 
                   Synechococcus  species JA-3-3Ab 
                 Cyanobacterium, Taxon: 321327 
               
               
                 Ssp-JA3 RIR1 
                   Synechococcus  species JA-3-3Ab 
                 Cyanobacterium, Taxon: 321327 
               
               
                 Ssp-PCC7002 DnaE-c 
                   Synechocystis  species, strain PCC 
                 Cyanobacterium, taxon: 32049 
               
               
                   
                 7002 
               
               
                 Ssp-PCC7002 DnaE-n 
                   Synechocystis  species, strain PCC 
                 Cyanobacterium, taxon: 32049 
               
               
                   
                 7002 
               
               
                 Ssp-PCC7335 RIR1 
                   Synechococcus  sp. PCC 7335 
                 Taxon: 91464 
               
               
                 StP-Twort ORF6 
                   Staphylococcus  phage Twort 
                 Phage, taxon 55510 
               
               
                 Susp-NBC371 DnaB 
                   Sulfurovum  sp. NBC37-1 
                 taxon: 387093 
               
               
                 intein 
               
               
                 Taq-Y51MC23 DnaE 
                   Thermus aquaticus  Y51MC23 
                 Taxon: 498848 
               
               
                 Taq-Y51MC23 RIR1 
                   Thermus aquaticus  Y51MC23 
                 Taxon: 498848 
               
               
                 Tcu-DSM43183 RecA 
                   Thermomonospora curvata  DSM 
                 Taxon: 471852 
               
               
                   
                 43183 
               
               
                 Tel DnaE-c 
                   Thermosynechococcus elongatus  BP-1 
                 Cyanobacterium, taxon: 197221 
               
               
                 Tel DnaE-n 
                   Thermosynechococcus elongatus  BP-1 
                 Cyanobacterium, 
               
               
                 Ter DnaB-1 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter DnaB-2 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter DnaE-1 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter DnaE-2 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter DnaE-3c 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter DnaE-3n 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter GyrB 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter Ndse-1 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter Ndse-2 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter RIR1-1 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter RIR1-2 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter RIR1-3 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter RIR1-4 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter Snf2 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Ter ThyX 
                   Trichodesmium erythraeum  IMS101 
                 Cyanobacterium, taxon: 203124 
               
               
                 Tfus RecA-1 
                   Thermobifida fusca  YX 
                 Thermophile, taxon: 269800 
               
               
                 Tfus RecA-2 
                   Thermobifida fusca  YX 
                 Thermophile, taxon: 269800 
               
               
                 Tfus Tfu2914 
                   Thermobifida fusca  YX 
                 Thermophile, taxon: 269800 
               
               
                 Thsp-K90 RIR1 
                   Thioalkalivibrio  sp. K90mix 
                 Taxon: 396595 
               
               
                 Tth-DSM571 RIR1 
                 
                   Thermoanaerobacterium 
                 
                 Taxon: 580327 
               
               
                   
                   thermosaccharolyticum  DSM 571 
               
               
                 Tth-HB27 DnaE-1 
                   Thermus thermophilus  HB27 
                 thermophile, taxon: 262724 
               
               
                 Tth-HB27 DnaE-2 
                   Thermus thermophilus  HB27 
                 thermophile, taxon: 262724 
               
               
                 Tth-HB27 RIR1-1 
                   Thermus thermophilus  HB27 
                 thermophile, taxon: 262724 
               
               
                 Tth-HB27 RIR1-2 
                   Thermus thermophilus  HB27 
                 thermophile, taxon: 262724 
               
               
                 Tth-HB8 DnaE-1 
                   Thermus thermophilus  HB8 
                 thermophile, taxon: 300852 
               
               
                 Tth-HB8 DnaE-2 
                   Thermus thermophilus  HB8 
                 thermophile, taxon: 300852 
               
               
                 Tth-HB8 RIR1-1 
                   Thermus thermophilus  HB8 
                 thermophile, taxon: 300852 
               
               
                 Tth-HB8 RIR1-2 
                   Thermus thermophilus  HB8 
                 thermophile, taxon: 300852 
               
               
                 Tvu DnaE-c 
                 
                   Thermosynechococcus vulcanus 
                 
                 Cyanobacterium, taxon: 32053 
               
               
                 Tvu DnaE-n 
                 
                   Thermosynechococcus vulcanus 
                 
                 Cyanobacterium, taxon: 32053 
               
               
                 Tye RNR-1 
                 
                   Thermodesulfovibrio yellowstonii 
                 
                 taxon: 289376 
               
               
                   
                 DSM 11347 
               
               
                 Tye RNR-2 
                 
                   Thermodesulfovibrio yellowstonii 
                 
                 taxon: 289376 
               
               
                   
                 DSM 11347 
               
            
           
           
               
            
               
                 Archaea 
               
            
           
           
               
               
               
            
               
                 Ape APE0745 
                   Aeropyrum pernix  K1 
                 Thermophile, taxon: 56636 
               
               
                 Cme-boo Pol-II 
                 
                   Candidatus Methanoregula boonei 
                 
                 taxon: 456442 
               
               
                   
                 6A8 
               
               
                 Fac-Fer1 RIR1 
                   Ferroplasma acidarmanus , 
                 strain Fer1, eats iron 
               
               
                   
                 taxon: 97393 and taxon 261390 
               
               
                 Fac-Fer1 SufB (Fac 
                 
                   Ferroplasma acidarmanus 
                 
                 strain fer1, eats 
               
               
                 Pps1) 
                   
                 iron, taxon: 97393 
               
               
                 Fac-TypeI RIR1 
                   Ferroplasma acidarmanus  type I, 
                 Eats iron, taxon 261390 
               
               
                 Fac-typeI SufB (Fac 
                 
                   Ferroplasma acidarmanus 
                 
                 Eats iron, taxon: 261390 
               
               
                 Pps1) 
               
               
                 Hma CDC21 
                   Haloarcula marismortui  ATCC 
                 taxon: 272569, 
               
               
                   
                 43049 
               
               
                 Hma Pol-II 
                   Haloarcula marismortui  ATCC 
                 taxon: 272569, 
               
               
                   
                 43049 
               
               
                 Hma PolB 
                   Haloarcula marismortui  ATCC 
                 taxon: 272569, 
               
               
                   
                 43049 
               
               
                 Hma TopA 
                   Haloarcula marismortui  ATCC 
                 taxon: 272569 
               
               
                   
                 43049 
               
               
                 Hmu-DSM12286 
                   Halomicrobium mukohataei  DSM 
                 taxon: 485914 (Halobacteria) 
               
               
                 MCM 
                 12286 
               
               
                 Hmu-DSM12286 PolB 
                   Halomicrobium mukohataei  DSM 
                 Taxon: 485914 
               
               
                   
                 12286 
               
               
                 Hsa-R1 MCM 
                   Halobacterium salinarum  R-1 
                 Halophile, 
               
               
                   
                   
                 taxon: 478009, strain = “R1; 
               
               
                   
                   
                 DSM 671” 
               
               
                 Hsp-NRC1 CDC21 
                   Halobacterium  species NRC-1 
                 Halophile, taxon: 64091 
               
               
                 Hsp-NRC1 Pol-II 
                   Halobacterium salinarum  NRC-1 
                 Halophile, taxon: 64091 
               
               
                 Hut MCM-2 
                   Halorhabdus utahensis  DSM 12940 
                 taxon: 519442 
               
               
                 Hut-DSM12940 MCM- 
                   Halorhabdus utahensis  DSM 12940 
                 taxon: 519442 
               
               
                 1 
               
               
                 Hvo PolB 
                   Haloferax volcanii  DS70 
                 taxon: 2246 
               
               
                 Hwa GyrB 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa MCM-1 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa MCM-2 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa MCM-3 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa MCM-4 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa Pol-II-1 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa Pol-II-2 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa PolB-1 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa PolB-2 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa PolB-3 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa RCF 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa RIR1-1 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa RIR1-2 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa Top6B 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Hwa rPol A″ 
                   Haloquadratum walsbyi  DSM 16790 
                 Halophile, taxon: 362976, 
               
               
                   
                   
                 strain: DSM 16790 = 
               
               
                   
                   
                 HBSQ001 
               
               
                 Maeo Pol-II 
                   Methanococcus aeolicus  Nankai-3 
                 taxon: 419665 
               
               
                 Maeo RFC 
                   Methanococcus aeolicus  Nankai-3 
                 taxon: 419665 
               
               
                 Maeo RNR 
                   Methanococcus aeolicus  Nankai-3 
                 taxon: 419665 
               
               
                 Maeo-N3 Helicase 
                   Methanococcus aeolicus  Nankai-3 
                 taxon: 419665 
               
               
                 Maeo-N3 RtcB 
                   Methanococcus aeolicus  Nankai-3 
                 taxon: 419665 
               
               
                 Maeo-N3 UDP GD 
                   Methanococcus aeolicus  Nankai-3 
                 taxon: 419665 
               
               
                 Mein-ME PEP 
                   Methanocaldococcus infernus  ME 
                 thermophile, Taxon: 573063 
               
               
                 Mein-ME RFC 
                   Methanocaldococcus infernus  ME 
                 Taxon: 573063 
               
               
                 Memar MCM2 
                   Methanoculleus marisnigri  JR1 
                 taxon: 368407 
               
               
                 Memar Pol-II 
                   Methanoculleus marisnigri  JR1 
                 taxon: 368407 
               
               
                 Mesp-FS406 PolB-1 
                   Methanocaldococcus  sp. FS406-22 
                 Taxon: 644281 
               
               
                 Mesp-FS406 PolB-2 
                   Methanocaldococcus  sp. FS406-22 
                 Taxon: 644281 
               
               
                 Mesp-FS406 PolB-3 
                   Methanocaldococcus  sp. FS406-22 
                 Taxon: 644281 
               
               
                 Mesp-FS406-22 LHR 
                   Methanocaldococcus  sp. FS406-22 
                 Taxon: 644281 
               
               
                 Mfe-AG86 Pol-1 
                   Methanocaldococcus fervens  AG86 
                 Taxon: 573064 
               
               
                 Mfe-AG86 Pol-2 
                   Methanocaldococcus fervens  AG86 
                 Taxon: 573064 
               
               
                 Mhu Pol-II 
                   Methanospirillum hungateii  JF-1 
                 taxon 323259 
               
               
                 Mja GF-6P 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja Helicase 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja Hyp-1 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja IF2 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja KlbA 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja PEP 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja Pol-1 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja Pol-2 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja RFC-1 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja RFC-2 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja RFC-3 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja RNR-1 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja RNR-2 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja RtcB (Mja Hyp-2) 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja TFIIB 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja UDP GD 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja r-Gyr 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja rPol A′ 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mja rPol A″ 
                 
                   Methanococcus jannaschii 
                 
                 Thermophile, DSM 2661, 
               
               
                   
                 ( Methanocaldococcus jannaschii   
                 taxon: 2190 
               
               
                   
                 DSM 2661) 
               
               
                 Mka CDC48 
                   Methanopyrus kandleri  AV19 
                 Thermophile, taxon: 190192 
               
               
                 Mka EF2 
                   Methanopyrus kandleri  AV19 
                 Thermophile, taxon: 190192 
               
               
                 Mka RFC 
                   Methanopyrus kandleri  AV19 
                 Thermophile, taxon: 190192 
               
               
                 Mka RtcB 
                   Methanopyrus kandleri  AV19 
                 Thermophile, taxon: 190192 
               
               
                 Mka VatB 
                   Methanopyrus kandleri  AV19 
                 Thermophile, taxon: 190192 
               
               
                 Mth RIR1 
                 
                   Methanothermobacter 
                 
                 Thermophile, delta H strain 
               
               
                   
                 
                   thermautotrophicus 
                 
               
               
                   
                 ( Methanobacterium   
               
               
                   
                   thermoautotrophicum ) 
               
               
                 Mvu-M7 Helicase 
                   Methanocaldococcus vulcanius  M7 
                 Taxon: 579137 
               
               
                 Mvu-M7 Pol-1 
                   Methanocaldococcus vulcanius  M7 
                 Taxon: 579137 
               
               
                 Mvu-M7 Pol-2 
                   Methanocaldococcus vulcanius  M7 
                 Taxon: 579137 
               
               
                 Mvu-M7 Pol-3 
                   Methanocaldococcus vulcanius  M7 
                 Taxon: 579137 
               
               
                 Mvu-M7 UDP GD 
                   Methanocaldococcus vulcanius  M7 
                 Taxon: 579137 
               
               
                 Neq Pol-c 
                   Nanoarchaeum equitans  Kin4-M 
                 Thermophile, taxon: 228908 
               
               
                 Neq Pol-n 
                   Nanoarchaeum equitans  Kin4-M 
                 Thermophile, taxon: 228908 
               
               
                 Nma-ATCC43099 
                   Natrialba magadii  ATCC 43099 
                 Taxon: 547559 
               
               
                 MCM 
               
               
                 Nma-ATCC43099 
                   Natrialba magadii  ATCC 43099 
                 Taxon: 547559 
               
               
                 PolB-1 
               
               
                 Nma-ATCC43099 
                   Natrialba magadii  ATCC 43099 
                 Taxon: 547559 
               
               
                 PolB-2 
               
               
                 Nph CDC21 
                   Natronomonas pharaonis  DSM 2160 
                 taxon: 348780 
               
               
                 Nph PolB-1 
                   Natronomonas pharaonis  DSM 2160 
                 taxon: 348780 
               
               
                 Nph PolB-2 
                   Natronomonas pharaonis  DSM 2160 
                 taxon: 348780 
               
               
                 Nph rPol A″ 
                   Natronomonas pharaonis  DSM 2160 
                 taxon: 348780 
               
               
                 Pab CDC21-1 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab CDC21-2 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab IF2 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab KlbA 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab Lon 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab Moaa 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab Pol-II 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab RFC-1 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab RFC-2 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab RIR1-1 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab RIR1-2 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab RIR1-3 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab RtcB (Pab Hyp-2) 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Pab VMA 
                 
                   Pyrococcus abyssi 
                 
                 Thermophile, strain Orsay, 
               
               
                   
                   
                 taxon: 29292 
               
               
                 Par RIR1 
                   Pyrobaculum arsenaticum  DSM 
                 taxon: 340102 
               
               
                   
                 13514 
               
               
                 Pfu CDC21 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, taxon: 186497, 
               
               
                   
                   
                 DSM3638 
               
               
                 Pfu IF2 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, taxon: 186497, 
               
               
                   
                   
                 DSM3638 
               
               
                 Pfu KlbA 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, taxon: 186497, 
               
               
                   
                   
                 DSM3638 
               
               
                 Pfu Lon 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, taxon: 186497, 
               
               
                   
                   
                 DSM3638 
               
               
                 Pfu RFC 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, DSM3638, 
               
               
                   
                   
                 taxon: 186497 
               
               
                 Pfu RIR1-1 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, taxon: 186497, 
               
               
                   
                   
                 DSM3638 
               
               
                 Pfu RIR1-2 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, taxon: 186497, 
               
               
                   
                   
                 DSM3638 
               
               
                 Pfu RtcB (Pfu Hyp-2) 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, taxon: 186497, 
               
               
                   
                   
                 DSM3638 
               
               
                 Pfu TopA 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, taxon: 186497, 
               
               
                   
                   
                 DSM3638 
               
               
                 Pfu VMA 
                 
                   Pyrococcus furiosus 
                 
                 Thermophile, taxon: 186497, 
               
               
                   
                   
                 DSM3638 
               
               
                 Pho CDC21-1 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho CDC21-2 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho IF2 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho KlbA 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho LHR 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho Lon 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho Pol I 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho Pol-II 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho RFC 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho RIR1 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho RadA 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho RtcB (Pho Hyp-2) 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho VMA 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Pho r-Gyr 
                   Pyrococcus horikoshii  OT3 
                 Thermophile, taxon: 53953 
               
               
                 Psp-GBD Pol 
                   Pyrococcus  species GB-D 
                 Thermophile 
               
               
                 Pto VMA 
                   Picrophilus torridus , DSM 9790 
                 DSM 9790, taxon: 263820, 
               
               
                   
                   
                 Thermoacidophile 
               
               
                 Smar 1471 
                   Staphylothermus marinus  F1 
                 taxon: 399550 
               
               
                 Smar MCM2 
                   Staphylothermus marinus  F1 
                 taxon: 399550 
               
               
                 Tac-ATCC25905 VMA 
                   Thermoplasma acidophilum , ATCC 
                 Thermophile, taxon: 2303 
               
               
                   
                 25905 
               
               
                 Tac-DSM1728 VMA 
                   Thermoplasma acidophilum , 
                 Thermophile, taxon: 2303 
               
               
                   
                 DSM1728 
               
               
                 Tag Pol-1 (Tsp-TY Pol-1) 
                 
                   Thermococcus aggregans 
                 
                 Thermophile, taxon: 110163 
               
               
                 Tag Pol-2 (Tsp-TY Pol-2) 
                 
                   Thermococcus aggregans 
                 
                 Thermophile, taxon: 110163 
               
               
                 Tag Pol-3 (Tsp-TY Pol-3) 
                 
                   Thermococcus aggregans 
                 
                 Thermophile, taxon: 110163 
               
               
                 Tba Pol-II 
                   Thermococcus barophilus  MP 
                 taxon: 391623 
               
               
                 Tfu Pol-1 
                 
                   Thermococcus fumicolans 
                 
                 Thermophilem, taxon: 46540 
               
               
                 Tfu Pol-2 
                 
                   Thermococcus fumicolans 
                 
                 Thermophile, taxon: 46540 
               
               
                 Thy Pol-1 
                 
                   Thermococcus hydrothermalis 
                 
                 Thermophile, taxon: 46539 
               
               
                 Thy Pol-2 
                 
                   Thermococcus hydrothermalis 
                 
                 Thermophile, taxon: 46539 
               
               
                 Tko CDC21-1 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko CDC21-2 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko Helicase 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko IF2 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko KlbA 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko LHR 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko Pol-1 (Pko Pol-1) 
                 
                   Pyrococcus/Thermococcus 
                 
                 Thermophile, taxon: 69014 
               
               
                   
                   kodakaraensis  KOD1 
               
               
                 Tko Pol-2 (Pko Pol-2) 
                 
                   Pyrococcus/Thermococcus 
                 
                 Thermophile, taxon: 69014 
               
               
                   
                   kodakaraensis  KOD1 
               
               
                 Tko Pol-II 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko RFC 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko RIR1-1 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko RIR1-2 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko RadA 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko TopA 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tko r-Gyr 
                   Thermococcus kodakaraensis  KOD1 
                 Thermophile, taxon: 69014 
               
               
                 Tli Pol-1 
                 
                   Thermococcus litoralis 
                 
                 Thermophile, taxon: 2265 
               
               
                 Tli Pol-2 
                 
                   Thermococcus litoralis 
                 
                 Thermophile, taxon: 2265 
               
               
                 Tma Pol 
                 
                   Thermococcus marinus 
                 
                 taxon: 187879 
               
               
                 Ton-NA1 LHR 
                   Thermococcus onnurineus  NA1 
                 Taxon: 523850 
               
               
                 Ton-NA1 Pol 
                   Thermococcus onnurineus  NA1 
                 taxon: 342948 
               
               
                 Tpe Pol 
                   Thermococcus peptonophilus  strain 
                 taxon: 32644 
               
               
                   
                 SM2 
               
               
                 Tsi-MM739 Lon 
                   Thermococcus sibiricus  MM 739 
                 Thermophile, Taxon: 604354 
               
               
                 Tsi-MM739 Pol-1 
                   Thermococcus sibiricus  MM 739 
                 Taxon: 604354 
               
               
                 Tsi-MM739 Pol-2 
                   Thermococcus sibiricus  MM 739 
                 Taxon: 604354 
               
               
                 Tsi-MM739 RFC 
                   Thermococcus sibiricus  MM 739 
                 Taxon: 604354 
               
               
                 Tsp AM4 RtcB 
                   Thermococcus  sp. AM4 
                 Taxon: 246969 
               
               
                 Tsp-AM4 LHR 
                   Thermococcus  sp. AM4 
                 Taxon: 246969 
               
               
                 Tsp-AM4 Lon 
                   Thermococcus  sp. AM4 
                 Taxon: 246969 
               
               
                 Tsp-AM4 RIR1 
                   Thermococcus  sp. AM4 
                 Taxon: 246969 
               
               
                 Tsp-GE8 Pol-1 
                   Thermococcus  species GE8 
                 Thermophile, taxon: 105583 
               
               
                 Tsp-GE8 Pol-2 
                   Thermococcus  species GE8 
                 Thermophile, taxon: 105583 
               
               
                 Tsp-GT Pol-1 
                   Thermococcus  species GT 
                 taxon: 370106 
               
               
                 Tsp-GT Pol-2 
                   Thermococcus  species GT 
                 taxon: 370106 
               
               
                 Tsp-OGL-20P Pol 
                   Thermococcus  sp. OGL-20P 
                 taxon: 277988 
               
               
                 Tthi Pol 
                 
                   Thermococcus thioreducens 
                 
                 Hyperthermophile 
               
               
                 Tvo VMA 
                   Thermoplasma volcanium  GSS1 
                 Thermophile, taxon: 50339 
               
               
                 Tzi Pol 
                 
                   Thermococcus zilligii 
                 
                 taxon: 54076 
               
               
                 Unc-ERS PFL 
                 uncultured archaeon Gzfos13E1 
                 isolation_source = “Eel River 
               
               
                   
                   
                 sediment”, 
               
               
                   
                   
                 clone = “GZfos13E1”, 
               
               
                   
                   
                 taxon: 285397 
               
               
                 Unc-ERS RIR1 
                 uncultured archaeon GZfos9C4 
                 isolation_source = “Eel River 
               
               
                   
                   
                 sediment”, taxon: 285366, 
               
               
                   
                   
                 clone = “GZfos9C4” 
               
               
                 Unc-ERS RNR 
                 uncultured archaeon GZfos10C7 
                 isolation_source = “Eel River 
               
               
                   
                   
                 sediment”, 
               
               
                   
                   
                 clone = “GZfos10C7”, 
               
               
                   
                   
                 taxon: 285400 
               
               
                 Unc-MetRFS MCM2 
                 uncultured archaeon (Rice Cluster I) 
                 Enriched methanogenic 
               
               
                   
                   
                 consortium from rice field 
               
               
                   
                   
                 soil, taxon: 198240 
               
               
                   
               
            
           
         
       
     
     Inteins of the disclosed compositions or that can be used in the disclosed methods can be modified, or mutated, inteins. A modified intein can comprise additional amino acids at the N-terminus of a naturally occurring intein, or can be at the C-terminus, or within the intein. In a non-limiting example, there can be one, two, three, four, five, or more additional amino acids at the N-terminus of the naturally occurring intein. In some aspects, the first amino acid at the N-terminal end of the intein can be alanine, histidine, cysteine or glycine. 
     In addition to the modification of comprising a reversible ZBM, an intein can also include other modifications that can make it useful for various applications. In other words, an intein with a ZBM can also comprise other modifications. For example, an intein can comprise another tunable motif that is useful for modifying behavior. For example, inteins can be encoded by a temperature-sensitive intein allele (Adam et al. 2002, Mol. Microbiol. Biotechnol. 4 479-487; Cann et al. 2004, Appl. Environ. Microbiol. 70 3158-3162; Zeidler et al. 2004, Nat. Biotechnol. 22 871-876), inteins can incorporate nonnative amino acids whose activity is regulated by photolysis (Cook et al. 1995 Angew. Chem. Int. Ed. Engl. 34 1629-1630), or the activity of inteins can be ligand-controlled (Mills et al. 2001, J. Biol. Chem. 276 10832-10838; Mootz et al. 2002, J. Am. Chem. Soc. 124 9044-9045; Mootz et al. 2003, J. Am. Chem. Soc. 125 10561-10569). It has also been shown that controllable intein splicing in cis can be used for various applications that will be appreciated by those skilled in the art. In the first case, regulation of protein activity was accomplished with the isolation and use of temperature-sensitive variants of the  Saccharomyces cerevisiae  vacuolar ATPase subunit intein (Zeidler et al. 2004) and, in the second case, with the construction of a novel chimeric intein whose splicing activity is triggered by the addition of 4-hydroxytamoxifen (Buskirk et al. Proc. Natl. Acad. Sci. 101 10505-10510). 
     Another example of modified inteins is a protein engineering approach for generating inteins whose splicing activity is regulated in vivo by the presence of human thyroid hormone. Designed according to other engineered allosteric enzyme prototypes (Baird et al. 1999, Proc. Natl. Acad. Sci. 96 11241-11246; Doi et al. 1999, FEBS Lett. 453 305-307; Tucker et al. 2001, Nat. Biotechnol. 19 1042-1046; Guntas et al, 2004, J. Mol. Biol. 336 263-273), an artificial intein chimera was created by fusing a thyroid-hormone-binding domain within a previously engineered mini-intein. The insertion of the binding domain abolished the splicing activity of the intein but allowed it to be later restored by addition of thyroid hormone or synthetic analogs. The resulting allosteric intein was then used to conditionally activate a variety of different proteins in  Escherichia coli  in a dose-dependent manner. Finally, a combination of directed evolution and genetic selection was employed to engineer an additional controllable intein whose splicing activity is inhibited by the presence of an orthogonal set of synthetic estrogen ligands. 
     Further examples of modified inteins include, but are not limited to, those found in U.S. Pat. No. 7,026,526, such as mutated inteins of  Pyrococcus  species GB-D DNA polymerase, and those created artificially by removing the endonuclease domains from endonuclease containing inteins (Xu, M-Q &amp; Perler, F. B. EMBO Journal, 1996, 15, 5146-5153; Chong, et al. J. Biol. Chem. 1997, 272, 15587-15590). In a further example, an intein can be selected so that it consists of the minimal number of amino acids needed to perform the splicing function, such as the intein from the  Mycobacterium xenopi  GyrA protein (Telenti, A., et al., J. Bacteriol. 1997, 179, 6378-6382). In another aspect, an intein without endonuclease activity can be selected, such as the intein from the  Mycobacterium  xenopi GyrA protein or the  Saccharomyces cerevisiae  VMA intein that has been modified to remove endonuclease domains (Chong, 1997). 
     In some aspects, the first residue of the C-terminal extein is engineered to contain a glycine or alanine, a modification that was shown to prevent extein ligation with the  Pyrococcus  species GB-D DNA polymerase (Xu, M-Q &amp; Perler, F. B. EMBO Journal, 1996, 15, 5146-5153). In this embodiment, preferred C-terminal exteins contain coding sequences that naturally contain a glycine or an alanine residue following the N-terminal methionine in the native amino acid sequence. Fusion of the glycine or alanine of the extein to the C-terminus of the intein can provide the native amino acid sequence after processing of the polypeptide. In another embodiment, an artificial glycine or alanine is created on the C-terminal extein either by altering the native sequence or by adding an additional amino acid residue onto the N-terminus of the native sequence. In this example, the native amino acid sequence of the protein is altered by one amino acid after polypeptide processing. 
     A further example of a modified intein includes the Pch PRP8 mini-intein of  Penicillium chrysogenum , which was modified to include: 1) aminoglycoside phosphotransferase; 2) imidazoleglycerol-phosphate dehydratase, His5 from  S. pombe  3) hygromycin B phosphotransferase; and 4) the transcriptional activator LexA-VP16 (Muller et al., BMC Biotechnology 2011, 11:71). 
     U.S. Pat. No. 5,834,247 teaches that modification of inteins can be accomplished in a number of ways and is hereby incorporated by reference in its entirety for its teaching of methods that can be used to modify inteins. For example, the sequence surrounding the amino acid residue to be modified may be altered to create a biological phosphorylation site allowing it to be a substrate for specific kinases and phosphatases. Examples of protein kinases include, for example, casein kinase II, cAMP-dependent protein kinase, cdc2, and pp60c-src (Pearson and Kemp, Methods in Enzymology 200:62 (1991)). Examples of phosphatases include, for example, protein phosphatase 2A, lambda phosphatase, and the yop phosphatase from  Yersinia  (Tonks, Current Opinion in Cell Biology, 2:1114 (1990)). 
     Additionally, critical splice junction residues can be modified chemically such that the splicing reaction is blocked until the modification is reversed. This can be accomplished by using, for example, unnatural amino acid mutagenesis (Noren, et al., Science 244:182 (1989); Ellman, et al., Methods in Enzymology 202:301 (1991)). Using this method, one of the amino acids involved in the splicing reaction can be replaced, during translation, by a synthetic derivative in which the side chain functionality of the side chain is “masked” by a chemically or photolytically removable group. 
     In another example, certain host cells may not be able to tolerate the gene product of the CIPS, and thus, in some embodiments it may be preferable to inactivate the endonuclease function. It has been shown that protein splicing can occur when the CIPS endonuclease function has been inactivated. Such inactivation can be accomplished in a variety of ways, including for example, random mutagenesis, deletion or insertional inactivation, or site directed mutagenesis. In one example, the endonuclease function is inactivated by site directed mutagenesis. l-Tli-l shares sequence similarity with other “homing endonucleases” in the pair of characteristic dodecapeptide motifs (Cummings et al., Curr. Gent. 16:381 (1989)). Inactivation of endonuclease function has been shown to increase the stability of constructs carrying modified proteins. 
     Also disclosed in U.S. Pat. No. 6,933,362 (herein incorporated by reference in its entirety for its teaching concerning mutated inteins), are various modified inteins. For example, U.S. Pat. No. 6,933,362 discusses a non-naturally occurring intein having splicing activity and controllable cleavage activity or a non-naturally occurring compound having cleaving and/or cleaving and splicing activity, which is controllable. The intein can comprise a truncated intein. The cleavage activity can be controllable by varying oxidative potential. The intein can be obtained from random mutagenesis of a truncated intein, followed by selection based on growth phenotype. The intein can have C-terminal cleavage. The intein can be a truncated Mtu intein. The intein can have the endonuclease domain deleted. The intein can be a truncated Mtu intein with the endonuclease domain deleted, and V67L and/or D422G mutation(s) (relative to full-length Mtu intein). The intein can contain the C-terminal histidine-asparagine. (The presence of the C-terminal histidine residue is believed to confer pH sensitivity and thus it is advantageous that the C-terminal histidine be present; the final asparagine is believed useful for cleavage activity.) 
     Furthermore, one of skill in the art can readily discern useful modified inteins by the use of an effective screen for linking intein activity to an easily observable or selectable phenotype. Such screens are known in the art, for example those found in U.S. Pat. No. 6,933,362. Furthermore, the screen can allow for selection of desired traits under conditions that are relevant for the proposed application. An intein derivative exhibiting controllable cleavage activity has been isolated using rational and random mutagenesis followed by a genetic screen. 
     Affinity Tags 
     Disclosed herein are modified peptides comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible ZBM and an intein. 
     Affinity tags can be short peptide sequences or functional protein domains cloned in frame with protein coding sequences that change the protein&#39;s behavior. Affinity tags can be appended to the N- or C-terminus of proteins which can be used in methods of purifying a protein from cells. Cells expressing a peptide comprising an affinity tag can be pelleted, lysed, and applied to a column, resin or other solid support that displays a ligand to the affinity tags. The solid support can also be washed several times with buffer to eliminate unbound proteins. A protein of interest, if attached to an affinity tag, can be eluted from the solid support via a buffer that causes the affinity tag to dissociate from the ligand resulting in a purified protein. 
     Examples of affinity tags can be found in Kimple et al. Curr Protoc Protein Sci 2004 September; Arnau et al. Protein Expr Purif 2006 July; 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 June; 23(6) 316-20, all hereby incorporated by reference in their entirety for their teaching of examples of affinity tags. 
     Examples of affinity include, but are not limited to, maltose binding protein, which can bind to immobilized maltose to facilitate purification of the fused target protein; Chitin binding protein, which can bind to immobilized chitin; Glutathione S transferase, which can bind to immobilized chitin; Poly-histidine, which can bind to immobilized chelated metals; FLAG octapeptide, which can bind to immobilized anti-FLAG antibodies. 
     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 some aspects, affinity tags can be used that do not actually bind to a ligand. For example, an ELP tag, protein A or Protein G-binding domains can be used as affinity tags. 
     Linkers 
     The CIPSs disclosed herein can be attached to an affinity tag by a variety of means. For example, the CIPSs disclosed herein can be attached to an affinity tag through a linker sequence. The linker sequence can be designed to create distance between the intein and affinity tag, while providing minimal steric interference to the intein cleaving active site. It is generally accepted that linkers involve a relatively unstructured amino acid sequence, and the design and use of linkers are common in the art of designing fusion peptides. There is a variety of protein linker databases which one of skill in the art will recognize. This includes those found in Argos et al. J Mol Biol 1990 Feb. 20; 211(4) 943-58; Crasto et al. Protein Eng 2000 May; 13(5) 309-12; George et al. Protein Eng 2002 November; 15(11) 871-9; Arai et al. Protein Eng 2001 August; 14(8) 529-32; and Robinson et al. PNAS May 26, 1998 vol. 95 no. 11 5929-5934, hereby incorporated by reference in their entirety for their teaching of examples of linkers. 
     Examples of linkers include, but are not limited to: (1) poly-asparagine linker consisting of 4 to 15 asparagine residues, and (2) glycine-serine linker, consisting of various combinations and lengths of polypeptides consisting of glycine and serine. One of skill in the art can easily identify and use any linker that will successfully link the CIPS with an affinity tag. 
     Proteins of Interest 
     Proteins of interest, can include, for example, enzymes, toxins, cytokines, glycoproteins, growth factors, therapeutic proteins, such as antibodies as well as any other protein sought to be purified or used by one of skill in the art. The amino acid and nucleotide sequence of such proteins are easily available through many computer databases, for example, GenBank, EMBL and Swiss-Prot. Alternatively, the nucleotide or amino acid sequence of a protein of interest can be determined using routine procedures known in the art. 
     In some aspects disclosed herein, a protein of interest can be attached to a CIPS so that it can be purified. This is discussed in more detail herein. In some methods described herein, the protein of interest can be a protein that one wishes to purify and accumulate, such as an antibody. 
     Plasmids, Vectors, and Cell Lines 
     Disclosed herein are vectors comprising nucleic acids encoding the CIPSs disclosed herein, as well as cell lines comprising said vectors. Examples of CIPSs that can be encoded in the vectors disclosed herein can be found at least in  FIG. 17 , as well as throughout this disclosure. 
     As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as those encoding a CIPS and a peptide of interest, and/or an affinity tag, for example, into a cell without degradation and include a promoter yielding expression of the gene in the cells into which they can be delivered. In one example, a CIPS and peptide of interest are derived from either a virus or a retrovirus. Viral vectors can be, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also disclosed are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes; they are thermostable and can be stored at room temperature. Disclosed herein is a viral vector which has been engineered so as to suppress the immune response of a host organism, elicited by the viral antigens. Vectors of this type can carry coding regions for Interleukin 8 or 10. 
     Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promoter cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans. 
     The fusion DNA encoding a modified peptide can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used. For instance, when expressing a modified eukaryotic protein, it can be advantageous to use appropriate eukaryotic vectors and host cells. Expression of the fusion DNA results in the production of a modified protein. 
     Also disclosed herein are cell lines comprising the vectors or peptides disclosed herein. A variety of cells can be used with the vectors and plasmids disclosed herein. Non-limiting examples of such cells include somatic cells such as blood cells (erythrocytes and leukocytes), endothelial cells, epithelial cells, neuronal cells (from the central or peripheral nervous systems), muscle cells (including myocytes and myoblasts from skeletal, smooth or cardiac muscle), connective tissue cells (including fibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes and osteoblasts) and other stromal cells (e.g., macrophages, dendritic cells, thymic nurse cells, Schwann cells, etc.). Eukaryotic germ cells (spermatocytes and oocytes) can also be used, as can the progenitors, precursors and stem cells that give rise to the above-described somatic and germ cells. These cells, tissues and organs can be normal, or they can be pathological such as those involved in diseases or physical disorders, including, but not limited to, infectious diseases (caused by bacteria, fungi yeast, viruses (including HIV, or parasites); in genetic or biochemical pathologies (e.g., cystic fibrosis, hemophilia, Alzheimer&#39;s disease, schizophrenia, muscular dystrophy, multiple sclerosis, etc.); or in carcinogenesis and other cancer-related processes. 
     The eukaryotic cell lines disclosed herein can be animal cells, plant cells (monocot or dicot plants) or fungal cells, such as yeast. Animal cells include those of vertebrate or invertebrate origin. Vertebrate cells, especially mammalian cells (including, but not limited to, cells obtained or derived from human, simian or other non-human primate, mouse, rat, avian, bovine, porcine, ovine, canine, feline and the like), avian cells, fish cells (including zebrafish cells), insect cells (including, but not limited to, cells obtained or derived from  Drosophila  species, from  Spodoptera  species (e.g., Sf9 obtained or derived from  S. frugiperida , or HIGH FIVE™ cells) or from  Trichoplusa  species (e.g., MG1, derived from  T. ni )), worm cells (e.g., those obtained or derived from  C. elegans ), and the like. It will be appreciated by one of skill in the art, however, that cells from any species besides those specifically disclosed herein can be advantageously used in accordance with the vectors, plasmids, and methods disclosed herein, without the need for undue experimentation. 
     Examples of useful cell lines include, but are not limited to, HT1080 cells (ATCC CCL 121), HeLa cells and derivatives of HeLa cells (ATCC CCL 2, 2.1 and 2.2), MCF-7 breast cancer cells (ATCC BTH 22), K-562 leukemia cells (ATCC CCL 243), KB carcinoma cells (ATCC CCL 17), 2780AD ovarian carcinoma cells (see Van der Buick, A. M. et al., Cancer Res. 48:5927-5932 (1988), Raji cells (ATCC CCL 86), Jurkat cells (ATCC TIB 152), Namalwa cells (ATCC CRL 1432), HL-60 cells (ATCC CCL 240), Daudi cells (ATCC CCL 213), RPMI 8226 cells (ATCC CCL 155), U-937 cells (ATCC CRL 1593), Bowes Melanoma cells (ATCC CRL 9607), WI-38VA13 subline 2R4 cells (ATCC CLL 75.1), and MOLT-4 cells (ATCC CRL 1582), as well as heterohybridoma cells produced by fusion of human cells and cells of another species. Secondary human fibroblast strains, such as WI-38 (ATCC CCL 75) and MRC-5 (ATCC CCL 171 can also be used. Other mammalian cells and cell lines can be used in accordance with the present invention, including, but not limited to CHO cells, COS cells, VERO cells, 293 cells, PER-C6 cells, M1 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2/0 cells, BHK cells (including BHK-21 cells); these and other cells and cell lines are available commercially, for example from the American Type Culture Collection (P.O. Box 1549, Manassas, Va. 20108 USA). Many other cell lines are known in the art and will be familiar to the ordinarily skilled artisan; such cell lines therefore can be used equally well. 
     Also disclosed herein are cell-free protein expression systems comprising the vectors or peptides disclosed herein, and which also include the required cellular components for expression of a protein from isolated DNA or mRNA molecules. A variety of available cell-free expression systems can be used with the vectors and plasmids disclosed herein. Non-limiting examples of such systems include the Thermo Scientific Pierce Human In Vitro Protein Expression System (based on human cells), Rabbit Reticulocyte Lysate Systems (commercially available from Life Technologies and other suppliers and based on rabbit cells), Wheat Germ Extract Systems (available from several suppliers, and based on wheat germ cells),  E. coli  Cell-Free Systems (based on  E. coli  cell lysates and available from several suppliers), and the EasyXpress Insect Kit (available from Qiagen, and based on  Spodoptera frugiperda  insect cell extracts, with other versions also available from other suppliers). 
     Once obtained, the proteins of interest can be separated and purified by appropriate combination of known techniques. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation; methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis; methods utilizing a difference in electrical charge such as ion-exchange column chromatography; methods utilizing specific affinity such as affinity chromatography; methods utilizing a difference in hydrophobicity such as reverse-phase high performance liquid chromatography; and methods utilizing a difference in isoelectric point, such as isoelectric focusing electrophoresis. These are discussed in more detail below. 
     Methods of Purifying a Protein 
     Disclosed herein are methods of producing or purifying a protein of interest, the method comprising: producing a modified peptide with the structure:
 
X1-CIPS-X2
 
     wherein X1 is an affinity tag, and X2 comprises a protein of interest. The modified peptide comprising the affinity tag, protein of interest, and CIPS can then be exposed to a concentration of zinc which inhibits splicing or cleavage by the CIPS. The modified peptide can then be isolated, such as by protein purification, and the zinc removed, thereby allowing for splicing or cleavage of the protein of interest, thereby producing the protein of interest. The CIPS can be isolated by using the affinity tag. 
     Disclosed herein are methods of producing a modified peptide comprising a CIPS, the method comprising: (a) preparing a nucleic acid that encodes one or more peptides comprising a CIPS; (b) transforming a host cell with the nucleic acid; and (c) culturing the transformed host cell under conditions suitable for the expression of the modified peptide comprising a CIPS encoded by the nucleic acid. Such methods can further comprise isolating the modified peptide comprising a CIPS. 
     A peptide or protein produced using the methods disclosed herein can be a peptide or protein of interest or a therapeutic peptide or protein. Cell lines that can be used with the methods disclosed herein, include, but are not limited to mammalian cells such as CHO cells. 
     Disclosed herein are methods of producing a modified peptide comprising a CIPS, the method comprising: (a) preparing a nucleic acid that encodes one or more peptides comprising a CIPS; (b) transforming a host cell with the nucleic acid; and (c) culturing the transformed host cell under conditions suitable for the expression of the modified peptide comprising a CIPS encoded by the nucleic acid, further comprising the additional step of (d) exposing the modified peptide comprising the CIPS to a chemical reagent which inhibits splicing or cleavage. 
     Disclosed herein are methods of producing a modified peptide comprising a CIPS, the method comprising: (a) preparing a nucleic acid that encodes one or more peptides comprising a CIPS; (b) transforming a host cell with the nucleic acid; and (c) culturing the transformed host cell under conditions suitable for the expression of the modified peptide comprising a CIPS encoded by the nucleic acid, further comprising (d) exposing the modified peptide comprising the CIPS to a chemical reagent, such as zinc, which inhibits splicing or cleavage and (e) exposing the modified peptide to a chelating agent, a change in pH, a change in temperature, dialysis, or dilution, thereby removing the chemical reagent, for instance zinc. 
     Disclosed herein are methods of producing a modified peptide comprising a CIPS, the method comprising: (a) forming a nucleic acid encoding a peptide comprising a CIPS; (b) transforming a host cell with the nucleic acid of step (a); and (c) culturing the transformed host cell under conditions suitable for the expression of the modified peptide comprising the CIPS. 
     Disclosed herein are methods of producing a protein of interest comprising: (a) preparing a nucleic acid that encodes a modified peptide comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS-X2, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible zinc-binding motif and an intein, and wherein X2 comprises the protein of interest; (b) transforming a host cell with the nucleic acid; (c) culturing the transformed host cell under conditions suitable for the expression of the modified peptide; (d) exposing the modified peptide to a concentration of zinc which inhibits splicing or cleavage by the CIPS; (e) isolating the modified peptide; and (0 removing the zinc, thereby allowing for cleavage of the protein of interest from the modified peptide, thereby producing the substantially purified protein of interest. The protein of interest can be a therapeutic protein. 
     Disclosed herein are methods of producing a protein of interest comprising: (a) preparing a nucleic acid that encodes a modified peptide comprising a CIPS wherein the modified peptide comprises the structure: X1-CIPS-X2, wherein X1 is an affinity tag, and wherein the CIPS comprises a reversible zinc-binding motif and an intein, and wherein X2 comprises the protein of interest; (b) transforming a host cell with the nucleic acid; (c) culturing the transformed host cell under conditions suitable for the expression of the modified peptide; (d) exposing the modified peptide to zinc at a concentration of 200 μM or greater; (e) isolating the modified peptide; and (0 removing the zinc, thereby allowing for splicing or cleavage of the protein of interest from the modified peptide, thereby producing the substantially purified protein of interest. 
     Disclosed herein are methods for binding and eluting a phage-displayed polypeptide from a protein of interest comprising: (a) producing a modified peptide with the structure: X1-CIPS-X2, wherein X1 is an affinity tag, the CIPS comprises a reversible zinc-binding motif and an intein and X2 comprises a protein of interest; (b) binding the modified peptide of step (a) to a solid support; (c) contacting a phage-displayed polypeptide with the support-bound peptide of step (b), thereby permitting binding of the phage-displayed polypeptide with the peptide comprising the CIPS; (d) removing unbound phage-displayed polypeptides; and, (e) eluting the bound phage-displayed polypeptide by inducing cleavage of the protein of interest by the CIPS. 
     Also disclosed herein are methods for binding and eluting a phage-displayed polypeptide from a protein of interest, comprising: producing a modified peptide with the structure:
 
X1-CIPS-X2
 
     wherein X1 is an affinity tag, the CIPS comprises a reversible zinc-binding motif and an intein and X2 comprises a protein of interest. The modified peptide which comprises the CIPS can then be bound to a solid support. The phage-displayed polypeptide can then be contacted with the support-bound peptide (the peptide with the CIPS), thereby permitting binding of the phage-displayed polypeptide with the peptide comprising the CIPS. Any unbound phage-displayed polypeptide can then be removed, and the bound phage-displayed polypeptide is eluted by inducing cleavage of the protein of interest by the CIPS. 
     The modified peptide comprising the CIPS can be exposed to a chemical agent, such as zinc, which inhibits splicing or cleavage, until the phage-displayed polypeptide is bound. 
     The chemical agent (such as zinc) that inhibits splicing or cleavage can be removed by using a chelating agent, changing the temperature, changing the pH, dialysis, or dilution of the chemical agent. The phage-displayed polypeptide can comprise a library of at least two sequence variations of the displayed polypeptides. The phage can also be filamentous. 
     Fusion protein purification systems are well known to the skilled artisan. See, EPO 0 286 239 and N. M. Sassenfeld, TIBTECH, 8:88-93 (1990). Typically, in such systems, a binding protein and a target protein are joined by a linker having a protease recognition site. The fusion is then purified by affinity chromatography on a substrate having affinity for the binding protein. The binding protein and the target protein are then separated by contact with a protease, e.g., factor Xa. In these systems, in order to obtain a highly purified target protein, the protease must be separated from the target protein, thus adding an additional purification step, as well as the potential for contamination. The method disclosed herein, by using a CIPS, instead of a protease, avoids these and other problems encountered in currently used protein fusion purification systems. 
     Also disclosed are modified peptides comprising a fusion protein in which a CIPS is between the protein of interest and a protein or peptide having affinity for a substrate (binding protein or binding domain). Techniques for forming such fusion proteins are well known to the skilled artisan. See, EPO 0 286 239 and J. Sambrook, et al., Molecular Cloning: A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. p. 17.29-17.33. 
     Binding proteins which can be employed in the methods disclosed herein include, for example, sugar binding proteins, such as maltose or arabinose binding protein, receptor binding proteins, amino acid binding proteins and metal binding proteins. Another is a chitin binding protein, or the chitin binding domain of a chitinase. Other binding proteins are well known to the skilled artisan. See, EPO 0 286 239 and N. M. Sassenfeld, TIBTECH, supra. 
     A modified peptide can then be contacted with a substrate to which the binding protein has specific affinity, e.g., using affinity chromatography. A highly purified protein of interest can be liberated from the column by changing conditions, such as removing zinc, under which cleavage is initiated, for example, between the CIPS and the protein of interest. Alternatively, a purified fusion protein can be eluted from the column and liberated as above. 
     A CIPS disclosed herein can also be used to isolate modified peptides by use of affinity chromatography with antibodies specific to the CIPS. For example, monoclonal or polyclonal antibodies can be generated having binding affinity to a CIPS using standard techniques. These antibodies can then be utilized in affinity chromatography purification procedures to isolate a modified peptide. After purification, if desired, zinc can be removed, at which time the CIPS will undergo excision. 
     Methods of Producing a Peptide 
     Disclosed herein are methods of producing a modified peptide comprising a CIPS, the method comprising: forming a nucleic acid encoding a CIPS, and transforming a host cell with the nucleic acid; and culturing the transformed host cell under conditions suitable for the expression of the peptide comprising the CIPS. The modified protein can comprise a protein of interest, which can be a therapeutic protein, such as an antibody. The modified peptide can also comprise an affinity tag. 
     The methods disclosed herein can comprise the additional step of exposing the modified peptide comprising the CIPS to a chemical reagent which inhibits splicing or cleavage. This chemical reagent can be zinc, for example, and can be at a concentration below 1000 μM, for example. Additionally, the peptide comprising the CIPS can be exposed to a chelating agent, a change in pH, dialysis, or dilution, thereby removing zinc. 
     Kits 
     Disclosed herein are kits comprising a CIPS, or a CIPS linked to an affinity tag and/or a protein of interest. Also disclosed are kits comprising peptides comprising one or more of SEQ ID NOS: 11, 15, 19, 23, 27, or 31. Also disclosed are kits comprising nucleic acids, comprising one or more of SEQ ID NOS: 12, 16, 20, 24, 28, or 32. Also disclosed are kits comprising the vectors, plasmids, and/or the cell lines disclosed herein. In a further example, a kit can comprise zinc. Zinc, for example, can be at a concentration of 1000 μM or less. 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric. 
     EXAMPLES 
     Example 1 
     In Silico Design 
     Nearly all inteins end with a Histidine-Asparagine Dipeptide ( FIG. 3 ). The histidine is believed to be involved in the cleaving reaction, and the fact that the pH sensitivity of the cleaving reaction follows the pKa titration curve for Histidine also suggests this. One additional piece of evidence is that metal ions, and especially zinc, can suppress intein cleaving at high concentrations. This is presumably due to their binding to the Histidine. The development of stronger zinc-binding to this particular Histidine residue formed the basis for the rational intein design. 
     The tested zinc-binding motifs (ZBMs) are computationally designed to have high potential zinc-binding affinity and to interrupt intein activity through coordination of critical intein active site histidine residue side chains. Considered in the design were (1) the position to insert zinc-binding motif into intein and (2) the sequences to bind zinc with or without using existing residues that occur near the intein active site. Conceptually the intention was to adapt a minimal portion of previously reported ZBMs that did not require cysteine residues (for example 1ZE9). The subdomain was engineered proximal to the activity site. This is substantially different from previously reported work where control attempts were made using insertions into the distal ‘control loop’ region of the intein or through random mutagenesis approaches. A panel of related designs having subtle variations in predicted zinc coordination behaviors was tested. 
     In a notable design that is exemplary of the six initial designs, the last two naturally occurring intein amino acids were replaced and the short sub-domain/motif ‘xExHH’ was further appended to the intein N-terminus to give for example . . . x(E-2)x(H0)(H1)(L2)AEGT. This form can act through metal-coordination with H439, inhibiting H439&#39;s interactions with N440 in the active site. The related motifs subtly alter the orientation and distances of metal coordination bonds within the switch subdomain. The specific amino acid sequences of the six core inventive designs are: 
     
       
         
           
               
               
            
               
                   
                 LN001: 
               
               
                   
                 (SEQ ID NO: 39) 
               
               
                   
                 xGEGHHLAEGT 
               
               
                   
                   
               
               
                   
                 LN002: 
               
               
                   
                 (SEQ ID NO: 40) 
               
               
                   
                 xGEGHCLAEGT 
               
               
                   
                   
               
               
                   
                 LN003: 
               
               
                   
                 (SEQ ID NO: 41) 
               
               
                   
                 xGEGHHALAEGT 
               
               
                   
                   
               
               
                   
                 LN004: 
               
               
                   
                 (SEQ ID NO: 42) 
               
               
                   
                 xGEGHGCLAEGT 
               
               
                   
                   
               
               
                   
                 LN003B:  
               
               
                   
                 (SEQ ID NO: 43) 
               
               
                   
                 xGDGHGCLAEGT 
               
               
                   
                   
               
               
                   
                 LN004B:  
               
               
                   
                 (SEQ ID NO: 44) 
               
               
                   
                 xGDGHGCLAEGT 
               
            
           
         
       
     
     These sequences were subjected to computational modeling using Schrodinger Q-SITE and Impact packages run with explicit solvent and a quantum mechanical simulation set that included the zinc metal ion and the amino acid side chain, separated from the molecular modeling region using hydrogen caps between the alpha and beta carbons (See  FIG. 5A-F ). The simulations were run with and without a metal center to look for structures that were distorted upon insertion of the metal. The structures represented a range of conformations, all of which had potential to inhibit the intein active site upon zinc-binding. 
     Common wisdom predicted that the designs might result in a constitutively inactive intein due to steric and electronic disruption of the active site. However, it was shown that this was not the case for the disclosed amino acid sequences. 
     Example 2 
     In Vitro Testing 
     Previous attempts to modify the intein cleaving reaction and controllability through modifications to the body of the mini-intein ΔI-CM have been unsuccessful and generally lead to inactive inteins or inteins with very slow cleavage rates. For this reason, the focus of the disclosed inteins was on the sequence immediately preceding the N-terminus of the intein to generate a zinc-binding motif that includes the critical Histidine residue required for cleaving. This was done by appending the computationally designed sequences to the N-terminus of the mini intein ΔI-CM. 
     Several putative inteins were designed. Once tested, the most promising design was determined to be an intein that was denoted as LN-004b, for experimental purposes. This intein has a much stronger affinity for zinc than the original ΔI-CM intein, and the presence of zinc substantially suppressed the cleaving reaction, as shown in  FIG. 4 . 
     After extensive testing, it was determined that the zinc concentration required to decrease the cleaving rate by 50% had decreased from 100 μM in the ΔI-CM intein to 17 μM in the LN-004b intein. Thus, the concentration of zinc required to fully suppress intein cleaving also decreased by a factor of approximately 10 (from about 600 μM in  FIG. 4  to less than 100 μM). This change allows the safe use of zinc to suppress intein cleaving in CHO cell culture because CHO cells can easily tolerate 100 μM zinc, but suffer at 600 μM zinc. 
     Example 3 
     Therapeutic proteins are currently produced in large manufacturing plants using in vitro cell culture, where recombinant cells are grown over multiple days or weeks in &gt;1,000 liter bioreactors. The bioreactors are then harvested and the expressed proteins are recovered, purified, stabilized, and stored as an intermediate bulk drug substance. The drug substance is then formulated, filled into delivery devices and released by a quality assurance organization before shipment to a distribution warehouse for future delivery to the point-of-care. This is an enormously expensive, time consuming, and complex undertaking that takes months to execute. If starting from scratch, process development activities for a single therapeutic would ordinarily require 6 to 8 years per project to complete all of the characterization, process monitoring and validation activities, needed to meet regulatory authority approval. While companies continue to develop in vivo expression platforms for the manufacturing of proteins, there are no systems designed for the flexibility across proteins or the speed necessary for the rapid development of purification methods for arbitrary target proteins to be used in patient therapies. A novel mammalian in vitro protein expression technology and innovative purification technologies combined with advances in microfluidics and microelectronics to produce biologics on site can be used. Specifically, following technologies can be enhanced and integrated: 
     Protein Expression: Expression of the protein of interest can be made possible with a mammalian cell-free extract similar to the product currently commercialized by Thermo Scientific based on HeLa cell extracts. The Thermo Scientific 1-Step Human High-Yield In Vitro Translation (IVT) is a cell-free protein expression system that provides all of the essential components required for transcription and translation. The kits are optimized with Accessory Proteins and Reaction Mixes that support protein synthesis for up to 24 hours using a DNA template. The 1-Step Human High-Yield IVT Kits, with a continuous-feed device, enable the expression of functional proteins. The current system uses modified HeLa cell extracts to take advantage of the robust human translation machinery and generate functional full length proteins. The advantages of using the 1-Step Thermo High-Yield IVT Kits over traditional in vivo expression systems include (1) the ability to express toxic or insoluble proteins, (2) easily perform protein labeling with modified amino-acids, (3) reduce the time and cost of expressing human proteins in tissue culture cells, (4) 10 to 100 times greater expression than other mammalian IVT expression systems that are based on rabbit reticulocyte lysates, ability to carry out (5) post translational modifications (PTMs) including phosphorylation, and (6) lot-to-lot consistency as a cell culture based product. In this system, protein expression is performed in a proprietary mini dialysis device that allows a continuous supply of nucleotides, amino acids and energy generating substrates into the reaction while removing inhibitors of proteins synthesis. 
     Purification: A purification process platform can consist of two basic steps: (1) an affinity capture of the target protein and (2) a subsequent polishing step which the impurities present in the eluted product from the affinity step can be reduced to an acceptable level. The affinity capture step can be the use of a fusion product comprising an affinity tag, a self-cleaving intein, and a therapeutic target protein. This fusion is initially adsorbed onto a chromatography column and washed of impurities, after which the target protein is eluted by pH-induced self-cleavage of the intein-affinity tag. For the polishing step the use of membrane chromatography columns incorporating either conventional or mixed mode ligands used together with novel elution conditions can be used. The self-cleaving affinity tag used together with a suitable polishing step in this system represents a new platform for the purification of non-antibody proteins. A platform of this type has never been applied to general recombinant therapeutic proteins. 
     The intein purification method allows a product to be recovered directly from a cell free reactor and produce a purified therapeutic protein of interest in a form suitable for parenteral administration at a high product yield. 
     Development of Capture Methods Using an Intein Fusion Product: 
     In order to accomplish the intein-based affinity capture method proposed here, gene fusions can be constructed using conventional PCR cloning methods. The fusions can include different combinations of affinity tag, self-cleaving intein and target protein. The basic strategy for the intein based method is shown in  FIG. 2 . Three initial tags can be evaluated, including the chitin-binding domain (CBD), an Fc domain, and a quaternary amine binding protein. The CBD tag has been chosen due to its small size and proven track record with intein technologies, as well as its strong binding to its corresponding ligand. These characteristics provide very high purification factors with this tag, along with minimal impacts on overall expression. A disadvantage of this tag is that no pre-validated affinity resins are available, and the binding capacity of available resins is somewhat low. The Fc domain tag has been selected due to its small size, tight binding, and potential to bind to conventional Protein A affinity chromatography resin. Because Protein A resins are the current industry standard for producing therapeutic antibodies, they have an extensive track record with the FDA and are easily validated. Further, available Protein A resins have extremely high binding capacities (50 milligrams of IgG antibody per milliliter of bed volume) and can be regenerated over 100 times. This combination of characteristics indicates that Protein A resins can be optimal for the proposed device, as they will be both durable and highly productive in a relatively small volume. Disadvantages for Protein A are that they have not yet been evaluated with intein technologies, and it is not clear which Fc domain will be appropriate for this application. Finally, a novel affinity tag with highly specific binding to quaternary amines can be investigated. These amines are very commonly used in conventional chromatography resins used in the biopharmaceutical industry, and are readily available in a pre-validated form (e.g., Q-Sepharose). Non-specific binding is a concern due to the charged nature of these resins, but a properly designed purification method may actually capitalize on this aspect to enhance the intein-mediated purification step with some ion-exchange character. These tags can be combined with a variety of self-cleaving intein modules, each selected for high controllability that is appropriate to the expression system. The base case inteins can be the previously reported ΔI-CM and ΔI-SM inteins, which are tightly controlled by a combination of pH and temperature. These inteins can have sufficient control to allow a highly effective purification of the target proteins with one of the three aforementioned affinity tags. The development of more rapidly cleaving and controllable inteins is also being investigated. 
     Evaluations of the intein performance can rely on small-scale expression and quantification using conventional assays. As stated above, particular attention can be paid to expression level (productivity), premature cleaving and cleaving efficiency during purification (yield), and purity. Assessments can be made using SDS-PAGE for early experiments, and HPLC during later optimization. Final assessments can include specific activity assays and more advanced bioequivalence assays. 
     Ultimately, a combination of tag and intein can be selected for each target protein such that expression, cleaving and purity are maximized for downstream polishing. Based on previous work, there is potential to acquire greater than 80% yield in the intein-based affinity steps, which combine initial capture and rough purification of the initial target. Specific minimal performance metrics can be determined for each therapeutic target protein, and the optimal designs can be incorporated into the device DNA and methodology libraries. 
     Example 4 
     Background and Significance 
     The importance of highly purified biopharmaceutical proteins and enzymes cannot be overstated. Well-known blockbuster examples from industry include Remicade® for the treatment of Rheumatoid Arthritis (RA), Avastin® for metastatic cancers, Rituxan® for RA and Non-Hodgkin&#39;s Lymphoma, and dozens of other lifesaving recombinant antibodies and glycoproteins. Although some progress has been made in the expression of glycoproteins in microbial and yeast hosts, the vast majority of complex biopharmaceutical glycoproteins are still expressed in mammalian cell culture, where the most commonly used host is Chinese Hamster Ovary (CHO) cells. Further, dramatic increases in mammalian cell culture productivity, due to newly engineered cell lines and well-designed fed-batch processes, are making CHO and other hosts highly efficient for glycoprotein production at both laboratory and manufacturing scales. Monoclonal antibodies, which make up a large percentage of biologics, can be purified using the Protein A affinity platform, but increasingly important non-antibody glycoproteins cannot. These proteins require individual methods to be developed ( FIG. 27A ), which can slow the discovery and development of new therapeutics, while increasing their eventual costs. These developments have shifted research and manufacturing bottlenecks to protein purification, and have thus created an urgent need for reliable platform methods to purify new glycoproteins from mammalian cell culture. This study creates the first generally applicable self-cleaving tag purification platform for proteins expressed in mammalian cell culture, and particularly for those proteins that cannot be purified with existing Protein A pla ( FIG. 27B )tform methods. This platform can enable the rapid and inexpensive development of therapeutics at all scales. As such, it can have a critical impact on accessibility to personalized medicine, where small patient populations require development costs to be minimized. These methods can also enable high-throughput proteomic studies, which can accelerate the development of new pharmaceuticals. Moreover, the ability of this system to work with any given tag can facilitate the development of disposable technologies, which are becoming increasingly attractive for next generation multiproduct biopharmaceutical manufacturing facilities. Overall, this work can play a key role in the highly active field of high-capacity and low cost process development. 
     This approach is modeled after conventional affinity tag methods used ubiquitously in microbial recombinant protein expression. In this method, an easily purified affinity tag is genetically fused to the target protein to facilitate its purification ( FIG. 6A ). The purification method is then based entirely on the tag, and requires no target-dependent optimization, allowing virtually any target protein to be purified through a single, universal procedure. Thus, these methods lend themselves to the development of platform and high-throughput processes, and are potentially very appealing to the pharmaceutical industry. Commonly used tags include the maltose binding protein (MBP), glutathione S-transferase (GST), chitin binding protein (CBP), and the highly popular poly-histidine tag (His6). Each of these tags binds to its commercially available cognate affinity resin, allowing target proteins fused to the tag to be purified using a single, pre-optimized procedure. Although these methods are highly effective at laboratory scale, and are used in academic, government and industrial laboratories worldwide, they are never used in biopharmaceutical manufacturing, and are rarely used to purify products expressed in mammalian cell culture. This is primarily due to the potential immunogenicity of the tags, and the expense associated with removing them at multi-kilogram scale. In particular, the highly specific endopeptidases used for tag removal can add several thousand dollars per gram of purified target protein to their production cost, making these methods cost-prohibitive at even small biopharmaceutical manufacturing scales. 
     This study seeks to enable the use of tag methods for biopharmaceutical production by generating effective self-cleaving protein modules that can uniquely be used in mammalian cell culture ( FIG. 6B ). This controllable module can induce any conventional purification tag to self-cleave in response to a simple and innocuous process change (e.g., pH, temperature or ligand addition), allowing highly efficient tag removal at minimal expense. An important aspect of the proposed module is that its mechanism ensures precise cleavage of the target protein, so that unwanted or aberrant cleaving is impossible. The significance of this technology is evident from the number of self-cleaving modules that have been developed in the past several years. Each of these methods exhibits a variety of advantages and disadvantages, and unfortunately, none of them are universally applicable to mammalian cell culture products. Among the most significant disadvantages for many of these modules is a requirement for modification of the target protein amino acid sequence (Table 3,  Neisseria meningitides  FrpC protein,  Staph aureus  Sortase A transpeptidase, and  Vibrio cholerae  MARTX domain). These modifications are potentially immunogenic for therapeutic proteins, and can also interfere with protein activities in basic studies. The Classical Swine Fever Virus Npro Module requires a refolding procedure to induce cleaving, making this method impractical for mammalian cell culture as well. Two notable self-cleaving modules in this group belong to the commercially available IMPACT and IMPACTCN systems. These modules, currently marketed by New England Biolabs, are based on a naturally occurring self-splicing protein called an intein. These modified inteins have been combined with a chitin binding protein and chitin resin, and are induced to self-cleave at their N-terminus by addition of thiol compounds (e.g., dithiothreitol, 2-mercaptoethanol or 2-mercaptoethane sulfonate). In this case, the thiol compound chemically breaks a thioester intermediate bond between the target protein and tag, releasing the target protein from the intein and column-bound chitin binding tag. In the IMPACT-CN system, the peptide bonds at the N and C-termini of the intein are simultaneously induced to cleave by thiol addition. Unfortunately, disulfide bonds in antibodies and other therapeutic proteins are chemically similar to this labile thioester, causing them to also be broken by the IMPACT cleaving agent. This limitation effectively prevents the IMPACT system from being used with proteins containing disulfide bonds, which include all recombinant antibodies and a large majority of antibody fragments and other biologics currently in development. For these reasons, a controllable self-cleaving module with general applicability in eukaryotic host cells has yet to be found. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Summary existing self-cleaving modules and relative advantages and disadvantages 
               
            
           
           
               
               
               
               
            
               
                 Cleaving Method 
                 Cleaving Trigger 
                 Primary Advantages 
                 Primary Disadvantages 
               
               
                   
               
               
                 Conventional 
                 Protease Enzyme 
                 Well-established, with 
                 Expensive. 
               
               
                 Protease 
                 Addition, incubation 
                 many optimized 
                 Can require target 
               
               
                   
                 conditions depend on 
                 options available. No 
                 modification. Can cleave 
               
               
                   
                 protease used. 
                 premature cleaving. 
                 target unexpectedly. 
               
               
                   
                   
                   
                 Cleaving rate unpredictable. 
               
               
                 N-cleaving intein 
                 Thiol compounds at 
                 Most commonly used, 
                 Requires high concentrations 
               
               
                 (IMPACT ® 
                 30 to 100 mM. 
                 commercially available. 
                 of thiol compounds for 
               
               
                 System) 
                 Incubation at 4° C. to 
                 Very little premature 
                 cleaving. Breaks disulfide 
               
               
                   
                 37° C. for 2 hr to O/N. 
                 cleaving. Good low 
                 bonds in target. Expensive at 
               
               
                   
                   
                 temp cleaving rate. 
                 large scale. 
               
               
                 C-cleaving intein 
                 pH below 7.0, 
                 Safe for most proteins. 
                 Substantial premature 
               
               
                 (This proposal) 
                 incubation at room 
                 Most inexpensive and 
                 cleaving in vivo during 
               
               
                   
                 temp to 37° C., 4 to 24 hr. 
                 flexible cleaving 
                 expression. Slow cleaving at 
               
               
                   
                   
                 trigger. Tag can aid 
                 low temp. Cannot be used in 
               
               
                   
                   
                 solubility. 
                 CHO cells. 
               
               
                 N + C cleaving 
                 Same as N-cleaving 
                 Most commonly used, 
                 Requires high concentrations 
               
               
                 intein (IMPACT- 
                 inteins. 
                 commercially available. 
                 of thiol compounds for 
               
               
                 CN ® System) 
                   
                 Very little premature 
                 cleaving. Breaks disulfide 
               
               
                   
                   
                 cleaving. 
                 bonds in target. Expensive at 
               
               
                   
                   
                   
                 large scale. Requires dialysis 
               
               
                   
                   
                   
                 after cleaving. 
               
               
                 
                   Neisseria 
                 
                 10 mM Ca 2+ , 4° C. to 
                 Very little premature 
                 Requires addition of Asp 
               
               
                   meningitides  FrpC 
                 37° C., 4 to 8 hr (higher 
                 cleaving during 
                 residue to C-terminus of 
               
               
                 protein (N- 
                 temperatures 
                 expression. Fast 
                 target protein. May require 
               
               
                 cleaving) 
                 accelerate cleaving) 
                 cleaving upon Ca 2+   
                 EDTA and DTT in 
               
               
                   
                   
                 addition. 
                 purification buffers. 
               
               
                 
                   Staph aureus 
                 
                 5 mM Ca 2+ , 10 mM 
                 Fast cleaving. 
                 Requires addition of Gly 
               
               
                 Sortase A 
                 Gly3, incubation at 
                 Cleaving module 
                 residue to N-terminus of 
               
               
                 transpeptidase (C- 
                 25° C. for 4 to 6 hours. 
                 appears to increase 
                 target protein. Some 
               
               
                 cleaving) 
                   
                 solubility of target 
                 premature cleaving. 
               
               
                   
                   
                 protein. 
                 Unwanted cleaving in target 
               
               
                   
                   
                   
                 (?). 
               
               
                 
                   Vibrio cholerae 
                 
                 50-100 mM inositol 
                 Fast cleaving. 
                 Requires 1 to 4 a.a. to be 
               
               
                 MARTX domain 
                 hexakisphosphate 
                 Cleaving module 
                 added to C-terminus of target 
               
               
                 (N-cleaving) 
                 (InsP 6 ), 4° C. to room 
                 appears to increase 
                 protein. Small potential for 
               
               
                   
                 temp for 1 to 2 hr. 
                 solubility of target 
                 unwanted cleaving in target. 
               
               
                   
                   
                 increase solubility of 
               
               
                   
                   
                 target protein. 
               
               
                 Classical Swine 
                 In vitro refolding of 
                 High-level expression. 
                 Efficient refolding procedure 
               
               
                 Fever Virus N pro   
                 fusion protein under 
                 Protects target from 
                 for target must be available. 
               
               
                 Module 
                 kosmotropic 
                 protease. Good for 
               
               
                   
                 conditions. 
                 toxic targets. Native 
               
               
                   
                   
                 target N-terminus. 
               
               
                   
               
            
           
         
       
     
     The disclosed research effort has been the development of self-cleaving purification tags based on C-terminal cleaving inteins ( FIG. 6B ). These inteins are distinct from the N-terminal cleaving inteins of the IMPACT systems in that they are triggered to cleave by changes in pH and temperature alone, and most importantly, do not require thiol agents to induce thioester cleavage. These inteins are also capable of delivering a target protein without any additional amino acids on the N-terminus, allowing the production of fully native products. Further, the cleavage mechanism ensures that the cleavage reaction is strictly limited to the peptide bond immediately following the final conserved Asn residue of the intein. These aspects make C-terminal cleaving inteins uniquely attractive as a starting point for the development of self-cleaving tags that can be used with complex products produced in mammalian cell culture. The initial efforts produced an engineered mini-intein domain that cleaves cleanly at its C-terminus in response to a mild change in pH and/or temperature. This self-cleaving intein domain has been combined with a number of conventional and new nonchromatographic tags to create a set of powerful new methods for the purification of proteins expressed in  E. coli.    
     Intein-tag fusion proteins have been expressed in CHO cells and  Pichia pastoris . However, a major obstacle has become premature cleaving of the target protein during expression, which substantially decreases final yields of purified target. This is due to the fact that the cleaving reaction is solely dependent on pH and temperature. This dependence has been an asset in microbial expression hosts, but has become a liability when applying these systems to mammalian cell culture ( FIG. 7 ). The primary difference between microbial and mammalian expression systems is that the intein cleavage rate is slow under conditions experienced in microbial expression, while microbial expression rates are high. Conversely, the intein cleavage rate is rapid under conditions experienced in mammalian cell culture, while mammalian expression rates are lower. This difference allows an uncleaved target protein to be purified from  E. coli , but leads to substantial premature cleaving of the target protein before purification in mammalian hosts. This critical difference effectively prevents currently available self-cleaving tags from being applied to the production of therapeutic glycoproteins produced in mammalian hosts. This shortcoming is intrinsic to the cleaving mechanism of the intein, and thus is not trivial to overcome. Indeed, the development of a CHO-capable intein system has stymied several academic and industrial laboratories over the past decade. The disclosed approach combines rational protein engineering and directed evolution to create a next generation intein platform for the purification for complex biopharmaceutical glycoproteins at laboratory and manufacturing scales. 
     Potentially Transformative Aspects 
     The impact of this work is indicated by the success of two existing related technologies. The first is the use of conventional affinity tags in laboratories throughout the world for the purification of recombinant proteins expressed in microbial hosts. These methods have changed the way molecular biology is studied, and have impacted the development of countless therapeutic proteins on the market today. The second is the universal application of Protein A affinity methods to the manufacture and study of recombinant antibodies produced in mammalian cell culture. The popularity of this expensive, yet powerful method for antibody purification illustrates the appeal of simple affinity methods for the purification of mammalian cell culture products. However, Protein A affinity methods cannot be applied to non-antibody products, and conventional affinity tag methods have yet to penetrate mammalian cell culture processes. Thus, the disclosed work lies at the intersection of these two technologies. It seeks to provide a new platform method, with the compelling appeal of Protein A for purification, but with the universal applicability of conventional microbial affinity tags for virtually any target. The resulting methods can have a similar impact to the now ubiquitous affinity tags used in microbial expression, but applied to the production of complex glycoproteins in mammalian and other eukaryotic hosts. 
     Development of Self-Cleaving Tags for Mammalian Cell Culture Applications Overall Approach: 
     Rational active-site redesign can be combined with a yeast display-based evolutionary approach to develop a highly controllable self-cleaving intein for mammalian cell expression systems. This is a deviation from previously published approaches, which have relied on random mutagenesis with in vivo genetic selections for controlled intein cleaving. A critical shortcoming of these previous approaches is that direct selections for inteins sensitive to metal ions, small-molecule ligands, pH changes, strong temperature shifts, or any non-physiological condition, are impossible because these conditions cannot generally be manipulated inside a growing cell. Further, the use of random mutagenesis greatly decreases the probability that critical combinations of mutations will ever be observed. Thus, the disclosed approach derives from the specific focus on residues and structural features that are known to be critical to intein cleaving function, and from the selection of the resulting mutants in a fully flexible in vitro environment. This strategy can generate optimized mutant inteins, designed to be stable and uncleaved at the physiological conditions of a desired expression host, but that cleave rapidly under convenient downstream process conditions in vitro. 
     Introduction of engineered metal binding sites into the intein active center. Several lines of evidence indicate that intein cleavage can be controlled through the introduction of a metal binding site into the intein active center ( FIG. 8 ). The first is that intein splicing domains end with a highly conserved histidine-asparagine dipeptide at their C-terminus (H439/N440 in previously developed ΔI-CM intein;  FIG. 8A ). Cyclization of the C-terminal asparagine to form a succinimide ring cleaves the C-terminal peptide bond during the self-cleaving reaction, releasing the fused target protein. Plots of intein cleavage rates versus pH closely resemble that of the pKa curve for the histidine side chain (pKa ˜7.2), indicating that the protonation state of the highly conserved penultimate histidine residue can be critical for activation of intein cleaving ( FIG. 8B ). This is further supported by molecular simulations. Further, previous observations have shown that high concentrations of zinc ion can inhibit intein splicing and cleaving, indicating that this histidine residue is positioned to weakly bind divalent ions, and that binding can inhibit the cleaving reaction. Finally, crystallographic data indicate that the penultimate histidine residue of the previously developed ΔI-CM intein exists in an extended configuration, further indicating that this residue can be a target for allosteric intein control through metal binding ( FIG. 8C ). Based on these observations, a set of inteins with augmented metal binding sites can be assembled. 
     Histidine residues H429 and H439 of the ΔI-CM mini intein (residue numbering is relative to the full-length Mtu RecA intein) are critical to the intein active site. A metal binding site that includes one or both of these residues could sequester the enzymatically active imidazole nitrogens and prevent their interactions with N440, thus interfering with the cleaving reaction. The  Synechocystis  sp. PCC6803 DnaE intein and the Mtu RecA intein splicing activities are naturally and reversibly inhibited by as little as 2 mM divalent zinc. A crystal structure of a minimized Mtu RecA splicing intein, in complex with divalent zinc, showed cross-protein metal binding interactions with active site residues H429, H439, E424, and the cyclized N440 succinimide (Protein Data Bank: 2IMZ). Interaction of a cleaving intein mutant alone in free solution with Zn 2+  was studied using isothermal titration calorimetry. The binding constant for zinc was reported to be 6.8 nM, and likely involves specific metal coordination to H429 and H438. Unfortunately, this naturally occurring zinc binding site recruits ligands from two intein molecules aligned in a dimer within the protein crystal structure. Inteins with a fused N-terminal affinity tag and a C-terminal target protein fail to form this dimer structure and bind zinc efficiently, and thus fail to be inhibited. It is this binding site that can be augmented through protein engineering. In particular, an additional peptide segment with several metal coordinating side chains can be appended onto the N-terminus of the intein sequence, thus taking advantage of the proximity of the N and C-termini of the intein to “graft” on a new metal binding center that includes critical residues of the intein active site ( FIG. 9 ). Critically, this strategy does not interfere with the specific amino acids involved in the cleaving reaction, thereby maintaining the ability of the intein to release a native target protein after cleaving. Finally, release of the bound zinc, either by EDTA chelation or pH shift, can restore the histidine residues to their unbound state, thus allosterically activating the intein cleaving reaction. 
     Results 
     Rational protein design methods in conjunction with traditional template-driven PCR methods have been used to construct six Mtu RecA mutants in which divalent zinc binding sites recruit at least one critical active site residue. In each case, six or eight amino acids were added to the N-terminus of the intein to introduce the new metal-binding site. Once constructed, these engineered mutants were expressed in  E. coli  host cells for initial characterization of zinc binding and cleavage activity in vitro. Remarkably, several of these mutants exhibit significantly greater sensitivity to zinc than the original ΔI-CM parent intein. The most promising of these mutants, LN-004b ( FIG. 10A ), exhibits zinc binding almost an order of magnitude stronger than the original mini-intein (17.3 μM for LN-004b versus 100.3 μM for ΔI-CM), and surprisingly, exhibits significantly faster cleaving kinetics in the absence of zinc than the original mini-intein as well. In particular, the LN-004b intein exhibits no measurable cleaving over 24 hours at 37° C. and pH 7.0 in the presence of 100 μM zinc, while the original ΔI-CM intein requires fully 500 μM zinc to experience the same level of repression ( FIG. 10B ). Further, the zinc binding effect is abolished at pH lower than 6.8, allowing the zinc-binding inteins to be activated by a simple pH shift in a manner similar to the original mini-intein (without EDTA addition). Indeed, all of the constructed mutants exhibit pH sensitivity similar to the original ΔI-CM intein, with greatly enhanced controllability in the presence of zinc ion. Notably, some of the constructed mutants exhibit highly accelerated cleaving in vivo as well, and thus could not be well characterized in vitro since the uncleaved precursor could not be purified. Two of these inteins (LN-002 and LN-004b) are being expressed in CHO and cell-free expression systems in the presence of 300 μM zinc. 
     Experimental Plan 
     Stronger zinc binding can be beneficial for mammalian and other expression host applications. A two-pronged approach can be used to the optimization of the engineered intein mutants. The first approach can be to use the knowledge gained from the initial set of designs to inform a second round of rationally engineered metal binding centers. The initial six designed inteins have already been characterized and provide a clearer understanding of the strengths and weaknesses of the computational design methods. A reassuring result is that the LN-004b intein was predicted to have the strongest zinc binding, based on the angles and lengths of the predicted metal coordination bonds. These data indicate that an iterative rational approach, where each iteration is driven by experimental laboratory data, can generate highly optimized mutants. Another approach can be to target random mutagenesis of the residues surrounding the engineered binding center, coupled with the yeast display screen. In this case, beneficial random mutations can be screened, where the computational and structural models can be used to determine the specific residues targeted for mutagenesis. 
     Evolutionary methods can be applied to the optimization of the rationally designed inteins, as well as inteins designed for altered pH and temperature activity profiles. These approaches can include the development of an effective in vitro intein activity screening system based on yeast surface display, and a targeted mutant library, focused specifically on active site residues associated with control of the intein cleaving. 
     The impact of self-cleaving purification tags can be much greater if they can be used as a platform for biopharmaceutical manufacturing. To encourage their adoption, their performance can be demonstrated in the context of a cGMP process, with a focus on their incorporation into established conventional unit operations. Because CHO is the primary source of recombinant glycoproteins in the biopharmaceutical industry, it will be one of the primary targets. An important alternative is  Pichia pastoris , which has recently been engineered to produce human glycosylation patterns on secreted protein products. The intein fusions can initially be evaluated for overall target expression and suppression of premature cleaving in these cells, with the goal of generating an optimized intein toolbox for various mammalian and eukaryotic host organisms. A critical aspect of this work is the choice of affinity tag. In particular, there are no manufacturing scale bulk sources for practically any conventional affinity resins (such as amylose, chitin or glutathione), largely due to the fact that they are never used at manufacturing scales. Further, these resins have typically not been optimized for binding capacity as conventional biopharmaceutical purification resins have, leaving them unacceptably inefficient at large scale. For this reason, alternate tags, with specific affinities for established industry standard purification resins, are studied. An important candidate tag in this area is the Choline Binding Domain of the major autolysin (C-lytA) from  Streptococcus pneumoniae . This tag binds very tightly and specifically to the quaternary amine of choline, imparting it with strong and selective affinity for quaternary amine ion exchange resins. Most importantly, the Choline Binding Domain can bind strongly to Q-sepharose resins, even in the presence of 1.5 M NaCl. Most proteins will not bind Q-sepharose under these conditions, causing the choline binding tag to act effectively as an affinity tag for Q-sepharose, providing a very high degree of purity in a single column process ( FIG. 11 ). Further, because Q-sepharose is a highly optimized industry standard resin, it exhibits extremely high binding capacity (up to 100 grams per liter of bed volume), and is available from many commercial sources in bulk. 
     Results 
     In previous work with eukaryotic host cells, expression of intein-tagged fusion proteins in both CHO and  Pichia  was attempted. The ELP and chitin binding tags were used in these experiments, where they were combined with cleaving and non-cleaving control inteins to determine overall expression levels and product losses due to premature tag cleaving. These experiences (described below) allow for the incorporation of new inteins into the existing characterization framework, as well as the new choline binding tag. 
     The data shows that in both CHO and  Pichia , expression of intein-containing fusion proteins was possible with the original ΔI-CM intein, but unacceptable levels of premature cleaving were observed in both hosts. In CHO, an oncoM secretion leader sequence was used, followed by the ELP tag and intein genes, fused to secreted alkaline phosphatase as the target protein. A constitutive CMV promoter drove expression of the tagged fusion protein in a DG44 cell line, and in later experiments gene expression was amplified by methotrexate (MTX) addition. Gene expression could be amplified, although it typically reached only 10% to 50% of the untagged control protein. The use of a non-cleaving control intein with the ELP tag lead to the lowest observed levels, which were approximately 220 milligrams per liter of fusion protein, when expressed in shaker flask cultures at 400 nM MTX. The cleaving intein delivered approximately 800 mg/liter (by activity) under the same conditions. In  Pichia , expression was induced by methanol addition, but no significant product was secreted with the ELP tag fused to either intein (cleaving or non-cleaving). 
     Chitin binding domain-tagged proteins expressed well in  Pichia , and existing self-cleaving chitin-intein tags were used to purify both β-lactamase and β-galactosidase. In particular, the overall recovery of β-galactosidase using this method was 30% (by activity), with a 100-fold purification factor. Significant losses occurred due to excessive premature cleaving, however, leading to unacceptably low overall yields (milligrams per liter). 
     The choline binding tag exhibits excellent performance as a purification tag when expressed in  E. coli , but the binding capacity of the Q-sepharose resins is typically on the order of 2 to 3 mg/ml of bed volume. This is unacceptably low, and additional process conditions to increase this capacity are being performed. The choline binding tag has been combined with several secretion leader sequences and expressed in CHO-S in transient transfection with several control inteins. The results have been mixed, indicating zinc can affect both cleaving and secretion. 
     It has been established that intein fusions can be expressed in two important biopharmaceutical expression hosts. The ability to express highly tractable test proteins in  Pichia  and CHO indicates that performance benchmarks will be fairly easy to establish as new inteins become available. 
     Experimental Plan 
     Existing ΔI-CM intein and future intein mutants can be adapted for cGMP manufacturing processes. Combining these inteins with the choline binding module and other conventional affinity tags that might be incorporated into a cGMP purification process with minimal validation effort can be performed. Quaternary amine ion exchange resins in particular have been optimized for extremely high binding capacities, and they can be recycled many times. Most notably, Q-resins have an established track record with the FDA, and extensive tests are available to determine their effectiveness for a given target protein. Therefore, this resin can be adapted to an intein process with minimal cGMP validation. 
     A broad variety of target proteins can be expressed which have been selected based on the availability of convenient activity assays and antibodies for Western blots. These proteins have included β-galactosidase, β-lactamase, secreted alkaline phosphatase, green fluorescent protein, maltose-binding protein, carbonic anhydrase, S824 protein, and others. By expressing these test target proteins in combination with a variety of tags and inteins, the impact of the tag, intein, intein cleaving rate, and expression system on overall process yields can be evaluated. Particular attention can be paid to the impact of the purification tag on protein expression and secretion, which can be evaluated through the use of various tags and untagged target proteins. The rapidly cleaving ΔI-CM intein is also available, along with noncleaving control inteins, to evaluate the effects of the mutant tag-intein fusion combination on overall expression and secretion of the target protein. These experiments in particular, can provide benchmarks on the effects of intein tags on overall protein expression, which will translate directly to the attractiveness of these expression systems and their likely process economics. 
     Summary 
     These experiments allow for the ability to directly select for controllable cleaving activity under fully flexible in vitro conditions and allowing selected mutants to be targeted to industrially important protein expression platforms. This is a significant deviation from previously published approaches, where intein reporter and selection systems have been developed for in vivo intein splicing. Additionally, a lack of selectable reporter proteins that are strongly affected by intein cleaving (as opposed to splicing) has resulted in few selection systems for intein cleaving. For these reasons, these previous methods have not been successful in generating fully controllable cleaving. By generating selection systems directly for intein cleaving in vitro, the conditions to which the intein is exposed during selection can be specified. Further, the design of these systems provides a strong and quantitative readout for intein cleaving and cleavage rate, as well as an efficient selection method for isolating desirable mutants. These aspects can allow for unprecedented control over the selection conditions, and therefore the ultimate behavior of the evolved inteins. 
     This work is focused on developing inteins that are directly relevant to industrially important protein expression systems and downstream processes. First and foremost among these is the CHO cell expression system, which is the main industry workhorse for producing recombinant antibodies and other therapeutic glycoproteins at multi-kilogram scales. Previous approaches have focused been primarily on controllability at laboratory scales, without consideration of eventual large-scale bioprocess development (as with IMPACT system). Although the work proposed here draws on established methods in molecular biology, it is unique in its focus on delivering a directly useful product for large-scale bioprocess engineering. 
     This work provides a metal binding site design strategy for controlling intein function, and the potential for this strategy to be adapted for other protein targets. In particular, the strategy indicates that an engineered metal binding motif can be used to sequester active site residues in inteins or other target proteins. By establishing a platform for reliably engineering various control mechanisms into the inteins, a means to study a range of potential control methods for additional proteins can be provided, which can also include pH and temperature variations in the presence of metal ions. This knowledge will find utility in many applications, ranging from the development of allosteric proteins for metabolic engineering, to therapeutic proteins that can be activated in situ. 
     The work in protein purification focused on the development of two self-cleaving tag-based nonchromatographic protein methods. One method is based on the production of polyhydroxybutyrate (PHB) granules in  E. coli  that act as affinity carriers for a co-expressed, tagged target protein. In this way, the cells provide both the affinity matrix and tagged target protein for purification. Once the granules are recovered and washed, the bound purified target protein is cleaved from their surface by the self-cleaving intein. This method has been demonstrated in  E. coli  and a highly productive  Ralstonia  strain. An initial analysis indicates a substantial economical advantage over conventional affinity separations. Another method relies on an intein fusion with an elastin-like peptide (ELP). In this case, the ELP portion of the tag is designed to reversibly precipitate upon gentle heating (about 30° C.) at salt concentrations in the 1M range. This highly specific reversible precipitation forms the basis for the separation, allowing the uncleaved precursor protein to be captured, concentrated and purified by simple cycles of heating and centrifugation or filtration ( FIG. 12 ). Once the ELP fusion is purified, the intein is induced to cleave, releasing the native target protein from the intein-ELP tag. A final cycle of heating and centrifugation then precipitates and removes the cleaved tag, leaving the purified and native target alone in solution. 
     Example 5 
     A Self-Cleaving Purification Tag System for Use in Mammalian Cell Culture 
       FIG. 7  is a graph of the rate constant vs. pH and temperature for the ΔI-CM mini intein. It illustrates that the cleaving rate at pH 7.5 and about 20° C., which is seen inside  E. coli  cells during expression, is sufficiently slow enough to produce an uncleaved precursor. At the conditions of CHO cell culture (pH 7.0 and 37° C.), however, the cleaving rate is much faster, which causes the tag to cleave before the protein can be recovered and purified. 
       FIGS. 18-20  show a structure of the ΔI-CM mini intein, and the location on the structure of the conserved His residue that is involved in cleaving. These figures show the overall structure of the intein.  FIG. 19  shows the conserved His residue in ball-and-stick, to give its relative position to the cleavage site.  FIG. 20  shows how zinc could bind to the conserved His residue to inhibit cleaving. Since the binding involves only one interaction with the His residue, it is weak, and thus requires a high concentration of zinc to inhibit cleaving. 
       FIG. 21  shows that the parent ΔI-CM intein is slightly sensitive to zinc at pH 7.0 or above, but the zinc here is added at 1 mM concentration. This concentration can significantly decrease CHO cell productivity and viability. At low pH, the zinc has no effect since the His residue is protonated at this pH and will not bind zinc at all. 
       FIG. 22  is a close up of ΔI-CM intein, showing the location of the His residue.  FIG. 22A  shows how the zinc binding motif could be added to the N-terminus of the intein (blue end), and could partially surround the conserved His residue.  FIG. 22B  illustrates the logic and goal of designing a zinc binding motif at the N-terminus of the intein, that will not affect the C-terminus, except that it provides a multivalent binding motif for zinc ion. If properly designed, this motif will greatly increase the affinity for zinc by the intein, allowing a much higher sensitivity to zinc and suppressing cleaving at a much lower zinc concentration. This formed the basic strategy that was later realized through computer modeling. 
       FIG. 23  indicates that a platform purification system can be developed using this intein to create a self-cleaving tag. This platform is very similar to the Protein A platform that is used to purify pretty much every antibody therapeutic on the market today, but no equivalent platform exists for non-antibody products.