Patent Publication Number: US-2022235345-A1

Title: Methods for preparation of active separase

Description:
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS 
     The present patent application claims benefit of priority to U.S. Provisional Patent Application No. 62/865,611, filed Jun. 24, 2019, which is incorporated by reference for all purposes. 
    
    
     STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT 
     This invention was made with government support under Grant No. R35 GM118053 awarded by the National Institutes of Health. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 5, 2020, is named 081906-1191728-231610WO_SL.txt and is 176,274 bytes in size. 
     BACKGROUND OF THE INVENTION 
     The protease separase initiates chromosome segregation in anaphase by cleaving the kleisin subunit (Scc1/Rad21) of the cohesin protein complex, allowing the duplicated eukaryotic chromosomes to be segregated to opposite poles of the cell 1-3 . Tight regulation of separase function is critical, as premature cleavage of cohesin can lead to chromosome loss and genomic instability. 
     Separase is a large caspase-family cysteine protease (the human protein is 2,120 amino acids/233 kDa). Approximately one quarter of human separase is comprised of the C-terminal protease domain, which is conserved across eukaryotes and of which it is possible to make a structural model based on homology to orthologous structures 4-7 . The large N-terminal region is poorly conserved and there is currently no detailed structural model of this region in the human protein, although it is likely composed of superhelical repeats like those seen in structures of separase from budding yeast 5  and  C. elegans   6 . Between the helical N-terminal region and the C-terminal protease domain, human separase contains regions that are predicted to be intrinsically disordered. 
     Separase cleavage sites have a consensus motif of ExxR, with cleavage occurring after the arginine 1,2,8 . An acidic or phosphorylated residue immediately upstream of the ExxR promotes cleavage 4,8-10 . In the structure of the separase protease domain from  C. thermophilum , basic and acidic binding pockets accommodate, respectively, the glutamate and arginine of the consensus motif 4 . Two ExxR sites are thought to be cleaved in the human Scc1 substrate 8 . Human separase contains four ExxR sites in its central disordered region, three of which are subjected to autocleavage upon separase activation“. After autocleavage, the N- and C-terminal domains of separase remain bound, with no apparent loss of protease activity”.  C. elegans  separase has shorter but similarly located intrinsically disordered regions, and its structure reveals that association of the N- and C-terminal domains does not depend on the disordered polypeptide chain between them 6 . 
     In early mitosis, separase is inhibited by a high-affinity interaction with the protein securin. Securin is thought to be intrinsically disordered when free in solution 12 , and the structures of securin-separase complexes from budding yeast 5  and  C. elegans   6  reveal that securin binds as an extended polypeptide along the length of separase. A pseudosubstrate motif on securin interacts with the active site 4 , presumably blocking substrate interactions. Securin inhibition is relieved when the N-terminal region of securin is ubiquitinated by the APC/C in metaphase, targeting it for destruction by the proteasome. Other vertebrate-specific modes of separase regulation have been identified, including inhibition by cyclin B-Cdk1 binding to separase in a manner dependent on proline isomerization by Pin1 13 , but the specific molecular mechanism for this inhibition remains unknown. 
     The ExxR separase cleavage motif is ubiquitous in the proteome, but very few of these motifs are known to be cleaved by separase. Human Scc1 contains six ExxR motifs, for example, but only two are cleaved in mitosis 8 . Therefore, it seems likely that there are other as yet unidentified mechanisms governing separase activity at the substrate level. Many proteases contain exosites: protease regions distinct from the active site that bind substrate sequences away from the cleavage site, thereby enhancing reaction efficiency 14 . The only evidence for separase regulation by substrate engagement outside of the cleavage site is that the securin-separase complex binds to DNA, helping to localize it to chromosomes 15 . While this binding results in increased cleavage of DNA-associated substrates, DNA does not enhance the enzyme&#39;s catalytic rate, and this interaction is too general to explain the observed specificity of separase. 
     Separase was identified two decades ago 1,2,16  and its central role in cell division is well established. However, many basic questions about its biochemical behavior and regulation remain unanswered, in part because of the difficulty of producing active protein amenable for biochemical and biophysical studies. It is well established that soluble separase can only be obtained in recombinant systems by co-expression with securin, as securin appears to be a co-translational separase-folding chaperone in addition to being an inhibitory. Therefore, production of active separase typically begins with purification of the securin-separase complex, from which securin is removed using the APC/C-proteasome system (for human separase, an incubation with  Xenopus  egg extract serves this purpose) 13,18-20 . While this protocol is sufficient for certain experiments, it does not produce the quantities and purity of protein needed for detailed biophysical studies. 
     BRIEF SUMMARY OF THE INVENTION 
     Provided herein are polypeptide constructs containing a securin fused to a separase. In some embodiments the securin is a full-length securin. In some embodiments, the securin is a truncated securin. Polypeptide constructs containing a securin linked to an unfoldase recognition site are also provided. 
     Also provided herein are methods for identifying a separase modulator compound. The methods include: 
     (i) measuring a level or rate of peptide substrate cleavage by a polypeptide construct in the presence of a candidate compound, wherein the polypeptide construct comprises a securin fused to a separase; 
     (ii) measuring a level or rate of peptide substrate cleavage by the polypeptide construct in the absence of the candidate compound; and 
     (iii) identifying the candidate compound as a separase modulator compound when the level or rate of peptide substrate cleavage in step (i) is higher or lower than the level or rate of peptide substrate cleavage in step (ii). In some embodiments, the peptide substrate comprises an LPE motif. 
     Also provided herein are methods for obtaining an active separase. In some embodiments, the methods include: 
     (a) co-expressing a separase and a securin, wherein the securin is linked to an unfoldase recognition site; and 
     (b) combining the co-expressed separase and securin with an unfoldase-peptidase complex; 
     thereby removing the securin and obtaining the active separase. 
     In some embodiments, the methods for obtaining an active separase include: 
     (1) expressing a polypeptide comprising a securin fused to a separase; and 
     (2) removing the securin from the expressed polypeptide, thereby obtaining the active separase; wherein the active separase is substantially free of the securin. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  shows a cartoon diagram of the securin-separase complex. The vertical dashed line indicates an approximate delineation between the separase N-terminal helical domain and C-terminal protease domain containing the active site. The C-terminal region of securin binds in an antiparallel fashion along the length of separase, and begins with the pseudosubstrate motif bound in the separase active site. The N-terminal region of securin contains the APC/C degrons. 
         FIG. 1B  shows a diagram of the securin-separase fusion construct. Also depicted are the flexible Gly-Ser linker separating securin and separase and the regions of human separase predicted to be intrinsically disordered (IDR). 
         FIG. 1C  shows the analysis of purified securin-separase fusion protein by SDS-PAGE and stained with Coomassie Blue, using molecular weight markers as indicated. 
         FIG. 1D  shows the analysis of purified securin-separase (top) and apo (active) separase (bottom) by negative-stain EM. Five representative class averages of each preparation are shown. 
         FIG. 1E  shows the evaluation of securin-separase binding to fluorescein-labeled DNA by fluorescence polarization. Two 50 bp dsDNA molecules with the same base composition but different sequence were tested, as well as a 25 bp molecule. Data points indicate means (+/− SEM) of triplicate samples. 
         FIG. 1F  shows a schematic of separase activation by the ubiquitin-proteasome system, whereby securin is tagged for degradation and removed, which can be recapitulated using an N-terminal ClpX-specific sequence and the bacterial protease ClpXP. 
         FIG. 1G  shows the analysis of Scc1 fragment cleavage. Securin-separase fusion protein was incubated with TEV protease, ATP, and/or the ClpXP ATPase as indicated, and separase activity was measured by cleavage of an  35 S-labeled Scc1 fragment (residues 142-300) produced by translation in vitro. 
         FIG. 1H  shows a plot of initial velocity vs. peptide concentration. Michaelis-Menten analysis was performed with purified, active separase and the peptide DDREIMREGS (SEQ ID NO: 25), which includes cleavage site 1 in Scc1. The peptide sequence was flanked by the MCA fluorophore and DNP quencher, and cleavage was monitored by an increase in fluorescence. Initial velocity was normalized to enzyme concentration. Data points indicate means (+/− SEM) of triplicate samples. 
         FIG. 2A  shows a diagram of the Scc1 sequence, including the locations of two separase cleavage sites, LPE motif, and boundaries of truncated constructs evaluated in  FIG. 2B  and  FIG. 2C . Figure discloses SEQ ID NOS 27 and 28, respectively, in order of appearance. 
         FIG. 2B  shows the reaction products resulting from  35 S-labeled Scc1 fragments incubated with active or inactive separase as indicated, as analyzed by SDS-PAGE and Phosphorimaging. 
         FIG. 2C  shows the reaction products resulting from separase incubated with an  35 S-labeled Scc1 fragment (aa 142-300) in which the indicated residues were changed to alanines, as analyzed by SDS-PAGE and Phosphorimaging. The sequence of the relevant region of Scc1 is shown (SEQ ID NO: 29). 
         FIG. 3A  shows a schematic of the separase biosensor used to evaluate cleavage in vivo, which includes histone H2B, red fluorescent protein (RFP), the indicated Scc1 fragment, and green fluorescent protein (GFP) 28 . 
         FIG. 3B  shows the time course of wild-type (WT) biosensor cleavage by separase, showing green fluorescence (left), red fluorescence (center), and merged images (right). Time zero is the last time point before the onset of chromosome segregation. Biosensor cleavage is indicated by reduced green fluorescence relative to red fluorescence. 
         FIG. 3C  shows representative images demonstrating late anaphase fluorescence of biosensor variants carrying mutations in Scc1 (WT, wild-type; NC, non-cleavable mutations at sites 1 and 2; LP→AA, mutations of  255 LP; Δ10aa, deletion of aa 251 to 260, which contain the LPE motif). 
         FIG. 3D  shows the quantification of the loss of GFP fluorescence in the four biosensor variants shown in  FIG. 3C . Data points indicate means (+/− SEM) from between 15 and 30 cells. 
         FIG. 4A  shows a cartoon diagram of the securin-separase fusion protein containing the full separase-binding region of securin (left) or with securin truncated on the C-terminal side of the pseudosubstrate motif (right). The separase active site (circle) and pseudosubstrate motif (rectangle overlapping circle at left) are indicated. 
         FIG. 4B  shows a diagram of the human securin sequence, indicating the locations of the pseudosubstrate sequence (EIEKFFP (SEQ ID NO: 26)), all LP sites including the  130 LPE motif, and the positions of the three truncations tested. 
         FIG. 4C  shows a plot generated for Michaelis-Menten analysis performed with the three indicated securinΔ-separase fusion proteins, compared with purified separase lacking securin. Initial velocity was normalized by enzyme concentration. Data points indicate means (+/− SEM) of triplicate samples. 
         FIG. 4D  shows the reaction products resulting from incubation of  35 S-labeled Scc1 fragments (aa 142-300), with or without mutations in the  255 LPE motif, with the three indicated securinΔ-separase fusion proteins or with purified separase lacking securin, as analyzed by SDS-PAGE and Phosphorimaging. 
         FIG. 4E  shows that the pseudosubstrate motif in securin was converted to a separase cleavage site using two point mutations ( 118 FP to RE). Separase was incubated with an  35 S-labeled securin fragment (aa 93-150) containing these mutations as well as mutations in the indicated LP motifs. Reaction products were analyzed by SDS-PAGE and Phosphorimaging. 
         FIG. 4F  shows sequence alignment of securin pseudosubstrate motifs (EIE, DIE, or EVE at left), indicating the downstream conserved LPE motifs (highlighted at right) (SEQ ID NOS 30-37, respectively, in order of appearance). 
         FIG. 4G  shows a cartoon diagram of the securin-separase complex, illustrating the pseudosubstrate motif interaction with the active site and the LPE motif interaction with the separase exosite. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention was developed with the use of protein engineering for the generation of active separase. Using this active separase protein, it was discovered that rapid cleavage of Scc1 requires a sequence motif in Scc1 that is distinct from the cleavage motif, and which interacts with a docking site (exosite) on separase. It is demonstrated herein that securin binding interferes with separase engagement of the substrate docking motif, identifying a second mechanism by which securin inhibits cohesin cleavage by separase. The methods and polypeptide constructed provided herein allow for the production of large amounts of homogeneous, fully active enzyme for a variety of studies. 
     I. POLYPEPTIDE CONSTRUCTS 
     Provided herein are polypeptide constructs comprising a securin fused to a separase. As noted above, separase is a cysteine protease containing a large superhelical N-terminal region and a conserved C-terminal protease domain 4, 5, 6, 21  The protease domain includes a substrate binding domain and a caspase-like catalytic domain. The substrate binding domain is characterized by a mixed α/β fold, having a four-helix bundle packed against an RNase H-like β-sheet. This five-stranded, mostly anti-parallel β-sheet also contains a two-helix hairpin extension between strands 3 and 4. The caspase-like catalytic domain contains a central six-stranded, mostly parallel β-sheet flanked by α-helices. Catalytic cysteine and histidine residues are located in the β-sheet in loops following strands 3 and 4, respectively. The large N-terminal region adopts an extended conformation in species such as  H. sapiens  (UniProt Q14674) and  S. cerevisiae , while a closed conformation is adopted in species such as  C. elegans . The N-terminal region contains multiple HEAT repeat units (26 in  H. sapiens ), with each HEAT having a pair of anti-parallel α-helices linked by a flexible loop, and a disordered region between the HEAT repeat units and the C-terminal protease domain. Securin binds in an extended conformation along the length of separase. For example, two short helices are the only secondary structural features observed by X-ray crystallography in the  S. cerevisiae  securin-separase complex. Residues 258-269 of the  S. cerevisiae  securin (corresponding to residues 113-224 of  H. sapiens  securin; UniProt O95997) lie in the separase active site upon formation of the securin-separase complex. 
     In some embodiments, the separase comprises an amino acid sequence having at least 70% identity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to  H. sapiens  separase (SEQ ID NO:1),  M. musculus  separase (SEQ ID NO:7; UniProt P60330),  C. elegans  separase (SEQ IQ NO:8; UniProt G5ED39),  S. cerevisiae  separase (SEQ ID NO:9; UniProt Q03018), or  S. pombe  separase (SEQ ID NO:10; UniProt P18296). In some embodiments, the separase comprises an amino acid sequence having at least 80% identity to  H. sapiens  separase,  M. musculus  separase,  C. elegans  separase,  S. cerevisiae  separase, or  S. pombe  separase. In some embodiments, the separase comprises an amino acid sequence having at least 90% identity to  H. sapiens  separase,  M. musculus  separase,  C. elegans  separase,  S. cerevisiae  separase, or  S. pombe  separase. In some embodiments, the separase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:1. 
     Percentage of sequence identity can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence (e.g., a peptide of the invention) in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. 
     “Identical” and “identity,” in the context of two or more polypeptide sequences or nucleic acid sequences, refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. 
     For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990)  J. Mol. Biol.  215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always &gt;0) and N (penalty score for mismatching residues; always &lt;0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff,  Proc. Natl. Acad. Sci. USA  89:10915 (1989)). 
     The securin may be a full-length securin or a truncated securin. In some embodiments, the full-length securin comprises an amino acid sequence having at least 70% identity to  H. sapiens  securin (SEQ ID NO:2),  M. musculus  securin (SEQ ID NO:11; UniProt Q9CQJ7),  C. elegans  securin (SEQ IQ NO:12; UniProt Q18235),  S. cerevisiae  securin (SEQ ID NO:13; UniProt P40316), or  S. pombe  securin (SEQ ID NO:14; UniProt P21135). In some embodiments, the full-length securin comprises an amino acid sequence having at least 80% identity to  H. sapiens  securin,  M. musculus  securin,  C. elegans  securin,  S. cerevisiae  securin, or  S. pombe  securin. In some embodiments, the full-length securin comprises an amino acid sequence having at least 90% identity to  H. sapiens  securin,  M. musculus  securin,  C. elegans  securin,  S. cerevisiae  securin, or  S. pombe  securin. In some embodiments, the full-length securin comprises an amino acid sequence having at least 90% identity to SEQ ID NO:2. 
     In some embodiments, the truncated securin contains an amino acid sequence having at least 70% identity to residues 10-202 of SEQ ID NO:2, or a shorter polypeptide corresponding to, e.g., residues 20-202, or 30-202, or 40-202, or 50-202, or 60-202, or 70-202, or 80-202, or 90-202, or 100-202, or 110-202, or 120-202, or 130-202, or 140-202, or 150-202, or 160-202, or 170-202, or 180-202, or 190-202 of SEQ ID NO:2. 
     In some embodiments, the truncated securin contains an amino acid sequence having at least 70% identity to residues 10-199 of SEQ ID NO:11, or a shorter polypeptide corresponding to, e.g., residues 20-199, or 30-199, or 40-199, or 50-199, or 60-199, or 70-199, or 80-199, or 90-199, or 100-199, or 110-199, or 120-199, or 130-199, or 140-199, or 150-199, or 160-199, or 170-199, or 180-199, or 190-199 of SEQ ID NO:11. 
     In some embodiments, the truncated securin contains an amino acid sequence having at least 70% identity to residues 10-244 of SEQ ID NO:12, or a shorter polypeptide corresponding to, e.g., residues 20-244, or 30-244, or 40-244, or 50-244, or 60-244, or 70-244, or 80-244, or 90-244, or 100-244, or 110-244, or 120-244, or 130-244, or 140-244, or 150-244, or 160-244, or 170-244, or 180-244, or 190-244 of SEQ ID NO:12. 
     In some embodiments, the truncated securin contains an amino acid sequence having at least 70% identity to residues 10-373 of SEQ ID NO:13, or a shorter polypeptide corresponding to, e.g., residues 20-373, or 30-373, or 40-373, or 50-373, or 60-373, or 70-373, or 80-373, or 90-373, or 100-373, or 110-373, or 120-373, or 130-373, or 140-373, or 150-373, or 160-373, or 170-373, or 180-373, or 190-373, or 200-373, or 210-373, or 220-373, or 230-373, or 240-373, or 250-373, or 260-373, or 270-373, or 280-373, or 290-373, or 300-373, or 310-373, or 320-373, or 330-373, or 340-373, or 350-373, or 360-373 of SEQ ID NO:13. 
     In some embodiments, the truncated securin contains an amino acid sequence having at least 70% identity to residues 10-301 of SEQ ID NO:14, or a shorter polypeptide corresponding to, e.g., residues 20-301, or 30-301, or 40-301, or 50-301, or 60-301, or 70-301, or 80-301, or 90-301, or 100-301, or 110-301, or 120-301, or 130-301, or 140-301, or 150-301, or 160-301, or 170-301, or 180-301, or 190-301, or 200-301, or 210-301, or 220-301, or 230-301, or 240-301, or 250-301, or 260-301, or 270-301, or 280-301, or 290-301 of SEQ ID NO:14. 
     In some embodiments, the securin comprises an amino acid sequence having at least 90% identity to positions 93-202 of SEQ ID NO:2. In some embodiments, the securin consists of an amino acid sequence having at least 90% identity to positions 160-202 of SEQ ID NO:2. In some embodiments, the securin consists of an amino acid sequence having at least 90% identity to positions 138-202 of SEQ ID NO:2. In some embodiments, the securin consists of an amino acid sequence having at least 90% identity to positions 127-202 of SEQ ID NO:2. 
     In some embodiments, the securin is fused to the separase via a linker, e.g., a linker peptide. As used herein, the term “linker” refers to a peptidic moiety or a non-peptidic moiety that covalently connects one terminus of a securin to one terminus of a separase. In some embodiments, the linker covalently connects the C-terminus of the securin to the N-terminus of the separase. A number of linkers can be used for fusion of the securin to the separase including, for example, rigid, flexible, and cleavage linkers such as those described by Chen et al. ( Adv Drug Deliv Rev.  2013; 65(10): 1357-1369). In some embodiments, the linker contains a flexible peptide such as GGGGS (SEQ ID NO:15), (GGGGS) 2  (SEQ ID NO:16), (GGGGS) 3  (SEQ ID NO:17), (GGGGS) 4  (SEQ ID NO:18), GGGGGG (SEQ ID NO:19), GGGGGGGG (SEQ ID NO:20), GGSGGSGGGSGGGSG (SEQ ID NO:21), or the like. 
     In some embodiments, the polypeptide construct comprises a protease recognition site. A linker peptide in the polypeptide construct, for example, may contain one or more recognition sites for proteases such as those described by Waugh ( Protein Expr Purif.  2011; 80(2): 283-293). In some embodiments, the protease is a site-specific endopeptidase. Examples of suitable site-specific endopeptidases include, but are not limited to, FactorXa, enterokinase, α-thrombin, human rhinovirus 3C protease, Tobacco Vein Mottling Virus (TVMV) protease, and Tobacco Etch Virus (TEV) protease. In some embodiments, the protease is TEV protease. 
     In some embodiments, the polypeptide construct contains one or more affinity tags, e.g., for the purposes of detection or purification. A number of suitable tags can be included in the polypeptide constructs including, for example, those described by Kimple et al. ( Curr Protoc Protein Sci.  2013; 73(1): 9.9.1-9.9.23). Examples of affinity tags include, but are not limited to, a calmodulin binding peptide (CBP), a chitin binding domain (CBD), a dihyrofolate reductase (DHFR) moiety, a FLAG epitope, a glutathione S-transferase (GST) tag, a hemagglutinin (HA) tag; a maltose binding protein (MBP) moiety; a Myc epitope; a polyhistidine tag (e.g., HHHHHH, SEQ ID NO: 22); and streptavidin-binding peptides (e.g., those described in U.S. Pat. No. 5,506,121). An affinity tag may be included at one or more locations in the polypeptide construct. An affinity tag such as a streptavidin-binding peptide may reside, for example, at the N-terminus of the polypeptide construct or at the C-terminus of the polypeptide construct. In some embodiments, the linker peptide comprises an affinity tag, e.g., a FLAG epitope containing the sequence DYKDDDDK (SEQ ID NO: 23) with or without additional amino acid residues. 
     In some embodiments, the polypeptide construct further includes a recognition site for an unfoldase, e.g., an  E. coli  unfoldase, linked to the securin.  E. coli  have a collection of energy-dependent proteases that couple ATP hydrolysis to the translocation of a substrate protein to a sequestered proteolytic chamber. These include ClpXP, ClpAP, lon, HslUV, and FtsH. ClpXP is a complex of a hexamer of the ClpX unfoldase and the 14-mer ClpP protease. Upon substrate recognition, ClpX uses the energy from ATP hydrolysis to processively translocate along the substrate polypeptide chain, unfolding the substrate, and delivering the unfolded protein into the lumen of the ClpP structure where it encounters a high concentration of serine protease active sites. In some embodiments, the unfoldase recognition site is an  E. coli  ClpX recognition site. In some embodiments, the unfoldase recognition site contains the sequence TNTAKILNFGR (SEQ ID NO:24). In some embodiments, the unfoldase recognition site is linked to the securin via an affinity tag, e.g., a streptavidin-binding peptide. 
     Also provided herein are polypeptide constructs having a securin linked to an unfoldase recognition site. The securin may be linked to the unfoldase recognition site with any of the linkers described herein, and the construct may further contain any of the affinity tags and protease recognition sites described above. 
     In some embodiments, the polypeptide construct includes an amino acid sequence according to SEQ ID NO:3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6. The polypeptide constructs described herein, as well as specific securin portions and/or separase portions therein, can be used with or without N-terminal methionine residues (e.g., with or without the N-terminal methionine residues set forth in SEQ ID NOS:1-14). 
     II. COMPOSITIONS AND METHODS FOR IDENTIFYING SEPARASE MODULATORS 
     The securin-separase fusion constructs described herein can be used to facilitate basic studies of separase enzyme behavior, including its activity toward various substrates. The fusion constructs can be used, for example, as reagents for the mechanistic study of chromosome segregation. In addition, the fusion constructs can be used in the screening of chemical modulators (e.g., separase inhibitors) that may have research or therapeutic potential. Accordingly, some embodiments of the present disclosure provide a mixture comprising a polypeptide construct as described above and one or more test substances. In some embodiments, the test substance is an organic small-molecule separase inhibitor candidate. 
     Also provided herein are methods for identifying a separase modulator compound. The methods include: 
     (i) measuring a level or rate of peptide substrate cleavage by a polypeptide construct in the presence of a candidate compound, wherein the polypeptide construct comprises a securin fused to a separase; 
     (ii) measuring a level or rate of peptide substrate cleavage by the polypeptide construct in the absence of the candidate compound; and 
     (iii) identifying the candidate compound as a separase modulator compound when the level or rate of peptide substrate cleavage in step (i) is higher or lower than the level or rate of peptide substrate cleavage in step (ii). In some embodiments, the level or rate of peptide substrate cleavage in step (i) is lower than the level or rate of the peptide substrate cleavage in step (ii), and the candidate compound is identified as a separase inhibitor. 
     In some embodiments, the peptide substrate contains a cohesin Scc1 subunit sequence, such as an Scc1 site 1 sequence containing EIMR (SEQ ID NO: 27) (e.g., DDREIMREGS; SEQ ID NO:25). In addition to the Scc1 sequence, the peptide substrate may further contain a pair of fluorescence resonance energy transfer (FRET) partners to facilitate detection of substrate cleavage as described in more detail below. One non-limiting example of a FRET partner pair, for instance, is an Mca moiety (i.e., (7-methoxycoumarin-4-yl)acetyl) covalently bonded to a first terminus of the peptide substrate and a Dnp moiety (i.e., 2,4-dinitrophenyl) covalently bonded to the second terminus of the peptide substrate). A number of suitable FRET partners and other useful signal-generating moieties are described, for example, by Ong, et al. (Analyst, 2017, 142, 1867-1881). In some embodiments, the peptide substrate comprises an LPE motif (i.e., a leucine-proline-glutamic acid motif), which is present in the cohesin Scc1 subunit and also in the native securin sequence. 
     III. METHODS FOR PREPARING/OBTAINING ACTIVE SEPARASE 
     Also provided herein are methods for obtaining an active separase fusion protein. The methods include expressing a polypeptide comprising a truncated securin fused to a separase (e.g., a polypeptide construct having a sequence as set forth in SEQ ID NOS:4-6), thereby obtaining the active separase fusion protein. The methods may include the use of nucleic acids encoding a polypeptide construct as described above, as well as vectors containing the nucleic acids and host cells containing the nucleic acids and/or the vectors. 
     Nucleic acids encoding the polypeptide constructs can be obtained using routine techniques in the field of recombinant genetics. Basic texts disclosing such techniques include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994-1999). Nucleic acids encoding the polypeptide constructs may also be obtained through in vitro amplification methods such as those described herein and in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al., eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim &amp; Levinson (Oct. 1, 1990) C&amp;EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117. 
     One of skill will recognize that modifications can additionally be made without diminishing the biological activity of the securin or the separase. Some modifications may be made to facilitate the cloning, expression, or incorporation of a domain into a fusion protein. Such modifications include, for example, the addition of codons at either terminus of the polynucleotide that encodes the binding domain to provide, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences. 
     The fusion polypeptides as described herein can be expressed in a variety of host cells, including  E. coli , other bacterial hosts, yeasts, filamentous fungi, and various higher eukaryotic cells such as the Sf9, COS, CHO and HeLa cell lines and myeloma cell lines. There are many expression systems for producing the polypeptides that are well known to those of ordinary skill in the art. (See, e.g., Gene Expression Systems, Fernandex and Hoeffler, Eds. Academic Press, 1999; Sambrook and Russell, supra; and Ausubel et al., supra.) Typically, a polynucleotide that encodes the polypeptide is placed under the control of a promoter that is functional in the desired host cell. Many different promoters are available and known to one of skill in the art, and can be used in the expression vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active. Other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included. Constructs that include one or more of these control sequences are termed “expression cassettes.” 
     Eukaryotic expression systems for producing the polypeptide constructs—including insect cells, yeast, and mammalian cells—are well known in the art and are also commercially available. Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, p10 promoter, or other promoters shown effective for expression in eukaryotic cells. 
     Synthesis of heterologous proteins in yeast is well known and described in the literature. Methods in Yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory, (1982) is a well-recognized work describing the various methods available to produce the polypeptide constructs in yeast. In yeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2. Techniques for gene expression in various other microorganisms are described in, for example, Smith, Gene Expression in Recombinant Microorganisms (Bioprocess Technology, Vol. 22), Marcel Dekker, 1994. Examples of bacteria that are useful for expression include, but are not limited to,  Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla , and  Paracoccus . Filamentous fungi that are useful as expression hosts include, for example,  Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Mucor, Cochliobolus , and  Pyricularia . See, e.g., U.S. Pat. No. 5,679,543 and Stahl and Tudzynski, Eds., Molecular Biology in Filamentous Fungi, John Wiley &amp; Sons, 1992. 
     Commonly used prokaryotic control sequences, e.g., promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. (1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25); and the lambda-derived PL promoter and N-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128). The particular promoter system is not critical; any available promoter that functions in prokaryotes and provides the desired level of activity can be used. Standard bacterial expression vectors include plasmids such as pBR322-based plasmids, e.g., pBLUESCRIPT™, pSKF, pET23D, lambda-phage derived vectors, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc, HA-tag, 6-His tag (SEQ ID NO: 22), maltose binding protein, VSV-G tag, anti-DYKDDDDK tag (SEQ ID NO: 23), or any such tag, a large number of which are well known to those of skill in the art. 
     Either constitutive or regulated promoters can be used. Regulated promoters can be advantageous because the host cells can be grown to high densities before expression of the fusion polypeptides is induced. High level expression of heterologous proteins slows cell growth in some situations. An inducible promoter is a promoter that directs expression of a gene where the level of expression is alterable by environmental or developmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors and chemicals. For  E. coli  and other bacterial host cells, inducible promoters are known to those of skill in the art. These include, for example, the lac promoter, the bacteriophage lambda PL promoter, the hybrid trp-lac promoter (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc. Nat&#39;l. Acad. Sci. USA 80: 21), and the bacteriophage T7 promoter (Studier et al. (1986) J. Mol. Biol.; Tabor et al. (1985) Proc. Nat&#39;l Acad. Sci. USA 82: 1074-8). These promoters and their use are also discussed in Sambrook et al., supra. 
     Translational coupling may be used to enhance expression. The strategy uses a short upstream open reading frame derived from a highly expressed gene native to the translational system, which is placed downstream of the promoter, and a ribosome binding site followed after a few amino acid codons by a termination codon. Just prior to the termination codon is a second ribosome binding site, and following the termination codon is a start codon for the initiation of translation. The system dissolves secondary structure in the RNA, allowing for the efficient initiation of translation. See Squires, et. al. (1988), J. Biol. Chem. 263: 16297-16302. 
     The construction of securin-separase fusion proteins generally requires the use of vectors able to replicate in bacteria. Such vectors are commonly used in the art. Kits are commercially available for the purification of plasmids from bacteria (for example, EasyPrep™, FlexiPrep™, from Pharmacia Biotech; StrataClean™, from Stratagene; and, QIAexpress® Expression System, Qiagen). The isolated and purified plasmids can then be further manipulated to produce other plasmids, and used to transform cells. 
     The polypeptides described herein can be expressed intracellularly, or can be secreted from the cell. Intracellular expression often results in high yields. If necessary, the amount of soluble, active fusion polypeptide may be increased by performing refolding procedures (see, e.g., Sambrook et al., supra; Marston et al., Bio/Technology (1984) 2: 800; Schoner et al., Bio/Technology (1985) 3: 151 
     Once expressed, the polypeptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)). Substantially pure compositions of at least about 90 to 95% homogeneity (e.g., 98 to 99% or higher homogeneity) are provided in certain embodiments. Once purified, partially or to homogeneity as desired, the polypeptides may then be used (e.g., in an inhibitor screen or mechanistic study). 
     To facilitate purification of the polypeptides, the nucleic acids that encode the polypeptides can also include a coding sequence for an epitope or “tag” for which an affinity binding reagent is available. Examples of suitable epitopes include the myc and V-5 reporter genes; expression vectors useful for recombinant production of fusion polypeptides having these epitopes are commercially available (e.g., Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-His and pcDNA3.1/V5-His are suitable for expression in mammalian cells). Additional expression vectors suitable for attaching a tag to the fusion proteins of the invention, and corresponding detection systems are known to those of skill in the art, and several are commercially available (e.g., FLAG″ (Kodak, Rochester N.Y.)). Another example of a suitable tag is a polyhistidine sequence, which is capable of binding to metal chelate affinity ligands. Typically, six adjacent histidines (SEQ ID NO: 22) are used, although one can use more or less than six. Suitable metal chelate affinity ligands that can serve as the binding moiety for a polyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli, E. (1990) “Purification of recombinant proteins with metal chelating adsorbents” In Genetic Engineering: Principles and Methods, J. K. Setlow, Ed., Plenum Press, N.Y.; commercially available from Qiagen (Santa Clarita, Calif.)). 
     One of skill in the art would recognize that after biological expression or purification, the polypeptide constructs may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it may be necessary or desirable to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art (See, Debinski et al. (1993) J. Biol. Chem. 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem. 4: 581-585; and Buchner et al. (1992) Anal. Biochem. 205: 263-270). Debinski et al., for example, describe the denaturation and reduction of inclusion body proteins in guanidine-DTE. The protein is then refolded in a redox buffer containing oxidized glutathione and L-arginine. 
     In some embodiments, the methods for obtaining active separase include:
         (a) co-expressing a separase and a securin, wherein the securin is linked to an unfoldase recognition site; and   (b) combining the co-expressed separase and securin with an unfoldase-peptidase complex;   thereby removing the securin and obtaining the active separase.       

     In some embodiments, the methods for obtaining active separase include:
         (1) expressing a polypeptide comprising a securin fused to a separase; and   (2) removing the securin from the expressed polypeptide, thereby obtaining the active separase;   wherein the active separase is substantially free of the securin.       

     In some embodiments, the securin is fused to the separase via a linker comprising a protease recognition site, and removing the securin from the expressed polypeptide comprises cleaving the securin from the separase at the protease recognition site. In some embodiments, the polypeptide further comprises an unfoldase recognition site linked to the securin, and removing the securin from the expressed polypeptide comprises combining the expressed polypeptide with an unfoldase-peptidase complex. The methods of the present disclosure provide isolated active separase, which is substantially free of securin. 
     IV. EXAMPLES 
     Example. 1 Materials and Methods 
     Constructs, cloning and expression. Securin-separase fusion constructs were cloned into a pFastBac HT A vector with an L21 leader sequence added immediately upstream of the ORF 30 . DNA encoding the N-terminal region of each protein (containing all or a subset of the following: LambdaO ClpX sequence, 2× StrepII tag, securin, Gly-Ser linker, TEV protease cleavage site, 3× FLAG tag) was codon optimized for insect cell expression and synthesized as a gBlocks gene fragment by Integrated DNA Technologies (IDT). Separase was amplified from a human cDNA library, and mutations were made using either gBlocks gene fragments or fragment amplification and then assembled using Gibson assembly. All constructs contained the S1126A mutation to prevent proline isomerization and subsequent aggregation 13 . Catalytically-dead separase constructs contained the C2029S mutation. For all constructs with an intact active site, the autocleavage sites were mutated by reversing the E and R residues for each of the three sites 11 . All constructs were verified by full sequencing of the ˜7000 bp ORFs. The resulting plasmids were transformed into DH10Bac cells to generate bacmids through in vivo recombination. Purified bacmids were used to transfect Sf9 cells and generate P1 baculovirus. For protein expression, Sf9 cells were harvested 2-3 days after infection with P2 virus. 
       E. coli  ClpX and ClpP-6His expression constructs were a generous gift from Andreas Martin. ClpX is the full-length, AKH version 31 , which we modified with a C-terminal 2× StrepII tag. TEV protease construct pRK793 was a gift from David Waugh (Addgene plasmid #8827; http://n2t.net/addgene:8827; RRID:Addgene_8827) 32 . TEV protease and ClpX were expressed in BL-21 DE3  E. coli  at 30° C. for 4 h after induction with IPTG. ClpP was expressed in a BL21 ClpP knockout strain at 25° C. for 4 h after induction with IPTG. 
     The separase biosensor was generated as described by Shindo et al. 28 . Specifically, Gibson cloning was performed to generate a final construct of pCMV-H2B-mRuby2-Scc1(142-467)-mNeonGreen in a plasmid backbone containing PGK-Neo. This was used as the template for all variations of the biosensor, which were also generated using Gibson cloning. 
     Protein purification. Securin-separase fusion protein and ClpX protein were purified on a StrepTrap column, with a lysis and wash buffer of 50 mM HEPES-KOH pH 7.8, 300 mM KCl, 0.1 mM EDTA-KOH, 0.5 mM TCEP, 10% glycerol. Proteins were eluted in one step in the same buffer containing 2.5 mM desthiobiotin. Securin-separase was used for ClpXP activation (see below) or buffer exchanged via PD-10 column into relevant buffers (see below), concentrated, frozen in aliquots of 100 μl or less in liquid nitrogen (LN 2 ), and stored at −80° C. Securin-separase used for negative-stain EM was additionally purified by size exclusion using a Superose 6 10/300 GL column pre-equilibrated in the following buffer: 25 mM HEPES pH 7.8, 75 mM KCl, 10 mM MgCl 2 , 0.5 mM TCEP, 5% glycerol. 
     TEV protease and ClpP were purified on a HisTrap column. TEV protease buffers were 50 mM Tris-HCl pH 8, 200 mM NaCl, 10% glycerol, 0.5 mM TCEP, with 25 mM imidazole in the lysis and wash buffers and 800 mM imidazole in the elution buffer. ClpP buffers were 50 mM HEPES pH 7.8, 100 mM KCl, 400 mM NaCl, 10% glycerol, 0.5 mM TCEP, with 20 mM imidazole in the lysis and wash buffers and 500 mM imidazole in the elution buffer. TEV protease, ClpX and ClpP were each dialyzed overnight into 50 mM HEPES-KOH pH 7.5, 200 mM KCl, 25 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM TCEP, 10% glycerol. After dialysis, precipitate was pelleted by centrifugation and the supernatant frozen in aliquots of 250 μl or less in LN 2  and stored at −80° C. 
     Separase activation and purification. Securin-separase fusion was purified as described above. Eluted fractions were stored at 4° C. overnight, and then pooled and concentrated to ˜1 ml (˜2.5 mg/ml). The concentrated protein was incubated with 1 ml TEV protease (˜2.5 mg/ml) and 10 μl Benzonase added to 11.1 ml of 25 mM HEPES pH 7.8, 100 mM KCl, 10 mM MgCl 2 , 10% glycerol for 1 h at 30° C. ClpX (1.7 ml, ˜1.6 mg/ml) and ClpP (830 μl, ˜2 mg/ml) were mixed and pre-incubated at 25° C. for over 30 min After the TEV protease incubation, 830 μl 100 mM ATP (in 25 mM HEPES pH 7.8, 100 mM KCl, 10 mM MgCl 2 , 10% glycerol) was added to the securin-separase reaction mixture, followed by the pre-incubated ClpXP. After 1.5 h at 30° C., the mixture was filtered (0.2 μm) and run on a HisTrap column to remove ClpP and TEV protease. The flow-through was pooled, concentrated to less than 2.5 ml, and run over a PD-10 column to change the buffer to 50 mM HEPES-KOH pH 7.8, 300 mM KCl, 0.1 mM EDTA-KOH, 0.5 mM TCEP 10% glycerol. The protein was run on a StrepTrap column to remove ClpX and also any separase still bound by securin. The flow-through was pooled and concentrated to less than 1 ml, and loaded on a Superose 6 10/300 GL column pre-equilibrated in the following buffer: 25 mM HEPES pH 7.8, 75 mM KCl, 10 mM MgCl 2 , 0.5 mM TCEP, 5% glycerol. The separase peak was pooled, concentrated, frozen in aliquots of 100 μl or less in LN 2  and stored at −80° C. 
     Electron Microscopy. Separase and the separase-securin complex were diluted to a nominal final concentration of 0.01 mg/ml in a buffer containing 25 mM HEPES-KOH pH 7.8, 75 mM KCl, 10 mM MgCl 2 , 0.5 mM TCEP. For both samples, 3 μl were applied to carbon-coated 200-mesh copper grids (Ted Pella, Redding, Calif.) which had been glow discharged for 30 s. Specimens were stained as previously described 33  with a solution containing 2% (w/v) uranyl formate. Data were acquired with a Tecnai F20 Twin transmission electron microscope (FEI, Hillsboro, Oreg.) operating at 200 kV using SerialEM 34  and a nominal range of 0.9-1.9 μm under focus. Images were recorded on a TemCam-F816 CMOS camera (TVIPS, Gauting, Germany) at a nominal magnification of 50,000×, which corresponds to 1.57 Å/px at the detector level. For the separase sample, 337 images were collected (28,540 particles picked, ˜80 particles per image) and for the separase-securin complex 75 images were collected (26,077 particles picked, ˜350 particles per image) Immediately following image acquisition, micrographs were binned by two to give a final pixel size of 3.14 Å/px. The CTF was estimated using GCTF 35 , and particles were picked using a reference free routine as implemented in Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch). Data were processed in a similar manner for each dataset, using Relion2 36  for 2D alignment and classification into 100 classes. 
     Analysis of DNA binding by fluorescence polarization. Double-stranded, 5′-fluorescein-labeled oligonucleotides were ordered from IDT. DNA was mixed with a dilution series of securin-separase C2029S with the following final conditions: 1 nM DNA in 25 mM HEPES pH 7.8, 50 mM KCl, 5 mM MgCl 2 , 0.5 mM TCEP. Samples were incubated 30 min at 25° C. prior to measurement. Fluorescence polarization was measured on a Biotek Synergy H4 plate reader using excitation/emission of 485/528 nm at a gain of 70. Signal from wells with no protein were used to blank subtract the data, then the blank-subtracted fluorescence polarization was normalized relative to the average value at the highest protein concentration. Data were fit to a one-site binding model using GraphPad Prism. 
     Scc1 cleavage assay.  35 S-methionine-labeled fragments of human Scc1 (and securin;  FIG. 4E ) were produced in rabbit reticulocyte lysates using the TnT Quick Coupled Transcription/Translation System (Promega). Variants were made by QuikChange mutagenesis or Gibson cloning. All variants included an N-terminal ZZ tag followed by a TEV protease cleavage site. Following translation in vitro, proteins were purified by immunoprecipitation on magnetic beads coated with anti-ZZ IgG, and eluted by TEV protease. Active separase (˜0.12 mg/ml) was mixed 1:1 with purified Scc1 substrate and incubated for 1 h at 25° C. Reaction products were analyzed by SDS-PAGE with BioRad 4-20% TXP gels and visualized with a Phosphorimager Gels were also stained with Coomassie Blue to confirm that enzyme concentration was the same in all reactions. 
     For experiments with securin-free separase, experiments were performed either with purified active separase or with activated separase but without downstream purification to remove TEV protease and ClpXP. The presence of ClpXP had no effect on the results. Additionally, in cases where ClpXP was present, apyrase was used to remove residual ATP and thereby prevent ClpXP activity. 
     Peptide cleavage assay. The following peptide, containing Scc1 site 1, was ordered from Genscript (&gt;90% purity): Mca-DDREIMREGS-Dnp (SEQ ID NO: 25). Peptide was dissolved in DMSO at a concentration of 47.5 mM. The peptide was serially diluted into buffer (25 mM HEPES pH 7.8, 25 mM KCl, 0.5 mM TCEP) and mixed with active separase (either securin-free separase purified after TEV protease/ClpXP incubation or purified securinΔ-separase) at 0.1-0.5 mg/ml in the buffer: 25 mM HEPES pH 7.8, 75 mM KCl, 10 mM MgCl 2 , 0.5 mM TCEP, 5% glycerol. The reaction was immediately monitored by fluorescence on a Biotek Synergy H4 plate reader, using an excitation of 328±20 nm and an emission filter of 393±20 nm (gain of 75). Fluorescence was monitored for 1 hour with 1 min reads. Data from 5-30 min was used for calculation of initial velocity. 
     To convert relative fluorescence units (RFU) to concentration of cleaved substrate, a standard curve was generated by incubating peptide with 0.1 mg/ml Trypsin for 2 h (to achieve full substrate cleavage) and then making a dilution series (in triplicate). Fluorescence was measured on the same day and at the same gain as in the kinetic assay. A plot of RFU vs concentration of cleaved peptide was fit with a linear regression and the slope taken as the conversion factor. 
     Separase concentrations were measured in triplicate on a Nanodrop spectrophotometer by absorbance at 280 nm, and evaluated using a theoretical extinction coefficient at A 280  (calculated according to the number of Trp and Tyr residues) 37 . The data for the Michaelis-Menten curves were normalized by enzyme concentration. Data were fit to the Michaelis-Menten equation using GraphPad Prism. Error for reported k cat  incorporates the error in protein concentration. 
     Biosensor expression and microscopy. Second-generation lentiviruses were generated by transient co-transfection of 293T cells in DMEM+10% FBS, using a three-plasmid combination: one well in a 6-well dish containing 1×10 6  293 T cells was transfected using PEI with 0.5 μg lentiviral vector, 0.5 μg psPAX and 0.5 μg pMD2.G. Supernatants were collected every 24 h between 24 and 72 h after transfection and frozen at −80° C. 
     For biosensor expression, U2OS cells growing in McCoy&#39;s media+10% FBS were plated in a 6-well dish at 1×10 6  cells per well. The following day, 0.5 ml lentivirus was added. After 48 h incubation, media was removed and cells were washed with PBS. Next, fresh media with 500 μg/ml Geneticin was added to the cells to select for transduced cells. After 1-2 weeks of selection, cell lines were expanded for FACS analysis: cells were re-suspended in FACS sorting buffer (PBS [Ca 2+ /Mg 2+ -free], 1 mM EDTA, 25 mM HEPES, 1% FBS) and filtered through a 50 μM filter. These cells were then sorted on a Sony SH800 Cell Sorter, selecting for cells with moderate levels of expression. 
     For microscopy, U2OS cells stably expressing the biosensor were plated in 24-well glass-bottom dishes (Mattek P24G-1.0-10-F) and allowed to adhere overnight. Media was removed and the cells were washed with PBS. Media was then replaced with Opti-Mem supplemented with 10% FBS. Cells were imaged at 37° C. with 5% CO 2  on a Nikon Ti inverted microscope equipped with CSU-22 spinning disk confocal and EMCCD camera. Mitotic cells were identified and time points were taken every 2.5 min. For data analysis, images were processed using ImageJ software as follows. Metaphase cells were identified by visual inspection of DNA labeled with H2B-mRuby2. The mean fluorescence intensities of GFP and RFP associated with DNA was then determined and the ratio of GFP to RFP was calculated. The ratio of fluorescent intensities was normalized to metaphase ratios, as it was assumed that the biosensor was intact at this stage. For each post-metaphase time point, the GFP:RFP ratio was determined for the brightest set of chromosomes and normalized against the GFP:RFP metaphase timepoint. 
     Example 2. A Novel Strategy to Produce Active Human Separase for Studies In Vitro 
     Production of active human separase protein at a purity and scale sufficient for biophysical characterization was sought, and expression in Sf9 insect cells with recombinant baculoviruses 21  was employed. First, a gene fusion between the securin C-terminus and the separase N-terminus, separated by a Gly-Ser linker ( FIG. 1B ) was created. There is evidence that securin is a folding chaperone of separase 17,22  and that these protein termini are co-localized 21 . Expression of the fusion construct led to protein levels that were significantly higher than those seen when securin and separase were co-expressed in Sf9 cells. Yield was improved further by N-terminal truncation of securin to remove its APC/C degrons and by elimination of the separase autocleavage sites by mutation. 
     Purified securin-separase ( FIG. 1C ) was characterized by negative-stain electron microscopy (EM) ( FIG. 1D , top). The sample was monodisperse, and class averages were consistent with existing EM data for human securin-separase 6,21 . 
     Human securin-separase has been demonstrated to bind DNA in a non-sequence specific manner 15 . The fusion securin-separase complex was evaluated for similar behavior. Binding of securin-separase to a fluorescently-labeled 50 base-pair double-stranded DNA molecule was evaluated by monitoring fluorescence polarization as a function of protein concentration ( FIG. 1E ). The data fit well to a one site specific-binding model with a K D  of 300 nM+/−100 nM. A DNA molecule with the same base composition but different sequence yielded a similar K D  (220 nM+/−60 nM). Because the separase-DNA interaction is not sequence-specific, it was expected that the measured affinity would depend on length, with shorter DNA molecules exhibiting lower affinities. Indeed, a 25 base-pair DNA molecule bound with a lower affinity (K D =800 nM+/−300 nM). 
     Next, a method for activating separase using purified components was developed, rather than the traditional method of using the APC/C-proteasome system in  Xenopus  egg extract. Analogous to the proteasome, the ClpXP protein complex consists of an unfoldase (the ATPase ClpX) and a peptidase (ClpP) 23 . However, whereas the proteasome interacts with ubiquitin to determine its targets, ClpXP engages with specific short amino acid sequences 23  ( FIG. 1F ). Additionally,  E. coli  ClpXP can be produced recombinantly much more readily than the proteasome. There is also precedent for the use of ClpXP to selectively remove a protein from a protein complex 24 . A ClpXP recognition site was added at the N-terminus of securin in the fusion construct, as well as a TEV protease cleavage site in the linker between securin and separase. Following purification and cleavage with TEV protease, incubation with purified ClpXP removed securin and activated separase, as evaluated by cleavage of an Scc1 fragment in vitro ( FIG. 1G ). Separase also cleaved a catalytically-dead separase with intact autocleavage sites, demonstrating that separase autocleavage can occur in trans. 
     The ClpXP-activated separase was re-purified to remove TEV protease, ClpXP, and any separase still bound by securin. This purification yielded sufficient active separase to measure protein concentration spectroscopically and to perform basic biophysical characterization. First, Michaelis-Menten analysis was used to analyze the kinetics of the interaction between the enzyme active site and a cleavage substrate. These experiments were performed with a substrate peptide encompassing the best-characterized separase cleavage site in human Scc1 ( 169 EIMR (SEQ ID NO: 27), or “site 1”) flanked by a FRET dye-quencher pair ( FIG. 1H ). The results fit well to a standard Michaelis-Menten curve, yielding a KM of 70±30 μM and a kcal of 3×10 −3 ±1×10 −3  sec −1  (or 10±3 hour −1 ). These results are consistent with a previous analysis of active separase reaction kinetics 25 . 
     Finally, the apo separase was evaluated using negative-stain EM ( FIG. 1D , bottom). The sample was monodisperse and indistinguishable from the securin-separase complex at this resolution, indicating that separase does not undergo a large-scale conformational change upon securin removal. 
     Example 3. Scc1 Residues Distant from the Separase Cleavage Site are Critical for Cleavage In Vitro 
     These studies revealed that separase activity toward a minimal cleavage site exhibits a very low catalytic rate 26 , suggesting that cleavage rate is somehow enhanced in the cell. Though it is possible that DNA binding ( FIG. 1E ) provides the extra affinity needed to boost function in vivo, this would be highly nonspecific if this were the only mechanism. The possibility that separase has a more specific substrate docking site was therefore explored. 
     The two separase cleavage sites in Scc1 are located within a large region of predicted disorder between the terminal regions that interact with the Smc3 and Smc1 subunits of cohesin 27 . To investigate whether local sequence context accelerates the cleavage of Scc1, a series of Scc1 truncations was evaluated with an in vitro cleavage assay ( FIG. 2A ). The starting point for this assay was an internal Scc1 fragment (amino acids 142-400), which was chosen after more robust cleavage of Scc1 by separase was observed when the terminal regions that interact with Smc3 and Smc1 were removed. This internal fragment does not contain site 2 ( 447 EPSR (SEQ ID NO: 28)), and so it is cleavage at site 1 ( 169 EIMR (SEQ ID NO: 27)) that was evaluated here. However, even when site 2 is present, it did not appear to be cleaved in this assay, perhaps because cleavage at site 2 requires other factors, such as adjacent phosphorylation by Plk1 10 . 
     An abrupt reduction in cleavage of the Scc1 fragment upon C-terminal truncation from residues 275 to 250 ( FIG. 2B ) was observed, suggesting the presence of a separase-binding motif in this region of Scc1. Note that this assay has lower sensitivity than the above peptide cleavage assay, explaining why cleavage of the smallest fragments is not observed even though they contain the peptide sequence. Alanine scanning revealed that the most critical residues for enhanced activity were a Leu-Pro sequence at residues 255 and 256, with a contribution from Glu 257 ( FIG. 2C ). These residues are referred to hereinafter as the LPE motif. 
     Example 4. The LPE Motif in Scc1 is Important for Cleavage by Separase In Vivo 
     Having demonstrated the importance of the LPE motif for separase cleavage of Scc1 in vitro, its importance in vivo was tested. A previously described separase biosensor in human U2OS cells ( FIG. 3A ) 28  was recreated for this purpose. With wild-type Scc1 (aa 142-467), efficient biosensor cleavage was observed during anaphase ( FIG. 3B ). Strikingly, the double point mutation 255 Leu-Pro 4 Ala-Ala reduced cleavage efficiency by 50% ( FIGS. 3C , D). A biosensor containing a 10 amino acid deletion centered on  255 Leu-Pro yielded identical results as the double alanine mutation, confirming that these two residues are key requirements for this interaction ( FIGS. 3C , D). Although separase cleavage site 2 is present in this biosensor, it was not cleaved in the assay, nor was it cleaved in a longer version of the biosensor that extended 123 amino acids beyond site 2. Therefore, the observation that  255 Leu-Pro promotes biosensor cleavage is specific to site 1. 
     Example 5. Securin Interferes with Separase Binding to the LPE Motif in Scc1 
     The results above suggest that an exosite on separase interacts with the LPE motif in Scc1, resulting in higher substrate affinity and more efficient cleavage. An intriguing possibility is that securin binding prevents this interaction, providing an additional mechanism by which securin inhibits Scc1 cleavage. To address this possibility, securin-separase fusion proteins were created in which securin was truncated after the pseudosubstrate sequence that binds the separase active site ( FIG. 4A ). The likely pseudosubstrate sequence ( 113 EIEKFFP (SEQ ID NO: 26)) was identified previously based on homology between human and  C. thermophilum  securins 4 . It was hypothesized that removal of the pseudosubstrate motif would relieve the inhibition caused by securin directly blocking the separase active site, but that it would retain any effect of securin binding elsewhere on separase. It was also hypothesized that the fusion approach would have the benefit of generating an extremely high local concentration of securin, compared to adding securin in trans. Three securin truncations were tested, having securin residues 127-202, 138-202 or 160-202 covalently linked to separase via a flexible glycine-serine linker. The constructs are referred to hereinafter as securinΔ127-separase, securinΔ138-separase, and securinΔ160-separase, respectively ( FIG. 4B ). 
     It was first asked whether removal of the securin pseudosubstrate region from the active site was sufficient to yield a cleavage-competent active site. Michaelis-Menten analyses with the peptide assay described above ( FIG. 1G ) showed that there were no significant differences between the peptide cleavage activities of the three securinΔ-separase proteins and separase with no securin bound ( FIG. 4C ). These results confirmed that the pseudosubstrate sequence blocks the active site, and they also suggested that securin binding outside the active site does not impair catalysis through some allosteric mechanism. 
     Experiments were then conducted to test whether the securinΔ-separase constructs were able to cleave the Scc1 fragment in the gel-based assay, and whether this cleavage was sensitive to mutation of the LPE motif ( FIG. 4D ). SecurinΔ138-separase and securinΔ160-separase exhibited efficient cleavage of the Scc1 substrate, and in both cases activity was reduced by mutation of  255 Leu-Pro. However, securinΔ127-separase exhibited no cleavage activity in this assay. This result strongly suggests that securin interferes with separase binding to the LPE motif on Scc1, and that this interference is localized to a region of securin between residues 127 and 138. Intriguingly, this region of securin contains an LPE motif (residues 130-132). 
     An approach for investigating the importance of  130 LPE for securin binding to separase was developed. It is known that fungal securin can be converted to a separase substrate by making mutations that convert the pseudosubstrate site into a cleavage site 4 . The equivalent mutations were made in human securin ( 118 FP to RE) and this securin RE  mutant was used to test the importance of  130 LPE for securin engagement with separase. An LP sequence a few residues further downstream ( 139 LP) was also tested. In initial experiments, a securin RE  fragment containing residues 93-202 was cleaved efficiently by separase, but mutation of either LP sequence had no effect, presumably because this fragment of securin makes too many contacts with separase for individual point mutations to significantly weaken affinity. A securin RE  fragment containing residues 93-150 was then tested. This fragment was 50% cleaved by separase, and mutation of  130 LP significantly impaired cleavage ( FIG. 4E ). Mutation of  139 LP had no effect, except when combined with mutation of  130 LP. These results indicate that the  130 LPE motif of securin interacts with separase. 
     Consistent with its importance in the regulation of separase, the LPE sequence immediately downstream of the pseudosubstrate motif is conserved in securin from vertebrates and in some lower eukaryotes ( FIG. 4F ). Budding yeast securin carries a VPE sequence at this location, and the crystal structure of the yeast separase-securin complex indicates that the valine and proline interact with the surface of separase adjacent to the catalytic domains. This region of the human separase differs from the yeast separase, precluding straightforward prediction of the precise LPE motif-binding site in the human protein. 
     The major current method for separase productions depends on removal of securin from small amounts of securin-separase complex using extracts of frog eggs. This generates very small amounts of impure enzyme and is not widely used. There are no currently-available commercial sources of separase, and no previous method could be scaled up to produce large amounts of homogeneous enzyme. As such, the biochemical behavior of the enzyme has remained largely unexplored in the 20 years since it was discovered. The separase constructs of the present disclosure allow for the new discovery of important enzyme characteristics such as the LPE motif described herein. 
     V. References 
     
         
         1. Uhlmann, et al.  Nature  400, 37-42, doi:10.1038/21831 (1999). 
         2. Uhlmann, et al.  Cell  103, 375-386 (2000). 
         3. Nasmyth &amp; Haering.  Annu Rev Genet  43, 525-558, doi:10.1146/annurev-genet-102108-134233 (2009). 
         4. Lin et al.  Nature  532, 131-134, doi:10.1038/nature17402 (2016). 
         5. Luo &amp; Tong.  Nature  542, 255-259, doi:10.1038/nature21061 (2017). 
         6. Boland, et al.  Nature Structural  &amp;  Molecular Biology  24, 414-418, doi:10.1038/nsmb.3386 (2017). 
         7. Luo &amp; Tong.  Curr Opin Struct Biol  49, 114-122, doi:10.1016/j.sbi.2018.01.012 (2018). 
         8. Hauf, et al.  Science  293, 1320-1323, doi:10.1126/science.1061376 (2001). 
         9. Alexandru, et al.  Cell  105, 459-472, doi:10.1016/S0092-8674(01)00362-2 (2001). 
         10. Hauf, et al.  PLoS biology  3, e69, doi:10.1371/journal.pbio.0030069 (2005). 
         11. Waizenegger, et al.  Current biology  12, 1368-1378 (2002). 
         12. Csizmok, et al.  Journal of the American Chemical Society  130, 16873-16879, doi:10.1021/ja805510b (2008). 
         13. Hellmuth, et al.  Molecular Cell  58, 1-12, doi:10.1016/j.molcel.2015.03.025 (2015). 
         14. Jabaiah, et al.  Biological Chemistry  393, 661-616, doi:10.1515/hsz-2012-0162 (2012). 
         15. Sun, et al.  Cell  137, 123-132, doi:10.1016/j.cell.2009.01.040 (2009). 
         16. Ciosk, et al.  Cell  93, 1067-1076, doi:10.1016/50092-8674(00)81211-8 (1998). 
         17. Hellmuth, et al.  Journal of Biological Chemistry  290, 8002-8010, doi:10.1074/jbc.M114.615310 (2015). 
         18. Stemmann, et al.  Cell  107, 715-726 (2001). 
         19. Gorr, et al.  Molecular Cell  19, 135-141, doi:10.1016/j.molcel.2005.05.022 (2005). 
         20. Hellmuth, et al.  The EMBO Journal  33, 1134-1147, doi:10.1002/embj.201488098 (2014). 
         21. Viadiu, et al.  Nature Structural  &amp;  Molecular Biology  12, 552-553, doi:10.1038/nsmb935 (2005). 
         22. Pfleghaar, et al.  PLoS biology  3, e416, doi:10.1371/journal.pbio.0030416 (2005). 
         23. Baker &amp; Sauer. BBA— Molecular Cell Research  1823, 15-28, doi:10.1016/j.bbamcr.2011.06.007 (2012). 
         24. Moore, et al.  Proc Natl Acad Sci USA  105, 11685-11690, doi:10.1073/pnas.0805633105 (2008). 
         25. Zhang, et al.  Journal of Biomolecular Screening  19, 878-889, doi:10.1177/1087057114520972 (2014). 
         26. Bar-Even, et al.  Biochemistry  50, 4402-4410, doi:10.1021/bi2002289 (2011). 
         27. Uhlmann.  Nature Reviews Molecular Cell Biology  17, 399-412, doi:10.1038/nrm.2016.30 (2016). 
         28. Shindo, et al.  Developmental Cell  23, 112-123, doi:10.1016/j.devce1.2012.06.015 (2012). 
         29. Kudo, et al.  J Cell Sci  122, 2686-2698, doi:10.1242/jcs.035287 (2009). 
         30. Sano, et al.  FEBS letters  532, 143-146 (2002). 
         31. Farrell, et al.  Molecular Cell  25, 161-166, doi:10.1016/j.molcel.2006.11.018 (2007). 
         32. Kapust, et al.  Protein Eng  14, 993-1000, doi:10.1093/protein/14.12.993 (2001). 
         33. Ohi, et al.  Biological procedures online  6, 23-34, doi:10.1251/bpo70 (2004). 
         34. Mastronarde.  J Struct Biol  152, 36-51, doi:10.1016/j.jsb.2005.07.007 (2005). 
         35. Zhang.  J Struct Biol  193, 1-12, doi:10.1016/j.jsb.2015.11.003 (2016). 
         36. Scheres.  J Struct Biol  180, 519-530, doi:10.1016/j.jsb.2012.09.006 (2012). 
         37. Edelhoch.  Biochemistry  6, 1948-1954 (1967). 
       
    
     Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate. 
     
       
         
           
               
               
            
               
                   
                 VI. INFORMAL SEQUENCE LISTING 
               
               
                   
                 Human ( Homo sapiens ) separase 
               
               
                   
                 SEQ ID NO: 1 
               
               
                   
                 MRSFKRVNFGTLLSSQKEAEELLPDLKEFLSNPPA 
               
               
                   
               
               
                   
                 GFPSSRSDAERRQACDAILRACNQQLTAKLACPRH 
               
               
                   
               
               
                   
                 LGSLLELAELACDGYLVSTPQRPPLYLERILFVLL 
               
               
                   
               
               
                   
                 RNAAAQGSPEATLRLAQPLHACLVQCSREAAPQDY 
               
               
                   
               
               
                   
                 EAVARGSFSLLWKGAEALLERRAAFAARLKALSFL 
               
               
                   
               
               
                   
                 VLLEDESTPCEVPHFASPTACRAVAAHQLFDASGH 
               
               
                   
               
               
                   
                 GLNEADADFLDDLLSRHVIRALVGERGSSSGLLSP 
               
               
                   
               
               
                   
                 QRALCLLELTLEHCRRFCWSRHHDKAISAVEKAHS 
               
               
                   
               
               
                   
                 YLRNTNLAPSLQLCQLGVKLLQVGEEGPQAVAKLL 
               
               
                   
               
               
                   
                 IKASAVLSKSMEAPSPPLRALYESCQFFLSGLERG 
               
               
                   
               
               
                   
                 TKRRYRLDAILSLFAFLGGYCSLLQQLRDDGVYGG 
               
               
                   
               
               
                   
                 SSKQQQSFLQMYFQGLHLYTVVVYDFAQGCQIVDL 
               
               
                   
               
               
                   
                 ADLTQLVDSCKSTVVWMLEALEGLSGQELTDHMGM 
               
               
                   
               
               
                   
                 TASYTSNLAYSFYSHKLYAEACAISEPLCQHLGLV 
               
               
                   
               
               
                   
                 KPGTYPEVPPEKLHRCFRLQVESLKKLGKQAQGCK 
               
               
                   
               
               
                   
                 MVILWLAALQPCSPEHMAEPVTFWVRVKMDAARAG 
               
               
                   
               
               
                   
                 DKELQLKTLRDSLSGWDPETLALLLREELQAYKAV 
               
               
                   
               
               
                   
                 RADTGQERFNIICDLLELSPEETPAGAWARATHLV 
               
               
                   
               
               
                   
                 ELAQVLCYHDFTQQTNCSALDAIREALQLLDSVRP 
               
               
                   
               
               
                   
                 EAQARDQLLDDKAQALLWLYICTLEAKIQEGIERD 
               
               
                   
               
               
                   
                 RRAQAPGNLEEFEVNDLNYEDKLQEDRFLYSNIAF 
               
               
                   
               
               
                   
                 NLAADAAQSKCLDQALALWKELLTKGQAPAVRCLQ 
               
               
                   
               
               
                   
                 QTAASLQILAALYQLVAKPMQALEVLLLLRIVSER 
               
               
                   
               
               
                   
                 LKDHSKAAGSSCHITQLLLTLGCPSYAQLHLEEAA 
               
               
                   
               
               
                   
                 SSLKHLDQTTDTYLLLSLTCDLLRSQLYWTHQKVT 
               
               
                   
               
               
                   
                 KGVSLLLSVLRDPALQKSSKAWYLLRVQVLQLVAA 
               
               
                   
               
               
                   
                 YLSLPSNNLSHSLWEQLCAQGWQTPEIALIDSHKL 
               
               
                   
               
               
                   
                 LRSIILLLMGSDILSTQKAAVETSFLDYGENLVQK 
               
               
                   
               
               
                   
                 WQVLSEVLSCSEKLVCHLGRLGSVSEAKAFCLEAL 
               
               
                   
               
               
                   
                 KLTTKLQIPRQCALFLVLKGELELARNDIDLCQSD 
               
               
                   
               
               
                   
                 LQQVLFLLESCTEFGGVTQHLDSVKKVHLQKGKQQ 
               
               
                   
               
               
                   
                 AQVPCPPQLPEEELFLRGPALELVATVAKEPGPIA 
               
               
                   
               
               
                   
                 PSTNSAPVLKTKPQPIPNFLSHSPTCDCSLCASPV 
               
               
                   
               
               
                   
                 LTAVCLRWVLVTAGVRLAMGHQAQGLDLLQVVLKG 
               
               
                   
               
               
                   
                 CPEAAERLTQALQASLNHKTPPSLVPSLLDEILAQ 
               
               
                   
               
               
                   
                 AYTLLALEGLNQPSNESLQKVLQSGLKFVAARIPH 
               
               
                   
               
               
                   
                 LEPWRASLLLIWALTKLGGLSCCTTQLFASSWGWQ 
               
               
                   
               
               
                   
                 PPLIKSVPGSEPSKTQGQKRSGRGRQKLASAPLSL 
               
               
                   
               
               
                   
                 NNTSQKGLEGRGLPCTPKPPDRIRQAGPHVPFTVF 
               
               
                   
               
               
                   
                 EEVCPTESKPEVPQAPRVQQRVQTRLKVNFSDDSD 
               
               
                   
               
               
                   
                 LEDPVSAEAWLAEEPKRRGTASRGRGRARKGLSLK 
               
               
                   
               
               
                   
                 TDAVVAPGSAPGNPGLNGRSRRAKKVASRHCEERR 
               
               
                   
               
               
                   
                 PQRASDQARPGPRIMETIPEEELTDNWRKMSFRIL 
               
               
                   
               
               
                   
                 EGSDGEDSASGGKTPAPGPEAASGEWRLLELDSSK 
               
               
                   
               
               
                   
                 KKLPSPCPDKESDKDLGPRLQLPSAPVATGLSTLD 
               
               
                   
               
               
                   
                 SICDSLSVAFRGISHCPPSGLYAHLCRFLALCLGH 
               
               
                   
               
               
                   
                 RDPYATAFLVTESVSITCRHQLLTHLHRQLSKAQK 
               
               
                   
               
               
                   
                 HRGSLEIADQLQGLSLQEMPGDVPLARIQRLFSFR 
               
               
                   
               
               
                   
                 ALESGHFPQPEKESFQERLALIPSGVTVCVLALAT 
               
               
                   
               
               
                   
                 LQPGTVGNTLLLTRLEKDSPPVSVQIPTGQNKLHL 
               
               
                   
               
               
                   
                 RSVLNEFDAIQKAQKENSSCTDKREWWTGRLALDH 
               
               
                   
               
               
                   
                 RMEVLIASLEKSVLGCWKGLLLPSSEEPGPAQEAS 
               
               
                   
               
               
                   
                 RLQELLQDCGWKYPDRTLLKIMLSGAGALTPQDIQ 
               
               
                   
               
               
                   
                 ALAYGLCPTQPERAQELLNEAVGRLQGLTVPSNSH 
               
               
                   
               
               
                   
                 LVLVLDKDLQKLPWESMPSLQALPVTRLPSFRFLL 
               
               
                   
               
               
                   
                 SYSIIKEYGASPVLSQGVDPRSTFYVLNPHNNLSS 
               
               
                   
               
               
                   
                 TEEQFRANFSSEAGWRGVVGEVPRPEQVQEALTKH 
               
               
                   
               
               
                   
                 DLYIYAGHGAGARFLDGQAVLRLSCRAVALLFGCS 
               
               
                   
               
               
                   
                 SAALAVHGNLEGAGIVLKYIMAGCPLFLGNLWDVT 
               
               
                   
               
               
                   
                 DRDIDRYTEALLQGWLGAGPGAPLLYYVNQARQAP 
               
               
                   
               
               
                   
                 RLKYLIGAAPIAYGLPVSLR 
               
               
                   
               
               
                   
                 Human ( Homo sapiens ) securin 
               
               
                   
                 SEQ ID NO: 2 
               
               
                   
                 MATLIYVDKENGEPGTRVVAKDGLKLGSGPSIKAL 
               
               
                   
               
               
                   
                 DGRSQVSTPRFGKTFDAPPALPKATRKALGTVNRA 
               
               
                   
               
               
                   
                 TEKSVKTKGPLKQKQPSFSAKKMTEKTVKAKSSVP 
               
               
                   
               
               
                   
                 ASDDAYPEIEKFFPFNPLDFESFDLPEEHQIAHLP 
               
               
                   
               
               
                   
                 LSGVPLMILDEERELEKLFQLGPPSPVKMPSPPWE 
               
               
                   
               
               
                   
                 SNLLQSPSSILSTLDVELPPVCCDIDI 
               
               
                   
               
               
                   
                 Securin-separase 
               
               
                   
                 SEQ ID NO: 3 
               
               
                   
                 MTNTAKILNFGRWSHPQFEKGSAGSAAGSGAGWSH 
               
               
                   
               
               
                   
                 PQFEKGSASMTEKTVKAKSSVPASDDAYPEIEKFF 
               
               
                   
               
               
                   
                 PFNPLDFESFDLPEEHQIAHLPLSGVPLMILDEER 
               
               
                   
               
               
                   
                 ELEKLFQLGPPSPVKMPSPPWESNLLQSPSSILST 
               
               
                   
               
               
                   
                 LDVELPPVCCDIDIGGSGGSGGGSGGGSGENLYFQ 
               
               
                   
               
               
                   
                 GDYKDHDGDYKDHDIDYKDDDDKSGPMRSFKRVNF 
               
               
                   
               
               
                   
                 GTLLSSQKEAEELLPDLKEFLSNPPAGFPSSRSDA 
               
               
                   
               
               
                   
                 ERRQACDAILRACNQQLTAKLACPRHLGSLLELAE 
               
               
                   
               
               
                   
                 LACDGYLVSTPQRPPLYLERILFVLLRNAAAQGSP 
               
               
                   
               
               
                   
                 EATLRLAQPLHACLVQCSREAAPQDYEAVARGSFS 
               
               
                   
               
               
                   
                 LLWKGAEALLERRAAFAARLKALSFLVLLEDESTP 
               
               
                   
               
               
                   
                 CEVPHFASPTACRAVAAHQLFDASGHGLNEADADF 
               
               
                   
               
               
                   
                 LDDLLSRHVIRALVGERGSSSGLLSPQRALCLLEL 
               
               
                   
               
               
                   
                 TLEHCRRFCWSRHHDKAISAVEKAHSYLRNTNLAP 
               
               
                   
               
               
                   
                 SLQLCQLGVKLLQVGEEGPQAVAKLLIKASAVLSK 
               
               
                   
               
               
                   
                 SMEAPSPPLRALYESCQFFLSGLERGTKRRYRLDA 
               
               
                   
               
               
                   
                 ILSLFAFLGGYCSLLQQLRDDGVYGGSSKQQQSFL 
               
               
                   
               
               
                   
                 QMYFQGLHLYTVVVYDFAQGCQIVDLADLTQLVDS 
               
               
                   
               
               
                   
                 CKSTVVWMLEALEGLSGQELTDHMGMTASYTSNLA 
               
               
                   
               
               
                   
                 YSFYSHKLYAEACAISEPLCQHLGLVKPGTYPEVP 
               
               
                   
               
               
                   
                 PEKLHRCFRLQVESLKKLGKQAQGCKMVILWLAAL 
               
               
                   
               
               
                   
                 QPCSPEHMAEPVTFWVRVKMDAARAGDKELQLKTL 
               
               
                   
               
               
                   
                 RDSLSGWDPETLALLLREELQAYKAVRADTGQERF 
               
               
                   
               
               
                   
                 NIICDLLELSPEETPAGAWARATHLVELAQVLCYH 
               
               
                   
               
               
                   
                 DFTQQTNCSALDAIREALQLLDSVRPEAQARDQLL 
               
               
                   
               
               
                   
                 DDKAQALLWLYICTLEAKIQEGIERDRRAQAPGNL 
               
               
                   
               
               
                   
                 EEFEVNDLNYEDKLQEDRFLYSNIAFNLAADAAQS 
               
               
                   
               
               
                   
                 KCLDQALALWKELLTKGQAPAVRCLQQTAASLQIL 
               
               
                   
               
               
                   
                 AALYQLVAKPMQALEVLLLLRIVSERLKDHSKAAG 
               
               
                   
               
               
                   
                 SSCHITQLLLTLGCPSYAQLHLEEAASSLKHLDQT 
               
               
                   
               
               
                   
                 TDTYLLLSLTCDLLRSQLYWTHQKVTKGVSLLLSV 
               
               
                   
               
               
                   
                 LRDPALQKSSKAWYLLRVQVLQLVAAYLSLPSNNL 
               
               
                   
               
               
                   
                 SHSLWEQLCAQGWQTPEIALIDSHKLLRSIILLLM 
               
               
                   
               
               
                   
                 GSDILSTQKAAVETSFLDYGENLVQKWQVLSEVLS 
               
               
                   
               
               
                   
                 CSEKLVCHLGRLGSVSEAKAFCLEALKLTTKLQIP 
               
               
                   
               
               
                   
                 RQCALFLVLKGELELARNDIDLCQSDLQQVLFLLE 
               
               
                   
               
               
                   
                 SCTEFGGVTQHLDSVKKVHLQKGKQQAQVPCPPQL 
               
               
                   
               
               
                   
                 PEEELFLRGPALELVATVAKEPGPIAPSTNSAPVL 
               
               
                   
               
               
                   
                 KTKPQPIPNFLSHSPTCDCSLCASPVLTAVCLRWV 
               
               
                   
               
               
                   
                 LVTAGVRLAMGHQAQGLDLLQVVLKGCPEAAERLT 
               
               
                   
               
               
                   
                 QALQASLNHKTPPSLVPSLLDEILAQAYTLLALEG 
               
               
                   
               
               
                   
                 LNQPSNESLQKVLQSGLKFVAARIPHLEPWRASLL 
               
               
                   
               
               
                   
                 LIWALTKLGGLSCCTTQLFASSWGWQPPLIKSVPG 
               
               
                   
               
               
                   
                 SEPSKTQGQKRSGRGRQKLASAPLSLNNTSQKGLE 
               
               
                   
               
               
                   
                 GRGLPCTPKPPDRIRQAGPHVPFTVFEEVCPTESK 
               
               
                   
               
               
                   
                 PEVPQAPRVQQRVQTRLKVNFSDDSDLEDPVSAEA 
               
               
                   
               
               
                   
                 WLAEEPKRRGTASRGRGRARKGLSLKTDAVVAPGS 
               
               
                   
               
               
                   
                 APGNPGLNGRSRRAKKVASRHCEERRPQRASDQAR 
               
               
                   
               
               
                   
                 PGPRIMETIPEEELTDNWRKMSFRILEGSDGEDSA 
               
               
                   
               
               
                   
                 SGGKTPAPGPEAASGEWRLLELDSSKKKLPSPCPD 
               
               
                   
               
               
                   
                 KESDKDLGPRLQLPSAPVATGLSTLDSICDSLSVA 
               
               
                   
               
               
                   
                 FRGISHCPPSGLYAHLCRFLALCLGHRDPYATAFL 
               
               
                   
               
               
                   
                 VTESVSITCRHQLLTHLHRQLSKAQKHRGSLEIAD 
               
               
                   
               
               
                   
                 QLQGLSLQEMPGDVPLARIQRLFSFRALESGHFPQ 
               
               
                   
               
               
                   
                 PEKESFQERLALIPSGVTVCVLALATLQPGTVGNT 
               
               
                   
               
               
                   
                 LLLTRLEKDSPPVSVQIPTGQNKLHLRSVLNEFDA 
               
               
                   
               
               
                   
                 IQKAQKENSSCTDKREWWTGRLALDHRMEVLIASL 
               
               
                   
               
               
                   
                 EKSVLGCWKGLLLPSSEEPGPAQEASRLQELLQDC 
               
               
                   
               
               
                   
                 GWKYPDRTLLKIMLSGAGALTPQDIQALAYGLCPT 
               
               
                   
               
               
                   
                 QPERAQELLNEAVGRLQGLTVPSNSHLVLVLDKDL 
               
               
                   
               
               
                   
                 QKLPWESMPSLQALPVTRLPSFRFLLSYSIIKEYG 
               
               
                   
               
               
                   
                 ASPVLSQGVDPRSTFYVLNPHNNLSSTEEQFRANF 
               
               
                   
               
               
                   
                 SSEAGWRGVVGEVPRPEQVQEALTKHDLYIYAGHG 
               
               
                   
               
               
                   
                 AGARFLDGQAVLRLSCRAVALLFGCSSAALAVHGN 
               
               
                   
               
               
                   
                 LEGAGIVLKYIMAGCPLFLGNLWDVTDRDIDRYTE 
               
               
                   
               
               
                   
                 ALLQGWLGAGPGAPLLYYVNQARQAPRLKYLIGAA 
               
               
                   
               
               
                   
                 PIAYGLPVSLR 
               
               
                   
               
               
                   
                 SecurinΔ127-separase 
               
               
                   
                 SEQ ID NO: 4 
               
               
                   
                 MHWSHPQFEKGSAGSAAGSGAGWSHPQFEKGSSTA 
               
               
                   
               
               
                   
                 SENLYFQGSFDLPEEHQIAHLPLSGVPLMILDEER 
               
               
                   
               
               
                   
                 ELEKLFQLGPPSPVKMPSPPWESNLLQSPSSILST 
               
               
                   
               
               
                   
                 LDVELPPVCCDIDIGGSGGSGGGSGGGSGGGSGGD 
               
               
                   
               
               
                   
                 YKDHDGDYKDHDIDYKDDDDKSGPMRSFKRVNFGT 
               
               
                   
               
               
                   
                 LLSSQKEAEELLPDLKEFLSNPPAGFPSSRSDAER 
               
               
                   
               
               
                   
                 RQACDAILRACNQQLTAKLACPRHLGSLLELAELA 
               
               
                   
               
               
                   
                 CDGYLVSTPQRPPLYLERILFVLLRNAAAQGSPEA 
               
               
                   
               
               
                   
                 TLRLAQPLHACLVQCSREAAPQDYEAVARGSFSLL 
               
               
                   
               
               
                   
                 WKGAEALLERRAAFAARLKALSFLVLLEDESTPCE 
               
               
                   
               
               
                   
                 VPHFASPTACRAVAAHQLFDASGHGLNEADADFLD 
               
               
                   
               
               
                   
                 DLLSRHVIRALVGERGSSSGLLSPQRALCLLELTL 
               
               
                   
               
               
                   
                 EHCRRFCWSRHHDKAISAVEKAHSYLRNTNLAPSL 
               
               
                   
               
               
                   
                 QLCQLGVKLLQVGEEGPQAVAKLLIKASAVLSKSM 
               
               
                   
               
               
                   
                 EAPSPPLRALYESCQFFLSGLERGTKRRYRLDAIL 
               
               
                   
               
               
                   
                 SLFAFLGGYCSLLQQLRDDGVYGGSSKQQQSFLQM 
               
               
                   
               
               
                   
                 YFQGLHLYTVVVYDFAQGCQIVDLADLTQLVDSCK 
               
               
                   
               
               
                   
                 STVVWMLEALEGLSGQELTDHMGMTASYTSNLAYS 
               
               
                   
               
               
                   
                 FYSHKLYAEACAISEPLCQHLGLVKPGTYPEVPPE 
               
               
                   
               
               
                   
                 KLHRCFRLQVESLKKLGKQAQGCKMVILWLAALQP 
               
               
                   
               
               
                   
                 CSPEHMAEPVTFWVRVKMDAARAGDKELQLKTLRD 
               
               
                   
               
               
                   
                 SLSGWDPETLALLLREELQAYKAVRADTGQERFNI 
               
               
                   
               
               
                   
                 ICDLLELSPEETPAGAWARATHLVELAQVLCYHDF 
               
               
                   
               
               
                   
                 TQQTNCSALDAIREALQLLDSVRPEAQARDQLLDD 
               
               
                   
               
               
                   
                 KAQALLWLYICTLEAKIQEGIERDRRAQAPGNLEE 
               
               
                   
               
               
                   
                 FEVNDLNYEDKLQEDRFLYSNIAFNLAADAAQSKC 
               
               
                   
               
               
                   
                 LDQALALWKELLTKGQAPAVRCLQQTAASLQILAA 
               
               
                   
               
               
                   
                 LYQLVAKPMQALEVLLLLRIVSERLKDHSKAAGSS 
               
               
                   
               
               
                   
                 CHITQLLLTLGCPSYAQLHLEEAASSLKHLDQTTD 
               
               
                   
               
               
                   
                 TYLLLSLTCDLLRSQLYWTHQKVTKGVSLLLSVLR 
               
               
                   
               
               
                   
                 DPALQKSSKAWYLLRVQVLQLVAAYLSLPSNNLSH 
               
               
                   
               
               
                   
                 SLWEQLCAQGWQTPEIALIDSHKLLRSIILLLMGS 
               
               
                   
               
               
                   
                 DILSTQKAAVETSFLDYGENLVQKWQVLSEVLSCS 
               
               
                   
               
               
                   
                 EKLVCHLGRLGSVSEAKAFCLEALKLTTKLQIPRQ 
               
               
                   
               
               
                   
                 CALFLVLKGELELARNDIDLCQSDLQQVLFLLESC 
               
               
                   
               
               
                   
                 TEFGGVTQHLDSVKKVHLQKGKQQAQVPCPPQLPE 
               
               
                   
               
               
                   
                 EELFLRGPALELVATVAKEPGPIAPSTNSAPVLKT 
               
               
                   
               
               
                   
                 KPQPIPNFLSHSPTCDCSLCASPVLTAVCLRWVLV 
               
               
                   
               
               
                   
                 TAGVRLAMGHQAQGLDLLQVVLKGCPEAAERLTQA 
               
               
                   
               
               
                   
                 LQASLNHKTPPSLVPSLLDEILAQAYTLLALEGLN 
               
               
                   
               
               
                   
                 QPSNESLQKVLQSGLKFVAARIPHLEPWRASLLLI 
               
               
                   
               
               
                   
                 WALTKLGGLSCCTTQLFASSWGWQPPLIKSVPGSE 
               
               
                   
               
               
                   
                 PSKTQGQKRSGRGRQKLASAPLSLNNTSQKGLEGR 
               
               
                   
               
               
                   
                 GLPCTPKPPDRIRQAGPHVPFTVFEEVCPTESKPE 
               
               
                   
               
               
                   
                 VPQAPRVQQRVQTRLKVNFSDDSDLEDPVSAEAWL 
               
               
                   
               
               
                   
                 AEEPKRRGTASRGRGRARKGLSLKTDAVVAPGSAP 
               
               
                   
               
               
                   
                 GNPGLNGRSRRAKKVASRHCEERRPQRASDQARPG 
               
               
                   
               
               
                   
                 PRIMETIPEEELTDNWRKMSFRILEGSDGEDSASG 
               
               
                   
               
               
                   
                 GKTPAPGPEAASGEWRLLELDSSKKKLPSPCPDKE 
               
               
                   
               
               
                   
                 SDKDLGPRLQLPSAPVATGLSTLDSICDSLSVAFR 
               
               
                   
               
               
                   
                 GISHCPPSGLYAHLCRFLALCLGHRDPYATAFLVT 
               
               
                   
               
               
                   
                 ESVSITCRHQLLTHLHRQLSKAQKHRGSLEIADQL 
               
               
                   
               
               
                   
                 QGLSLQEMPGDVPLARIQRLFSFRALESGHFPQPE 
               
               
                   
               
               
                   
                 KESFQERLALIPSGVTVCVLALATLQPGTVGNTLL 
               
               
                   
               
               
                   
                 LTRLEKDSPPVSVQIPTGQNKLHLRSVLNEFDAIQ 
               
               
                   
               
               
                   
                 KAQKENSSCTDKREWWTGRLALDHRMEVLIASLEK 
               
               
                   
               
               
                   
                 SVLGCWKGLLLPSSEEPGPAQEASRLQELLQDCGW 
               
               
                   
               
               
                   
                 KYPDRTLLKIMLSGAGALTPQDIQALAYGLCPTQP 
               
               
                   
               
               
                   
                 ERAQELLNEAVGRLQGLTVPSNSHLVLVLDKDLQK 
               
               
                   
               
               
                   
                 LPWESMPSLQALPVTRLPSFRFLLSYSIIKEYGAS 
               
               
                   
               
               
                   
                 PVLSQGVDPRSTFYVLNPHNNLSSTEEQFRANFSS 
               
               
                   
               
               
                   
                 EAGWRGVVGEVPRPEQVQEALTKHDLYIYAGHGAG 
               
               
                   
               
               
                   
                 ARFLDGQAVLRLSCRAVALLFGCSSAALAVHGNLE 
               
               
                   
               
               
                   
                 GAGIVLKYIMAGCPLFLGNLWDVTDRDIDRYTEAL 
               
               
                   
               
               
                   
                 LQGWLGAGPGAPLLYYVNQARQAPRLKYLIGAAPI 
               
               
                   
               
               
                   
                 AYGLPVSLR 
               
               
                   
               
               
                   
                 SecurinΔ138-separase 
               
               
                   
                 SEQ ID NO: 5 
               
               
                   
                 MHWSHPQFEKGSAGSAAGSGAGWSHPQFEKGSSTA 
               
               
                   
               
               
                   
                 SHLPLSGVPLMILDEERELEKLFQLGPPSPVKMPS 
               
               
                   
               
               
                   
                 PPWESNLLQSPSSILSTLDVELPPVCCDIDIGGSG 
               
               
                   
               
               
                   
                 GSGGGSGGGSGENLYFQGDYKDHDGDYKDHDIDYK 
               
               
                   
               
               
                   
                 DDDDKSGPMRSFKRVNFGTLLSSQKEAEELLPDLK 
               
               
                   
               
               
                   
                 EFLSNPPAGFPSSRSDAERRQACDAILRACNQQLT 
               
               
                   
               
               
                   
                 AKLACPRHLGSLLELAELACDGYLVSTPQRPPLYL 
               
               
                   
               
               
                   
                 ERILFVLLRNAAAQGSPEATLRLAQPLHACLVQCS 
               
               
                   
               
               
                   
                 REAAPQDYEAVARGSFSLLWKGAEALLERRAAFAA 
               
               
                   
               
               
                   
                 RLKALSFLVLLEDESTPCEVPHFASPTACRAVAAH 
               
               
                   
               
               
                   
                 QLFDASGHGLNEADADFLDDLLSRHVIRALVGERG 
               
               
                   
               
               
                   
                 SSSGLLSPQRALCLLELTLEHCRRFCWSRHHDKAI 
               
               
                   
               
               
                   
                 SAVEKAHSYLRNTNLAPSLQLCQLGVKLLQVGEEG 
               
               
                   
               
               
                   
                 PQAVAKLLIKASAVLSKSMEAPSPPLRALYESCQF 
               
               
                   
               
               
                   
                 FLSGLERGTKRRYRLDAILSLFAFLGGYCSLLQQL 
               
               
                   
               
               
                   
                 RDDGVYGGSSKQQQSFLQMYFQGLHLYTVVVYDFA 
               
               
                   
               
               
                   
                 QGCQIVDLADLTQLVDSCKSTVVWMLEALEGLSGQ 
               
               
                   
               
               
                   
                 ELTDHMGMTASYTSNLAYSFYSHKLYAEACAISEP 
               
               
                   
               
               
                   
                 LCQHLGLVKPGTYPEVPPEKLHRCFRLQVESLKKL 
               
               
                   
               
               
                   
                 GKQAQGCKMVILWLAALQPCSPEHMAEPVTFWVRV 
               
               
                   
               
               
                   
                 KMDAARAGDKELQLKTLRDSLSGWDPETLALLLRE 
               
               
                   
               
               
                   
                 ELQAYKAVRADTGQERFNIICDLLELSPEETPAGA 
               
               
                   
               
               
                   
                 WARATHLVELAQVLCYHDFTQQTNCSALDAIREAL 
               
               
                   
               
               
                   
                 QLLDSVRPEAQARDQLLDDKAQALLWLYICTLEAK 
               
               
                   
               
               
                   
                 IQEGIERDRRAQAPGNLEEFEVNDLNYEDKLQEDR 
               
               
                   
               
               
                   
                 FLYSNIAFNLAADAAQSKCLDQALALWKELLTKGQ 
               
               
                   
               
               
                   
                 APAVRCLQQTAASLQILAALYQLVAKPMQALEVLL 
               
               
                   
               
               
                   
                 LLRIVSERLKDHSKAAGSSCHITQLLLTLGCPSYA 
               
               
                   
               
               
                   
                 QLHLEEAASSLKHLDQTTDTYLLLSLTCDLLRSQL 
               
               
                   
               
               
                   
                 YWTHQKVTKGVSLLLSVLRDPALQKSSKAWYLLRV 
               
               
                   
               
               
                   
                 QVLQLVAAYLSLPSNNLSHSLWEQLCAQGWQTPEI 
               
               
                   
               
               
                   
                 ALIDSHKLLRSIILLLMGSDILSTQKAAVETSFLD 
               
               
                   
               
               
                   
                 YGENLVQKWQVLSEVLSCSEKLVCHLGRLGSVSEA 
               
               
                   
               
               
                   
                 KAFCLEALKLTTKLQIPRQCALFLVLKGELELARN 
               
               
                   
               
               
                   
                 DIDLCQSDLQQVLFLLESCTEFGGVTQHLDSVKKV 
               
               
                   
               
               
                   
                 HLQKGKQQAQVPCPPQLPEEELFLRGPALELVATV 
               
               
                   
               
               
                   
                 AKEPGPIAPSTNSAPVLKTKPQPIPNFLSHSPTCD 
               
               
                   
               
               
                   
                 CSLCASPVLTAVCLRWVLVTAGVRLAMGHQAQGLD 
               
               
                   
               
               
                   
                 LLQVVLKGCPEAAERLTQALQASLNHKTPPSLVPS 
               
               
                   
               
               
                   
                 LLDEILAQAYTLLALEGLNQPSNESLQKVLQSGLK 
               
               
                   
               
               
                   
                 FVAARIPHLEPWRASLLLIWALTKLGGLSCCTTQL 
               
               
                   
               
               
                   
                 FASSWGWQPPLIKSVPGSEPSKTQGQKRSGRGRQK 
               
               
                   
               
               
                   
                 LASAPLSLNNTSQKGLEGRGLPCTPKPPDRIRQAG 
               
               
                   
               
               
                   
                 PHVPFTVFEEVCPTESKPEVPQAPRVQQRVQTRLK 
               
               
                   
               
               
                   
                 VNFSDDSDLEDPVSAEAWLAEEPKRRGTASRGRGR 
               
               
                   
               
               
                   
                 ARKGLSLKTDAVVAPGSAPGNPGLNGRSRRAKKVA 
               
               
                   
               
               
                   
                 SRHCEERRPQRASDQARPGPRIMETIPEEELTDNW 
               
               
                   
               
               
                   
                 RKMSFRILEGSDGEDSASGGKTPAPGPEAASGEWR 
               
               
                   
               
               
                   
                 LLELDSSKKKLPSPCPDKESDKDLGPRLQLPSAPV 
               
               
                   
               
               
                   
                 ATGLSTLDSICDSLSVAFRGISHCPPSGLYAHLCR 
               
               
                   
               
               
                   
                 FLALCLGHRDPYATAFLVTESVSITCRHQLLTHLH 
               
               
                   
               
               
                   
                 RQLSKAQKHRGSLEIADQLQGLSLQEMPGDVPLAR 
               
               
                   
               
               
                   
                 IQRLFSFRALESGHFPQPEKESFQERLALIPSGVT 
               
               
                   
               
               
                   
                 VCVLALATLQPGTVGNTLLLTRLEKDSPPVSVQIP 
               
               
                   
               
               
                   
                 TGQNKLHLRSVLNEFDAIQKAQKENSSCTDKREWW 
               
               
                   
               
               
                   
                 TGRLALDHRMEVLIASLEKSVLGCWKGLLLPSSEE 
               
               
                   
               
               
                   
                 PGPAQEASRLQELLQDCGWKYPDRTLLKIMLSGAG 
               
               
                   
               
               
                   
                 ALTPQDIQALAYGLCPTQPERAQELLNEAVGRLQG 
               
               
                   
               
               
                   
                 LTVPSNSHLVLVLDKDLQKLPWESMPSLQALPVTR 
               
               
                   
               
               
                   
                 LPSFRFLLSYSIIKEYGASPVLSQGVDPRSTFYVL 
               
               
                   
               
               
                   
                 NPHNNLSSTEEQFRANFSSEAGWRGVVGEVPRPEQ 
               
               
                   
               
               
                   
                 VQEALTKHDLYIYAGHGAGARFLDGQAVLRLSCRA 
               
               
                   
               
               
                   
                 VALLFGCSSAALAVHGNLEGAGIVLKYIMAGCPLF 
               
               
                   
               
               
                   
                 LGNLWDVTDRDIDRYTEALLQGWLGAGPGAPLLYY 
               
               
                   
               
               
                   
                 VNQARQAPRLKYLIGAAPIAYGLPVSLR 
               
               
                   
               
               
                   
                 SecurinΔ160-separase 
               
               
                   
                 SEQ ID NO: 6 
               
               
                   
                 MHWSHPQFEKGSAGSAAGSGAGWSHPQFEKGSSTA 
               
               
                   
               
               
                   
                 SQLGPPSPVKMPSPPWESNLLQSPSSILSTLDVEL 
               
               
                   
               
               
                   
                 PPVCCDIDIGGSGGSGGGSGGGSGENLYFQGDYKD 
               
               
                   
               
               
                   
                 HDGDYKDHDIDYKDDDDKSGPMRSFKRVNFGTLLS 
               
               
                   
               
               
                   
                 SQKEAEELLPDLKEFLSNPPAGFPSSRSDAERRQA 
               
               
                   
               
               
                   
                 CDAILRACNQQLTAKLACPRHLGSLLELAELACDG 
               
               
                   
               
               
                   
                 YLVSTPQRPPLYLERILFVLLRNAAAQGSPEATLR 
               
               
                   
               
               
                   
                 LAQPLHACLVQCSREAAPQDYEAVARGSFSLLWKG 
               
               
                   
               
               
                   
                 AEALLERRAAFAARLKALSFLVLLEDESTPCEVPH 
               
               
                   
               
               
                   
                 FASPTACRAVAAHQLFDASGHGLNEADADFLDDLL 
               
               
                   
               
               
                   
                 SRHVIRALVGERGSSSGLLSPQRALCLLELTLEHC 
               
               
                   
               
               
                   
                 RRFCWSRHHDKAISAVEKAHSYLRNTNLAPSLQLC 
               
               
                   
               
               
                   
                 QLGVKLLQVGEEGPQAVAKLLIKASAVLSKSMEAP 
               
               
                   
               
               
                   
                 SPPLRALYESCQFFLSGLERGTKRRYRLDAILSLF 
               
               
                   
               
               
                   
                 AFLGGYCSLLQQLRDDGVYGGSSKQQQSFLQMYFQ 
               
               
                   
               
               
                   
                 GLHLYTVVVYDFAQGCQIVDLADLTQLVDSCKSTV 
               
               
                   
               
               
                   
                 VWMLEALEGLSGQELTDHMGMTASYTSNLAYSFYS 
               
               
                   
               
               
                   
                 HKLYAEACAISEPLCQHLGLVKPGTYPEVPPEKLH 
               
               
                   
               
               
                   
                 RCFRLQVESLKKLGKQAQGCKMVILWLAALQPCSP 
               
               
                   
               
               
                   
                 EHMAEPVTFWVRVKMDAARAGDKELQLKTLRDSLS 
               
               
                   
               
               
                   
                 GWDPETLALLLREELQAYKAVRADTGQERFNIICD 
               
               
                   
               
               
                   
                 LLELSPEETPAGAWARATHLVELAQVLCYHDFTQQ 
               
               
                   
               
               
                   
                 TNCSALDAIREALQLLDSVRPEAQARDQLLDDKAQ 
               
               
                   
               
               
                   
                 ALLWLYICTLEAKIQEGIERDRRAQAPGNLEEFEV 
               
               
                   
               
               
                   
                 NDLNYEDKLQEDRFLYSNIAFNLAADAAQSKCLDQ 
               
               
                   
               
               
                   
                 ALALWKELLTKGQAPAVRCLQQTAASLQILAALYQ 
               
               
                   
               
               
                   
                 LVAKPMQALEVLLLLRIVSERLKDHSKAAGSSCHI 
               
               
                   
               
               
                   
                 TQLLLTLGCPSYAQLHLEEAASSLKHLDQTTDTYL 
               
               
                   
               
               
                   
                 LLSLTCDLLRSQLYWTHQKVTKGVSLLLSVLRDPA 
               
               
                   
               
               
                   
                 LQKSSKAWYLLRVQVLQLVAAYLSLPSNNLSHSLW 
               
               
                   
               
               
                   
                 EQLCAQGWQTPEIALIDSHKLLRSIILLLMGSDIL 
               
               
                   
               
               
                   
                 STQKAAVETSFLDYGENLVQKWQVLSEVLSCSEKL 
               
               
                   
               
               
                   
                 VCHLGRLGSVSEAKAFCLEALKLTTKLQIPRQCAL 
               
               
                   
               
               
                   
                 FLVLKGELELARNDIDLCQSDLQQVLFLLESCTEF 
               
               
                   
               
               
                   
                 GGVTQHLDSVKKVHLQKGKQQAQVPCPPQLPEEEL 
               
               
                   
               
               
                   
                 FLRGPALELVATVAKEPGPIAPSTNSAPVLKTKPQ 
               
               
                   
               
               
                   
                 PIPNFLSHSPTCDCSLCASPVLTAVCLRWVLVTAG 
               
               
                   
               
               
                   
                 VRLAMGHQAQGLDLLQVVLKGCPEAAERLTQALQA 
               
               
                   
               
               
                   
                 SLNHKTPPSLVPSLLDEILAQAYTLLALEGLNQPS 
               
               
                   
               
               
                   
                 NESLQKVLQSGLKFVAARIPHLEPWRASLLLIWAL 
               
               
                   
               
               
                   
                 TKLGGLSCCTTQLFASSWGWQPPLIKSVPGSEPSK 
               
               
                   
               
               
                   
                 TQGQKRSGRGRQKLASAPLSLNNTSQKGLEGRGLP 
               
               
                   
               
               
                   
                 CTPKPPDRIRQAGPHVPFTVFEEVCPTESKPEVPQ 
               
               
                   
               
               
                   
                 APRVQQRVQTRLKVNFSDDSDLEDPVSAEAWLAEE 
               
               
                   
               
               
                   
                 PKRRGTASRGRGRARKGLSLKTDAVVAPGSAPGNP 
               
               
                   
               
               
                   
                 GLNGRSRRAKKVASRHCEERRPQRASDQARPGPRI 
               
               
                   
               
               
                   
                 METIPEEELTDNWRKMSFRILEGSDGEDSASGGKT 
               
               
                   
               
               
                   
                 PAPGPEAASGEWRLLELDSSKKKLPSPCPDKESDK 
               
               
                   
               
               
                   
                 DLGPRLQLPSAPVATGLSTLDSICDSLSVAFRGIS 
               
               
                   
               
               
                   
                 HCPPSGLYAHLCRFLALCLGHRDPYATAFLVTESV 
               
               
                   
               
               
                   
                 SITCRHQLLTHLHRQLSKAQKHRGSLEIADQLQGL 
               
               
                   
               
               
                   
                 SLQEMPGDVPLARIQRLFSFRALESGHFPQPEKES 
               
               
                   
               
               
                   
                 FQERLALIPSGVTVCVLALATLQPGTVGNTLLLTR 
               
               
                   
               
               
                   
                 LEKDSPPVSVQIPTGQNKLHLRSVLNEFDAIQKAQ 
               
               
                   
               
               
                   
                 KENSSCTDKREWWTGRLALDHRMEVLIASLEKSVL 
               
               
                   
               
               
                   
                 GCWKGLLLPSSEEPGPAQEASRLQELLQDCGWKYP 
               
               
                   
               
               
                   
                 DRTLLKIMLSGAGALTPQDIQALAYGLCPTQPERA 
               
               
                   
               
               
                   
                 QELLNEAVGRLQGLTVPSNSHLVLVLDKDLQKLPW 
               
               
                   
               
               
                   
                 ESMPSLQALPVTRLPSFRFLLSYSIIKEYGASPVL 
               
               
                   
               
               
                   
                 SQGVDPRSTFYVLNPHNNLSSTEEQFRANFSSEAG 
               
               
                   
               
               
                   
                 WRGVVGEVPRPEQVQEALTKHDLYIYAGHGAGARF 
               
               
                   
               
               
                   
                 LDGQAVLRLSCRAVALLFGCSSAALAVHGNLEGAG 
               
               
                   
               
               
                   
                 IVLKYIMAGCPLFLGNLWDVTDRDIDRYTEALLQG 
               
               
                   
               
               
                   
                 WLGAGPGAPLLYYVNQARQAPRLKYLIGAAPIAYG 
               
               
                   
               
               
                   
                 LPVSLR 
               
               
                   
               
               
                   
                 Mouse ( Mus musculus ) separase 
               
               
                   
                 SEQ ID NO: 7 
               
               
                   
                 MRNFKGVNFATLLCSKEETQQLLPDLKEFLSRSRT 
               
               
                   
               
               
                   
                 DFPSSRTDAERRQICDTILRACTQQLTAKLDCPGH 
               
               
                   
               
               
                   
                 LRSILDLAELACDGYLLSTPQRPPLYLERILFILL 
               
               
                   
               
               
                   
                 RNGSTQGSPDTVLRLAQPLHACLVQNSGEAAPQDY 
               
               
                   
               
               
                   
                 EAVTRGSFSLFWKGAEALLERRAAFSTRLNALSFL 
               
               
                   
               
               
                   
                 VLLEDGSVPCEVPHFASPTACRLVAAYQLYDATGQ 
               
               
                   
               
               
                   
                 GLDEADADFLYEVLSRHLIRVLVGEGGSSPGPLSP 
               
               
                   
               
               
                   
                 QRALCLLEITLEHCRRLCWNHHHRQAARAVERARN 
               
               
                   
               
               
                   
                 HLEKTSVAPSLQLCQMGVELLEAVEERPGAVAQLL 
               
               
                   
               
               
                   
                 RKAAAVLINSIEAPSPPLRALYDSCQFFLSGLERG 
               
               
                   
               
               
                   
                 IRRHCGLDAILSLFAFLGGYSSLVRHLREVSEASS 
               
               
                   
               
               
                   
                 KQQQCLLQMHFQGFHLFTGIVYDFAQGCQATELAQ 
               
               
                   
               
               
                   
                 LVDGCRSAAVWMLEALEGLSGGELADYLSMTASYT 
               
               
                   
               
               
                   
                 SNLAYSFFSQKLYEEACVISEPVCQHLGSATSGAC 
               
               
                   
               
               
                   
                 PEVPPEKLHRCFRLHVESLKKLGKQAQGCKMVTLW 
               
               
                   
               
               
                   
                 LAALKPYSLEHMVEPVTFWVRVKMDASRAGDKELQ 
               
               
                   
               
               
                   
                 LQTLRDSLSCWDPETQSLLLREELRAYKSVRADTG 
               
               
                   
               
               
                   
                 QERFNIICDLLELSPEETAAGAWARATYLVELAQV 
               
               
                   
               
               
                   
                 LCYHNFTQQTNCSALDAVQEALQLLESVSPEAQEQ 
               
               
                   
               
               
                   
                 DRLLDDKAQALLWLYICTLEAKMQEGIERDRRAQA 
               
               
                   
               
               
                   
                 PSNLEEFEVNDLNYEDKLQEDRFLYSSIAFNLAAD 
               
               
                   
               
               
                   
                 AAQSKCLDQALTLWKEVLTKGRAPAVRCLQQTAAS 
               
               
                   
               
               
                   
                 LQILAAVYQLVAKPLQALETLLLLQIVSKRLQDHA 
               
               
                   
               
               
                   
                 KAASSSCQLTQLLLNLGCPSYAQLYLEEAESSLRS 
               
               
                   
               
               
                   
                 LDQTSDACQLLSLTCALLGSQLCWACQKVTAGVSL 
               
               
                   
               
               
                   
                 LLSVLRDPALQKSSKAWYLLRVQALQVLAFYLSLS 
               
               
                   
               
               
                   
                 SNLLSSALREQLWDQGWQTPETALIDAHKLLRSII 
               
               
                   
               
               
                   
                 ILLMGSDVLSIQKAATESPFLDYGENLVQKWQVLT 
               
               
                   
               
               
                   
                 EVLTCSERLVGRLGRLGNVSEAKAFCLEALKLTTK 
               
               
                   
               
               
                   
                 LQIPRQCALFLVLKGELELARGDIDLCQSDLQQVL 
               
               
                   
               
               
                   
                 FLLESSTEFGVVTQHPDSVKKVHTQKGKHKAQGPC 
               
               
                   
               
               
                   
                 FPPLSEEEPFLKGPALELVDTVLNEPGPIQSSVNS 
               
               
                   
               
               
                   
                 SPVLKTKPPPNPGFLSHLPSCDCLLCASPALSAVC 
               
               
                   
               
               
                   
                 LRWVLVTAGVRLATGHKAQGLDLLQAVLTRCPAAT 
               
               
                   
               
               
                   
                 KRFTQSLQASLNHRTTPSCVPSLFDEIMAQVYTHL 
               
               
                   
               
               
                   
                 ALEFLNQTSEKSLGKVLASGLKFVATRIQSLEIWR 
               
               
                   
               
               
                   
                 AHLLLVQALAKLAHFSCCTSELFASSWGWHPPLVK 
               
               
                   
               
               
                   
                 SLPVLEPAKIRRQKCSGRGRRRIASVPPPLHNSSQ 
               
               
                   
               
               
                   
                 KGLEEEGPPCTPKPPGRARQAGPRVPFTIFEEVHP 
               
               
                   
               
               
                   
                 TKSKLQVPLAPRVHRRAQTRLKVIFSDDSDLEDLV 
               
               
                   
               
               
                   
                 SADTQLVEEPKRRGTASRTRGQTRKGRSLKTDAVV 
               
               
                   
               
               
                   
                 AIESTPGHSSVSGRTRRARKVASRNCEEESPKAPL 
               
               
                   
               
               
                   
                 CVWASQGPEIMRSIPEEEPVDNHLEKSFEILRGSD 
               
               
                   
               
               
                   
                 GEDSASGEKAAAADTGLPVGECEVLRRDSSKAERP 
               
               
                   
               
               
                   
                 VLYSDTEANSDPSPWLPPFSVPAPIDLSTLDSISD 
               
               
                   
               
               
                   
                 SLSIAFRGVSHCPPSGLYAHLCRFLALCLGHRDPY 
               
               
                   
               
               
                   
                 ATAFLVAESISITCRHQLLTHLHRQLSKAQKQQES 
               
               
                   
               
               
                   
                 PELAEHLQRLDLKERPGGVPLARIQRLFSFKALGS 
               
               
                   
               
               
                   
                 GCFPQAEKESFQERLALIPSGVTVCVLALATLQPG 
               
               
                   
               
               
                   
                 TLSNTLLLTRLEKDNPPITVKIPTAQNKLPLSAVL 
               
               
                   
               
               
                   
                 KEFDAIQKDQKENSSCTEKRVWWTGRLALDQRMEA 
               
               
                   
               
               
                   
                 LITALEEQVLGCWRGLLLPCSADPSLAQEASKLQE 
               
               
                   
               
               
                   
                 LLRECGWEYPDSTLLKVILSGARILTSQDVQALAC 
               
               
                   
               
               
                   
                 GLCPAQPDRAQVLLSEAVGQVQSQEAPRSQHLVLV 
               
               
                   
               
               
                   
                 LDKDLQKLPWESTPILQAQPVTRLPSFRFLLSYTV 
               
               
                   
               
               
                   
                 TKEAGASSVLSQGVDPQNTFYVLNPHSNLSSTEER 
               
               
                   
               
               
                   
                 FRASFSSETGWKGVIGEVPSLDQVQAALTERDLYI 
               
               
                   
               
               
                   
                 YAGHGAGARFLDGQAVLRLSCRAVALLFGCSSAAL 
               
               
                   
               
               
                   
                 AVHGNLEGAGIVLKYIMAGCPLFLGNLWDVTDRDI 
               
               
                   
               
               
                   
                 DRYTEALLQGWLGAGPGAPFLYYASQARQAPRLKY 
               
               
                   
               
               
                   
                 LIGAAPVAYGLPISLQTP 
               
               
                   
               
               
                   
                 Nematode 
               
               
                   
                 ( Caenorhabditis   elegans ) separase 
               
               
                   
                 SEQ ID NO: 8 
               
               
                   
                 MKITNKSVDKQHIEKLDELRKNVSCTVIGFAEQTA 
               
               
                   
               
               
                   
                 ELQQEISELFIAEFGVNGPIDMNSLSKLARITSYY 
               
               
                   
               
               
                   
                 ASSEYFQGLAKYQRTACKMFITWQT 
               
               
                   
               
               
                   
                 LRKEAMECRSKDREIFASIPAKLCFFYFYNGELCR 
               
               
                   
               
               
                   
                 AVVCLLDYIDLSDDTLAKEAALRWLMFLGETELIE 
               
               
                   
               
               
                   
                 KKLKTWKMDKSSKDMFSATEFAMNYLKKSEYRVEM 
               
               
                   
               
               
                   
                 LEKLMKLRDKVKSDPTRSFSRYELASYVSWLCSTL 
               
               
                   
               
               
                   
                 SNVPVGSALRECEFPDRVSHIQEAALKSDSLVRNR 
               
               
                   
               
               
                   
                 IPGLASSQFDNSVNASIWPFLDGHQEDSNYYVHIG 
               
               
                   
               
               
                   
                 STIAWHFEMRRECALVNVTTAQTRDSMSAMILNLR 
               
               
                   
               
               
                   
                 VALKSASFFRVLQTTNTLAYYSSIIEEAGSEKNAK 
               
               
                   
               
               
                   
                 LMRVSCVNLLSSNPIIVRCSTPKETGATSRAHTPM 
               
               
                   
               
               
                   
                 AGSSVSEKQNTMRPDLADLLGDLELLDEQSFHPIT 
               
               
                   
               
               
                   
                 RSCVCNVCTIYPLHSSFAAEYMMSYAIHSDFSQLS 
               
               
                   
               
               
                   
                 IKHFNDEFARIRERGMSSQVLMHRDSSVRPRPNII 
               
               
                   
               
               
                   
                 QNEIFGMCVIRWLTKKLDSKESADEDTMEIFNNAL 
               
               
                   
               
               
                   
                 KIVRYLQQRTTDMILAVTQLGRQLEFPMECNYSWM 
               
               
                   
               
               
                   
                 RPTIRKPRVKATIDCAVDILRAVSPFGRRPKVEKL 
               
               
                   
               
               
                   
                 EKNLQPFDKERFEKVRLAMRNEMNHYGHILYREWR 
               
               
                   
               
               
                   
                 CRLFAYVGRTSRDPWEAAYAWAESTQIGARNAVQS 
               
               
                   
               
               
                   
                 RLEKCKRGLVTMSGHDRFKTCVQSMPDEMTLVQIA 
               
               
                   
               
               
                   
                 MADDKTIYLVKLHADRDPIIMPLAHYSQAVELMDK 
               
               
                   
               
               
                   
                 FTFLLDEDEMIAKYPGDITPEEFWKRRKIVDGRMM 
               
               
                   
               
               
                   
                 TFVDEVQKHFLGVAASLLMPSGQLGPKAAELAIKI 
               
               
                   
               
               
                   
                 HKLSKGGLLLGEAKEMVYQSKLMDAKSWEALILRF 
               
               
                   
               
               
                   
                 CEMRTTDEKFKSFLPLMHRNSVEVMNQDDSIVTEK 
               
               
                   
               
               
                   
                 KYTYLVICPHLSQFCWERLPIFDEYPYVGRQVSIH 
               
               
                   
               
               
                   
                 STFSQLEAMKSQEKQIPLQIDVQNAYYILDPDNNL 
               
               
                   
               
               
                   
                 GETQKRMVEYINKFNWEGTVGSAPKSNEISAALSQ 
               
               
                   
               
               
                   
                 RDAFFFIGHGSGSSVMPRSVLKQSTCNAISLLMGC 
               
               
                   
               
               
                   
                 GSVRTIPQALGFDGKTAILDYAMAKCPLIVGCLWT 
               
               
                   
               
               
                   
                 VTDGEIDRFLIRMIDDCFEDSKSLTGIDKLRQLSE 
               
               
                   
               
               
                   
                 AMHEARSKARLKYLTGAAVVMYGLPVVAKQTTPFV 
               
               
                   
               
               
                   
                 EKDQRNLPQTPKTSARTSMRMETVPKTPKQEFVTS 
               
               
                   
               
               
                   
                 KSVPMTPIFSNNENKSPSRARMPSRVLKTPRQVKT 
               
               
                   
               
               
                   
                 FQEEDDEAPKRSTTRQLKPLVAPPIPATPTTRTTR 
               
               
                   
               
               
                   
                 SSARTPSRSRNL 
               
               
                   
               
               
                   
                 Budding yeast 
               
               
                   
                 ( Saccharomyces   cerevisiae ) separase 
               
               
                   
                 SEQ ID NO: 9 
               
               
                   
                 MMVKQEEPLNEISPNTPMTSKSYLLNDTLSKVHHS 
               
               
                   
               
               
                   
                 GQTRPLTSVLSGDASSNSIGILAMHNNIIRDFTKI 
               
               
                   
               
               
                   
                 ASNNIDLAIEDITTVDHSLNSIYSLLKSHHMWGHI 
               
               
                   
               
               
                   
                 NSTVKQHLMIIVKLINNNALGLASSEIIFLFNETN 
               
               
                   
               
               
                   
                 LFQAHSLKNILLADFSTWNDYYLSNLKILALQIIL 
               
               
                   
               
               
                   
                 KRKLVDEYLPHILELFSHDKRYLLKDPNLKAHALT 
               
               
                   
               
               
                   
                 KIVLSFFSVTTSCKVLFGLKFLQYIKQFKLPFKKF 
               
               
                   
               
               
                   
                 ISNITVECFSKNLLHKNYLEMGPNKIYLNSFYLSY 
               
               
                   
               
               
                   
                 SMLYDGLDKIMLLDILSYEETTEVQRAIKSKKEFN 
               
               
                   
               
               
                   
                 EYCNMSENRLLWSCISVDDLNVILENATNFLQNKG 
               
               
                   
               
               
                   
                 KHISATLKCLVCLWSTIRLEGLPKNKDILRQFDCT 
               
               
                   
               
               
                   
                 VIYINSNIKSINDESAAALLSELLGVLSEICIDYK 
               
               
                   
               
               
                   
                 EPKRLSNIISVLFNASVLFKSHSFLLKTANLEISN 
               
               
                   
               
               
                   
                 VLISNDSKTSHRTILKFEKFISSAQSAQKKIEIFS 
               
               
                   
               
               
                   
                 CLFNVYCMLRNDTLSFVFDFCQNAFIHCFTRLKIT 
               
               
                   
               
               
                   
                 KFIEFSNSSEIMLSVLYGNSSIENIPSENWSQLSR 
               
               
                   
               
               
                   
                 MIFCSLRGIFDLDPLELNNTFDKLHLLNKYELLIR 
               
               
                   
               
               
                   
                 IVYLLNLDMSKHLTTNLSKITKLYINKWLQKSDEK 
               
               
                   
               
               
                   
                 AERISSFEMDFVKMLLCYLNFNNFDKLSIELSLCI 
               
               
                   
               
               
                   
                 KSKEKYYSSIVPYADNYLLEAYLSLYMIDDALMMK 
               
               
                   
               
               
                   
                 NQLQKTMNLSTAKIEQALLHASSLINVHLWDSDLT 
               
               
                   
               
               
                   
                 AFQIYFGKTLPAMKPELFDINNDHNLPMSLYIKVI 
               
               
                   
               
               
                   
                 LLNIKIFNESAKLNIKAGNVISAVIDCRKAQNLAL 
               
               
                   
               
               
                   
                 SLLKKKNKLSQGSRLALLKSLSFSFFQLIKIHIRI 
               
               
                   
               
               
                   
                 GSARDCEFYSKELSRIISDLEEPIIVYRCLHFLHR 
               
               
                   
               
               
                   
                 YYMITEQTCLQNITLGKANKAFDYLDAEADITSLT 
               
               
                   
               
               
                   
                 MFLYDNKEFVKLEQSLVLYFGDQLEKTFLPNLWKL 
               
               
                   
               
               
                   
                 HLGKDIDDSICLSEYMPKNVINRVHNMWQKVMSQL 
               
               
                   
               
               
                   
                 EEDPFFKGMFESTLGIPSSLPVIPSTMPNNILKTP 
               
               
                   
               
               
                   
                 SKHSTGLKLCDSPRSSSMTPRGKNIRQKFDRIAAI 
               
               
                   
               
               
                   
                 SKLKQMKELLESLKLDTLDNHELSKISSLSSLTLT 
               
               
                   
               
               
                   
                 ILSNITSIHNAESSLITNFSLTDLPRHMPLLFDKV 
               
               
                   
               
               
                   
                 LNNIDNKNYREFRVSSLIAPNNISTITESIRVSAA 
               
               
                   
               
               
                   
                 QKDLMESNLNINVITIDFCPITGNLLLSKLEPRRK 
               
               
                   
               
               
                   
                 RRTHLRLPLIRSNSRDLDEVHLSFPEATKKLLSII 
               
               
                   
               
               
                   
                 NESNQTTSVEVTNKIKTREERKSWWTTRYDLDKRM 
               
               
                   
               
               
                   
                 QQLLNNIENSWFNGVQGFFSPEVVDNSLFEKFKDK 
               
               
                   
               
               
                   
                 FYEILHQNLPSRKLYGNPAMFIKVEDWVIELFLKL 
               
               
                   
               
               
                   
                 NPQEIDFLSKMEDLIYFVLDILLFHGEENAYDEID 
               
               
                   
               
               
                   
                 FSMLHVQLEEQIKKYRATMTTNSIFHTFLVVSSSC 
               
               
                   
               
               
                   
                 HLFPWECLSFLKDLSITRVPSYVCLNKLLSRFHYQ 
               
               
                   
               
               
                   
                 LPLQVTIEDNISMILNPNGDLSRTESKFKGMFQKI 
               
               
                   
               
               
                   
                 IDAKPSSQLVMNEKPEEETLLKMLQNSNLFVYIGH 
               
               
                   
               
               
                   
                 GGGEQYVRSKEIKKCTKIAPSFLLGCSSAAMKYYG 
               
               
                   
               
               
                   
                 KLEPTGTIYTYLLGGCPMVLGNLWDVTDKDIDKFS 
               
               
                   
               
               
                   
                 EELFEKMGFRCNTDDLNGNSLSVSYAVSKSRGVCH 
               
               
                   
               
               
                   
                 LRYLNGAAPVIYGLPIKFVS 
               
               
                   
               
               
                   
                 Fission yeast 
               
               
                   
                 ( Schizosaccharomyces   pombe ) separase 
               
               
                   
                 SEQ ID NO: 10 
               
               
                   
                 MSTRSIVTSKVSWTPEKFISALSYPEHCSITLVKR 
               
               
                   
               
               
                   
                 LKASVKLKDLKQNISRDAPSWTFEHLFVAFKCAVS 
               
               
                   
               
               
                   
                 NLAKQWAELSTTDKEKTRRMFCTPSRLNTAHRPEV 
               
               
                   
               
               
                   
                 FYLLECCTYILEQMQVVTKNTSHLYDCIRSGVSIC 
               
               
                   
               
               
                   
                 NRLLDMEIFEPAISLLMKTHKNLIILLTYRDHDAI 
               
               
                   
               
               
                   
                 PTATLLNPTLDVSEIQLESCLFVPMVPASYFLNIG 
               
               
                   
               
               
                   
                 TIVVTFQLNVLRCLSLSQINGLSLNTINNLQSEDG 
               
               
                   
               
               
                   
                 PFQWIERSFPSQVQLANSRREILARLLTRFSMIQN 
               
               
                   
               
               
                   
                 NALQSFKLLILSIALWLNILSSQRADDKEFDVNQL 
               
               
                   
               
               
                   
                 ETRILQLFSKVVQLCKSEDIEGSILNKDMTQLHHL 
               
               
                   
               
               
                   
                 LENLSKESRLHILLQLSQLYYKYNDFQLSAAYVIR 
               
               
                   
               
               
                   
                 GYSLSFEDISFKLKFLLFSFRLSIRONSICFPFNL 
               
               
                   
               
               
                   
                 IQELSSLQQLFVENALPYSEALHLLDSIERSFRLF 
               
               
                   
               
               
                   
                 NDSTVFDDTVFALNISEILSWILSSVVRDILVEDE 
               
               
                   
               
               
                   
                 LLNLQLKIRKFLMFTFHIIRSFSELTKFQSSLEGC 
               
               
                   
               
               
                   
                 LNLAAYYEDAEFPQKLSNHLYNLCVKSSNVNYARE 
               
               
                   
               
               
                   
                 CISLSIKIAVSHKLTNDETYLLKILKNFQLRYHDS 
               
               
                   
               
               
                   
                 LQLQEKCDVLHTTFNQLDLYVGTTSVGKSSVLDNI 
               
               
                   
               
               
                   
                 LKRIFNSLTSINDSNIEKLLESISYSLLKLFFKCA 
               
               
                   
               
               
                   
                 NEGSRYNASAALSFKLSLMLHEKEEVLLLKTNVSC 
               
               
                   
               
               
                   
                 VLANHGYNDIKFEEMVLCVIKGDQNLLEHNSNNNA 
               
               
                   
               
               
                   
                 KLALNESLLCSWENLLCYRRAEDDSRILTIIESWT 
               
               
                   
               
               
                   
                 IFISRFSSVISRCSFTDFEINSILNFFFCFLHTVE 
               
               
                   
               
               
                   
                 PSGKLTFELAFLEIFYELFNCLLHLQFSKYLVIIG 
               
               
                   
               
               
                   
                 TLLSDKYMTLGFSGKAHLFYTKCYSYLRQCKSSPF 
               
               
                   
               
               
                   
                 INFWNVSYGKYLILTGNTDKGILQLKKYSLSSEED 
               
               
                   
               
               
                   
                 FNSNGLSRTVSLNLLLYERIQLSDALFQLGYTTVS 
               
               
                   
               
               
                   
                 LGFIMQNLKVIKGLFSKSSKEHFNGGKYITWRLFA 
               
               
                   
               
               
                   
                 VSAHSNVCAARIYEHMGQAREAEFFYRQACSISEK 
               
               
                   
               
               
                   
                 MPFSCFSATFQLRLCSLLTRAGKLEKGEKILFDLT 
               
               
                   
               
               
                   
                 EAMKSTDTYHKLLWNYGAAEVCATKSELDGAICHY 
               
               
                   
               
               
                   
                 SECVKLLEIIKSEYYLFFNRNREKSLTKGIKRLSL 
               
               
                   
               
               
                   
                 SSQPTFVTESNTTEFDDWSILQNTAANLLRLISMF 
               
               
                   
               
               
                   
                 ELKRGNLEIAKALMTDSTKCSIASFFNIVSANILK 
               
               
                   
               
               
                   
                 SKLIVCEADSTLFGDPVLRTLPDSVISLPGISHKF 
               
               
                   
               
               
                   
                 QKNQSKTKALGENTGFRKGSKRLDYLRERLKINLQ 
               
               
                   
               
               
                   
                 NVRLSCEIIFSNAYERSSVCVCREVNELISYSTIM 
               
               
                   
               
               
                   
                 QSALTTIGETTDVDSSSASFFLEIPKALGFHRRRE 
               
               
                   
               
               
                   
                 AQKFRNQHKELHFSSLEQILNSRLSIPDVRTFQDN 
               
               
                   
               
               
                   
                 FIDSLPSIWNVVSITINNSGEDLFISKIRKGHSPL 
               
               
                   
               
               
                   
                 IFRLPLQRHNSRDADEEILVFTKAQTELFRIISKS 
               
               
                   
               
               
                   
                 NQMAQNGKHYTRREDKETWWKERRHLDQCLQQLLE 
               
               
                   
               
               
                   
                 NIEISWLGGFKGIFNPHKIDTSLFAKFSSQFQNII 
               
               
                   
               
               
                   
                 AKNFNMDKKTPVPTLSPEILELFITLGKPGYEGYE 
               
               
                   
               
               
                   
                 QLLEDLIYFILDIFQFRGLHFAYDEIDTDQLSMDL 
               
               
                   
               
               
                   
                 QDALNAYFNNYVSEENRSHTVLVLDKSVHQFPWES 
               
               
                   
               
               
                   
                 LPCLNRQSVSRVPSLSILRDILSQSFVVNGEYVEV 
               
               
                   
               
               
                   
                 RKEAGSYILNPSLDLKHTQEMFEHKLVEGGWKGLI 
               
               
                   
               
               
                   
                 ASQPSNRDFIKMLSGNDFFLYFGHGGGEQYTTSYD 
               
               
                   
               
               
                   
                 LATLKRCAVTILMGCSSGALYECGSFEPWGTPLDY 
               
               
                   
               
               
                   
                 LSAGCPTLVANLWDVTDKDIDRFSLKMLESWGLFE 
               
               
                   
               
               
                   
                 NKAPFVNSTSICTAVSESRSCCHLRYLNGAAPVIY 
               
               
                   
               
               
                   
                 GIPAYIIP 
               
               
                   
               
               
                   
                 Mouse ( Mus   musculus ) securin 
               
               
                   
                 SEQ ID NO: 11 
               
               
                   
                 MATLIFVDKDNEEPGRRLASKDGLKLGTGVKALDG 
               
               
                   
               
               
                   
                 KLQVSTPRVGKVFNAPAVPKASRKALGTVNRVAEK 
               
               
                   
               
               
                   
                 PMKTGKPLQPKQPTLTGKKITEKSTKTQSSVPAPD 
               
               
                   
               
               
                   
                 DAYPEIEKFFPFNPLDFESFDLPEEHQISLLPLNG 
               
               
                   
               
               
                   
                 VPLMTLNEERGLEKLLHLGPPSPLKTPFLSWESDP 
               
               
                   
               
               
                   
                 LYSPPSALSTLDVELPPVCYDADI 
               
               
                   
               
               
                   
                 Nematode ( Caenorhabditis   elegans ) 
               
               
                   
                 securin 
               
               
                   
                 SEQ ID NO: 12 
               
               
                   
                 MEDLNFEERGSTQIPASLQQHFSAKLGRQNELEKT 
               
               
                   
               
               
                   
                 PSRGGLGLVVNSSKTPGGKSLQSLASACKVPPSTK 
               
               
                   
               
               
                   
                 KNTIPIAFECYEDETDDQIADVATIKKTEKHPCSP 
               
               
                   
               
               
                   
                 IDTANRCETFDSLAADIEDDMLNLEDQDVVLSEDR 
               
               
                   
               
               
                   
                 PYGDVIDPAESEAEALAELGVEEWDSYPPIDPASR 
               
               
                   
               
               
                   
                 IGDDFNYVLRTEDFAEEGDVKLEETRHRTVIADID 
               
               
                   
               
               
                   
                 EVKMSKAERNELFSMLADDLDSYDLLAEEANLPL 
               
               
                   
               
               
                   
                 Budding yeast 
               
               
                   
                 ( Saccharomyces   cerevisiae ) securin 
               
               
                   
                 SEQ ID NO: 13 
               
               
                   
                 MMPANEDKENNIVYTGNESSGINFPQTPAHLLKRS 
               
               
                   
               
               
                   
                 HSNILKPPVRLDQLKRDANSNNGNTLKYIQGGKEV 
               
               
                   
               
               
                   
                 SPTKRLHTHAQQQGRLPLAAKDNNRSKSFIFPETS 
               
               
                   
               
               
                   
                 NQSKDADLPQLQNTLSIRKNDQLRKLSQISRSRSR 
               
               
                   
               
               
                   
                 ANHNDLLSNSRKLQKYGSVLGYNALPKMKSLVLKD 
               
               
                   
               
               
                   
                 LADSGKNEESSDDDEGNEDSESKLGKKLQSALLKQ 
               
               
                   
               
               
                   
                 DSSDGENELNGGLGLFNEQGGLQQLIKNSTKNEQK 
               
               
                   
               
               
                   
                 TKNDKSDKTDDYDIEIAPQRQEPLPYVPEGYSPFQ 
               
               
                   
               
               
                   
                 QDDIEKLKTFNSPYKLDLEDEDDTPDKVDLLPLEQ 
               
               
                   
               
               
                   
                 IDEEGEKDETECITRNQEEGAALPLLSKNFKEVAA 
               
               
                   
               
               
                   
                 VPTMELVYSEEGLDPEELEDLVT 
               
               
                   
               
               
                   
                 Fission yeast 
               
               
                   
                 ( Schizosaccharomyces   pombe ) securin 
               
               
                   
                 SEQ ID NO: 14 
               
               
                   
                 MLPRTMFSYGKENAFPVTPISNRNGTKGAGSKRAP 
               
               
                   
               
               
                   
                 LGSTKQSNAPSSVTVPRTVLGGKSTNISKFISAPS 
               
               
                   
               
               
                   
                 TKKMSPMDISMDSPTILEPNSQGISRSAVQERSKR 
               
               
                   
               
               
                   
                 LSASPRRSSLTDTPLPNELEEDIEYMPPPVHLDPI 
               
               
                   
               
               
                   
                 QSLGFDDVAIDCETLDPWPSMQNKATSVTIRNTPA 
               
               
                   
               
               
                   
                 SDFHVYKEFSDDDPIQFPLLSVDGDSPLTEKDTNL 
               
               
                   
               
               
                   
                 TTPATLKASDQQRKVLEKPSVSKQSSSRTRLSTVY 
               
               
                   
               
               
                   
                 RTKLASGKSIPRPLSHKLTRPRVTASGNSRRRPLS 
               
               
                   
               
               
                   
                 RSIHSLSSSRIDFSSLDTGLL 
               
               
                   
               
               
                   
                 SEQ ID NO: 15 
               
               
                   
                 GGGGS 
               
               
                   
               
               
                   
                 SEQ ID NO: 16 
               
               
                   
                 GGGGSGGGGS 
               
               
                   
               
               
                   
                 SEQ ID NO: 17 
               
               
                   
                 GGGGSGGGGSGGGGS 
               
               
                   
               
               
                   
                 SEQ ID NO: 18 
               
               
                   
                 GGGGSGGGGSGGGGSGGGGS 
               
               
                   
               
               
                   
                 SEQ ID NO: 19 
               
               
                   
                 GGGGGG 
               
               
                   
               
               
                   
                 SEQ ID NO: 20 
               
               
                   
                 GGGGGGGG 
               
               
                   
               
               
                   
                 SEQ ID NO: 21 
               
               
                   
                 GGSGGSGGGSGGGSG 
               
               
                   
               
               
                   
                 SEQ ID NO: 22 
               
               
                   
                 HHHHHH 
               
               
                   
               
               
                   
                 SEQ ID NO: 23 
               
               
                   
                 DYKDDDDK 
               
               
                   
               
               
                   
                 SEQ ID NO: 24 
               
               
                   
                 TNTAKILNFGR 
               
               
                   
               
               
                   
                 SEQ ID NO: 25 
               
               
                   
                 DDREIMREGS