# Patent

## Title
Chimeric autoprocessing polypeptides and uses thereof

## Abstract

A chimeric polypeptide comprising an autoprocessing segment having an amino acid sequence being capable of auto-cleavage, a polynucleotide encoding such a polypeptide, and uses of such a polypeptide and such a polynucleotide are provided.

## Background
RELATED PATENT APPLICATION
This application is a National Phase Application of PCT/IL03/00956 having International Filing Date of 12 Nov. 2003, which benefits of U.S. Provisional Patent Application No. 60/425,295 filed 12 Nov. 2002.


FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to polypeptides having the capacity to display auto-cleavage, polynucleotides encoding such polypeptides, and uses of such polypeptides and polynucleotides for reversibly binding proteins to specific substrates, reversibly binding specific substrates to each other, and for splicing amino acid sequences. More particularly, the present invention relates to chimeric polypeptides capable of auto-cleaving at defined locations, including auto-cleaving resulting in defined auto-splicing, to polynucleotides suitable for expressing such polypeptides, and to methods of using such polypeptides and polynucleotides for protein purification, affinity selection of display phages, and post-translational ligation of proteins.
Autoprocessing protein domains, such as inteins and Hogs, have the capacity to post-translationally auto-cleave or auto-splice flanking polypeptide sequences and thereby serve as unique and potent protein engineering tools useful in various applications, including protein purification, affinity selection of display phages, generation of cytotoxic proteins, segmental modification or labeling of proteins, protein or peptide cyclization, and generation of reactive polypeptide termini in expressed proteins for various biochemical reactions, including protein ligation (Perler and Adam, 2000. Curr Opin Biotechnol. 11, 377-83). However, the usefulness of the presently available repertoire of autoprocessing polypeptides is hampered by various limitations, as described in further detail hereinbelow.
Inteins are internal protein domains naturally occurring in a variety of host proteins (Hirata et al., 1990. J. Biol. Chem. 265, 6726-6733; Kane et al., 1990. Science 250, 651-657; Perler et al., 1994. Nucl. Acids Res. 22, 1125-1127; Noren et al., 2000. Angew. Chem. Int. Ed. 39, 450). Inteins have been found in organisms from all three domains of life, including in yeast and algal chloroplasts (eukaryotes), mycobacteria and cyanobacteria (bacteria), and thermophilic archaea (archaea). So far, no essential biological role has been shown for inteins, and all of their identified functions involve their own preservation and maintenance, with no apparent benefit to the host protein and organism (reviewed in Pietrokovski, 2000. Trends in Genetics 17, 465-472). At least some inteins are multifunctional, being able to both catalyze their own protein splicing and to home a copy of their gene into intein-less alleles (Gimble and Thorner, 1992. Nature 357, 301; Chong et al., 1996. J Biol. Chem. 271, 22159). Hogs are protein domains found in Hedgehogs which are proteins composed of an amino terminal Hedge protein domain and a carboxy terminal Hog protein region (Aspock G., 1999. Genome Res. 9, 909; Hammerschmidt et al., 1997. Trends Genet. 13, 14). Other protein domains, such as various Caenorhabditis elegans carboxy terminal domains, are believed to autocatalytically cleave themselves from host proteins, thereby modulating the activity of the amino terminal parts (Burglin, 1996. Curr Biol. 6, 1047; Porter et al., 1996. Cell 86, 21), similarly to Hogs.
Members of the intein and Hog protein domain families share the capacity to autocatalytically cleave the peptide bond joining them to polypeptides flanking their amino terminal ends (“amino terminal cleavage”). Inteins have the further capacity to cleave the peptide bond joining them to polypeptides flanking their carboxy terminal ends (“carboxy terminal cleavage”) while splicing polypeptides flanking their amino and carboxy terminal ends (termed “exteins”), resulting in self-excision of the intein from the host protein, and concomitant ligation of the flanking extein domains with a peptide bond. Thus, intein-containing host proteins undergo a switch from an intein-containing state to an intein-less state via such a process. Most reported inteins furthermore also contain an endonuclease domain whose function is to mediate the copying of the intein gene into specific unoccupied genomic insertion points, thereby enabling intein propagation.
Both inteins and Hogs share a similar structure fold and contain characteristic “Hint” consensus motifs which mediate the biochemical reactions involved in the autocatalytic activities of these protein domains (Hall T M., 1997. Cell 91, 85; Pietrokovski S., 1994. Protein Sci. 3, 2340; Pietrokovski S., 1998. Protein Sci. 7, 64; Paulus, 2000. Annu. Rev. Biochem. 69, 447). These Hint motif-mediated biochemical reactions are similar in both inteins and Hogs, but are involved in different biological processes (Dalgaard et al., 1997. J Comput Biol. 4, 193; Hall et al., 1997. Cell 91, 85; Pietrokovski S., 1998. Protein Sci. 7, 64; Xu and Perler, 1996. EMBO J. 15, 5146). The initial biochemical reactions of intein and Hog amino terminal cleavage are identical; the peptide bond attaching the amino terminal end of the Hint domain to an amino terminal-flanking sequence is converted into a thioester (or ester) bond, a trans-esterification reaction then covalently attaches the sequence flanking the carboxy terminal end of the intein, or a cholesterol molecule in the case of Hog proteins, to the amino terminal flanking sequence, thereby cleaving the bond attaching the amino terminal sequence to the Hint domain. In a process essential for organismal development, Hint-mediated autocatalytic excision of the carboxy terminal Hog protein domain from the amino terminal Hedge protein domain in Hedgehogs leads to covalent attachment of a cholesterol molecule to the carboxy end of the Hedge domain, leading to its activation and secretion from the cell (Porter J A. et al., 1996. Cell 86, 21; Porter, J A. et al., 1996. Science 274, 255). In the case of inteins, protein splicing is effected sequentially by cleavage of the bond attaching the intein amino terminal end to the carboxy terminal extein, ligation of the amino and carboxy terminal exteins, and cleavage of the bond attaching the intein carboxy terminal end to the carboxy terminal extein.
Mechanistic studies have determined the roles of highly conserved residues positioned near the intein/extein junctions in the splicing reaction (Chong et al., 1996. J. Biol. Chem. 271, 22159-22168; Xu et al., 1996. EMBO J. 15, 5146-5153; Stoddard et al., 1998. Nat. Struct. Biol. 5, 3). These residues include: the Cys, Ser or Thr residue forming the amino terminal end of the intein, which initiates splicing with an acyl shift; the conserved Cys, Ser or Thr residue flanking the carboxy terminal end of the intein, which ligates the exteins through nucleophilic attack; and the conserved Asn forming the carboxy terminal end of the intein, which releases the intein from the ligated exteins via succinimide formation. The amino terminal acyl shift and the carboxy terminal succinimide formation cleavage activities of the intein are separable. The amino terminal cleavage takes place in two separate steps. In the first step, as described above, the peptide bond between the intein and the amino terminal extein is converted to a thioester (or ester in some cases). In the second step, the thioester bond is cleaved by a nucleophilic attack from the side-chain of the residue flanking the carboxy terminal end of the intein, causing a transesterification reaction.
Because the structural information required for splicing exists entirely within inteins, and since the process of splicing has no energy requirements (for example hydrolysis of ATP), such protein domains can be used in a variety of applications involving intein insertion into foreign contexts. Various methods have been used in attempts to control and alter intein-mediated functions. Since endonuclease activity is not required for protein splicing, mini-inteins with accurate splicing activity have been generated by deletion of this central domain (Derbyshire et al., 1997. Proc. Natl. Acad. Sci. USA. 94, 11466; Chong et al., 1997. J. Biol. Chem. 272, 15587; and Shingledecker et al., 1998. Gene 207, 187). Also, mutation of residues near the intein/extein junctions has been used to alter intein activity, for example, to yield isolated cleavage at one or both of the intein-extein junctions (Chong et al., 1998. J. Biol. Chem. 273, 10567).
Thus, the ability to modulate the function of autoprocessing polypeptides such as inteins has broad potential application, as described above. In the case of protein purification where an autoprocessing polypeptide is used in conjunction with an affinity group to purify a desired target protein (Chong et al., 1997. Gene 192, 271-281; Chong et al., 1998. Nucl. Acids Res. 26, 5109), purification of a target protein is effected by co-expressing the target protein as a fusion protein containing a purification tag in one terminal segment, an internal autoprocessing polypeptide, and a target protein forming the other terminal segment. Such fusion proteins are exposed to affinity purification matrices designed to capture the tagged molecule. The target protein is then selectively released from the purification matrix by inducing autoprocessing polypeptide-mediated auto-cleavage of the peptide bond attaching the target protein to the autoprocessing polypeptide. Such a procedure is advantageous since autoprocessing polypeptide cleavage affects the fusion protein only, and thus non-specifically bound contaminant proteins are not released into the product stream. Furthermore, such a method does not employ contaminating and expensive proteases, such as those used in technologies employing protease-mediated cleavage of purification-tagged target proteins. The aforementioned strategy forms the basis of the protein purification systems such as the commercially available IMPACT-CN system (New England Biolabs, Beverly, Mass.).
However, prior art methods of using such autoprocessing polypeptides for applied uses have numerous drawbacks. In applied systems such as IMPACT-CN, the accessory molecule involved in cleavage of the thioester bond between the intein and the extein following amino terminal cleavage must be effected with a strong thiol-containing nucleophile such as 2-mercaptoethanol or dithiothreitol (DTT), both of which are strong reducing agents which modify the carboxy terminal end of the extein. In such systems, although initial thioester formation is mediated by the intein, the actual cleavage of the extein is effected via non-enzymatic chemical cleavage of a thioester bond by a small nucleophilic molecule, thereby severely limiting the maximal reaction rates achievable. While such systems allow carboxy terminal cleavage, such cleavage has the drawback of resulting in undesirable amino terminal cleavage, thereby requiring the amino terminal fragment to be removed in an additional purification step. Furthermore, despite insights into intein structure and function, modifications often result in unacceptably low activity, poor precursor stability, or insolubility (Derbyshire et al., 1997. Proc. Natl. Acad. Sci. USA. 94, 11466; Chong et al., 1997. Gene 192, 271-281; Shingledecker et al., 1998. Gene 207, 187; Chong et al., 1998. Nucl. Acids Res. 26, 5109).
Thus, all prior art approaches have failed to provide an adequate solution for providing autoprocessing polypeptides optimal for protein engineering applications.
There is thus a widely recognized need for, and it would be highly advantageous to have, autoprocessing polypeptides devoid of the above limitation.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided an chimeric polypeptide comprising an autoprocessing segment having an amino acid sequence set forth by SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106, the polypeptide being capable of auto-cleavage.
According to another aspect of the present invention there is provided a polynucleotide encoding a chimeric polypeptide comprising an autoprocessing segment having an amino acid sequence set forth by SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106, the polypeptide being capable of auto-cleavage.
According to further features in preferred embodiments of the invention described below, the chimeric polypeptide further comprises an affinity tag capable of specifically binding a substrate.
According to still further features in preferred embodiments, the substrate is selected from the group consisting of a molecule, a compound, a virus, and a cell.
According to yet another aspect of the present invention there is provided a nucleic acid construct comprising a nucleic acid sequence encoding a chimeric polypeptide comprising an autoprocessing segment having an amino acid sequence set forth by SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106, the chimeric polypeptide being capable of auto-cleavage.
According to further features in preferred embodiments of the invention described below, the nucleic acid construct further comprises a promoter sequence being for directing expression of the chimeric polypeptide in an expression system.
According to still further features in preferred embodiments, the chimeric polypeptide further comprises an affinity tag capable of specifically binding a specific substrate.
According to still another aspect of the present invention there is provided a method of generating a chimeric polypeptide capable of displaying auto-cleavage, the method comprising generating a chimeric amino acid sequence including an autoprocessing segment, the autoprocessing segment having an amino acid sequence set forth by SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106, thereby producing the chimeric polypeptide capable of displaying auto-cleavage.
According to further features in preferred embodiments of the invention described below, the chimeric polypeptide includes an affinity tag.
According to a further aspect of the present invention there is provided a method of purifying a protein, the method comprising: (a) generating a chimeric polypeptide including an autoprocessing segment having an amino acid sequence set forth by SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106, the autoprocessing segment being terminally attached to, or flanked by, an amino acid sequence of the protein, the chimeric polypeptide being capable of auto-cleavage when subjected to suitable conditions to thereby remove the amino acid sequence of the protein from the chimeric polypeptide thereby generating the protein; (b) immobilizing the chimeric polypeptide to a support; and (c) subjecting the chimeric polypeptide to the suitable conditions, thereby purifying the protein.
According to further features in preferred embodiments of the invention described below, the method of purifying a protein further comprises the step of separating the protein from the autoprocessing segment following step (c).
According to still further features in preferred embodiments, the support includes an antibody or antibody fragment capable of specifically binding the autoprocessing segment, and the immobilizing is via the autoprocessing segment.
According to still further features in preferred embodiments, the chimeric polypeptide further includes an affinity tag sequence, and the immobilizing is via the affinity tag sequence.
According to still further features in preferred embodiments, the support includes a specific ligand of the affinity tag sequence, and the immobilizing is via the specific ligand of the affinity tag sequence.
According to still further features in preferred embodiments, the generating the chimeric polypeptide is effected by synthesizing a polynucleotide encoding the chimeric polypeptide and expressing the polynucleotide in an expression system.
According to still further features in preferred embodiments, the expression system is a cellular expression system or a cell-free expression system.
According to still further features in preferred embodiments, the cellular expression system is an E. coli cellular expression system.
According to still further features in preferred embodiments, the cell-free expression system is an E. coli S30 extract expression system.
According to still further features in preferred embodiments, the polynucleotide comprises a promoter sequence being for directing the expression of the chimeric polypeptide.
According to still further features in preferred embodiments, the promoter sequence is inducible by isopropyl beta-D-thiogalactoside.
According to still further features in preferred embodiments, the auto-cleavage results in auto-splicing.
According to still further features in preferred embodiments, the auto-splicing is auto-splicing of segments of the chimeric polypeptide flanking the autoprocessing segment.
According to a further aspect of the present invention there is provided a method of reversibly attaching a first substrate to a second substrate, the method comprising: (a) providing a chimeric polypeptide including an autoprocessing segment having an amino acid sequence set forth by SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106 flanked by a first amino acid sequence capable of binding the first substrate and a second amino acid sequence capable of binding the second substrate, the chimeric polypeptide being capable of auto-cleavage when subjected to suitable conditions, to thereby release the first amino acid sequence from the second amino acid sequence; (b) exposing the first substrate and the second substrate to the chimeric polypeptide, thereby generating a complex including the first substrate attached via the chimeric polypeptide to the second substrate; and (c) subjecting the complex to the suitable conditions, thereby detaching the first substrate from the second substrate.
According to further features in preferred embodiments of the invention described below, each of the first and second substrates is independently selected from the group consisting of a molecule, a compound, a virus, and a cell.
According to still further features in preferred embodiments, the molecule is amylose or chitin.
According to still further features in preferred embodiments, the virus is a bacteriophage.
According to still further features in preferred embodiments, the chimeric polypeptide includes an affinity tag sequence, and the binding the first substrate or the binding the second substrate is via the affinity tag sequence.
According to still further features in preferred embodiments, the affinity tag sequence is a maltose-binding domain or a chitin-binding domain.
According to still further features in preferred embodiments, the autoprocessing segment is selected from the group consisting of BIL1_cloth, BIL2_cloth, BIL3_cloth, BIL4_cloth, BIL5_cloth, BIL6_cloth, BIL7_cloth, BIL9_cloth, BIL10_cloth, BIL11_cloth, 3875—87_magma, FhaB_manha, BIL2_neigo, BIL3_neigo, BIL5_neigo, BIL6_neigo, MafB1_neigo, MafB2_neigo, B0369+_neimeB, B0372+_neimeB, B0655+_neimeB, A2115_neime, BIL2_neimeC, BIL3_neimeC, BIL4_neimeC, BIL5_neimeC, BIL6_neimeC, MafB1_neimeC, FhaB1_psefl-PfO-1, FhaB1_psefl-SBW25, FhaB_psesy, SCP1.201_strco, 39—9_thefus, BIL1_gemob, BIL2_gemob, 0709_lepin, 3725_lepin, 3719_lepin, o665_myxxa, o1078_myxxa, o1070_myxxa, BIL1_strav, BIL2_strav, BIL3_strav, BIL1_pirsp, BIL1_chrvi, BIL1_glovi, BIL2_glovi, BIL3_glovi, BIL4_glovi, BIL5_glovi, BIL6_glovi, BIL7_glovi, o649_versp, o5687_versp, o3395_versp, II0519_brume, BIL2_magma, BIL3_magma, BIL4_magma, BIL5_magma, BIL6_magma, 06786_metex, 00126_rhoca, 00199_rhoca, 00459_rhoca, 00460_rhoca, 00746_rhoca, 00949_rhoca, 01216_rhoca, 01374_rhoca, 01523_rhoca, 01524_rhoca, 02710_rhoca, 03530_rhoca, 4825_rhosp, BIL2_rhosp, BIL1_silpo, BIL2_silpo, BIL3_silpo, BIL4_silpo, BIL5_silpo, BIL6_silpo, BIL7_silpo, BIL8_silpo, BIL9_silpo, BIL10_silpo, BIL11_silpo, BIL12_silpo, BIL13_silpo, BIL14_silpo, BIL15_silpo, BIL16_silpo, Bil1_rhile, BIL1_unknwn, and BIL2_unknwn.
According to still further features in preferred embodiments, the autoprocessing segment is derived from a protein of an organism belonging to a genus selected from the group consisting of Brucella, Clostridium, Magnetospirillum, Mannheimia, Methylobacterium, Neisseria, Pseudomonas, Rhodobacter, Silicibacter, Streptomyces, Thermobifida, Rhizobium, Chromobacterium, Myxococcus, Leptospira, Pirellula, Gemmata, Gloeobacter and Verrucomicrobium. 
According to still further features in preferred embodiments, the organism is selected from the group consisting of Rhodobacter capsulatus, Rhodobacter sphaeroides, Silicibacter pomeroyi, Brucella melitensis, Brucella suis, Magnetospirillum magnetotacticum, Methylobacterium extorquens, Rhizobium leguminosarum, Neisseria meningitidis, Neisseria meningitidis, Neisseria meningitidis, Neisseria gonorrhoeae, Chromobacterium violaceum, Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas fluorescens, Mannheimia haemolytica, Myxococcus xanthus, Leptospira interrogans, Streptomyces coelicolor, Streptomyces avermitilis, Thermobifida fusca, Clostridium thermocellum, Pirellula species 1, Gemmata obscuriglobus, Gloeobacter violaceus, and Verrucomicrobium spinosum. 
According to still further features in preferred embodiments, the auto-cleavage results in removal of a segment of the chimeric polypeptide adjacent to an amino terminal end or a carboxy terminal end of the autoprocessing segment.
According to still further features in preferred embodiments, the segment of the chimeric polypeptide adjacent to the autoprocessing segment is an amino terminal segment or a carboxy terminal segment of the chimeric polypeptide.
According to still further features in preferred embodiments, the segment of the chimeric polypeptide adjacent to the carboxy terminal end of the autoprocessing segment includes an amino acid residue comprising a nucleophilic group at an amino terminal end thereof.
According to still further features in preferred embodiments, the nucleophilic group is a hydroxyl group.
According to still further features in preferred embodiments, the amino acid residue is a threonine residue.
According to still further features in preferred embodiments, the segment of the chimeric polypeptide adjacent to the amino terminal end of the autoprocessing segment includes a serine amino acid residue at a carboxy terminal end thereof.
According to still further features in preferred embodiments, the chimeric polypeptide is capable of the auto-cleavage under a condition selected from the group consisting of a temperature selected from a range of 33° C. to 41° C., a pH selected from a range of pH 7.8 to pH 8.2, and a concentration of dithiothreitol selected from a range of 0.1 mM to 20 mM.
The present invention successfully addresses the shortcomings of the presently known configurations by providing novel chimeric polypeptides capable of defined auto-cleaving, including auto-cleaving resulting in defined auto-splicing, polynucleotides suitable for expressing such polypeptides, and methods of using such polypeptides and polynucleotides to purify proteins, affinity-select display phages, and post-translationally ligate proteins together.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
In the drawings:
FIG. 1a is a sequence alignment diagram of the amino acid sequences of various Type A BIL domains (SEQ ID NOs: 8-62). The names of the domains and the amino acid sequence coordinates of their amino terminal residues in their host proteins are indicated to the left of the sequence. Numbers indicated in parentheses indicate the amino acid residue length of an intervening amino acid sequence which may have any amino acid sequence. Dashed lines have been inserted to assist in visualizing the alignments.
FIG. 1b is a sequence alignment diagram of the amino acid sequences of various Type B BIL domains (SEQ ID NOs: 63-104). The names of the domains and the amino acid sequence coordinates of their amino terminal residues in their host proteins are indicated to the left of the sequence. Numbers indicated in parentheses indicate the amino acid residue length of an intervening amino acid sequence which may have any amino acid sequence. Dashed lines have been inserted to assist in visualizing the alignments.
FIG. 2a is a sequence diagram depicting an amino acid sequence motif (SEQ ID NO: 105) exclusively defining amino acid sequences of a subset of Type A BIL domains, including: 39—9_thefus, SCP1.201_strco, 3875—87_magma, B0372+_neimeB, B0655+_neimeB, A2115_neime, MafB1_neimeC, BIL2_neimeC, BIL4_neimeC, BIL5_neimeC, BIL6_neimeC, MafB1_neigo, BIL2_neigo, BIL3_neigo, MafB2_neigo, BIL5_neigo, BIL6_neigo, FhaB_psesy, FhaB_manha, FhaB1_psefl-PfO-1, FhaB1_psefl-SBW25, BIL6_cloth, BIL5_cloth, BIL2_cloth, BIL4_cloth, BIL1_cloth, BIL8_cloth, BIL9_cloth, BIL1_gemob, BIL2_gemob, 0709_lepin, 3725_lepin, o1078_myxxa, o1070_myxxa, BIL1_strav, BIL2_strav, BIL3_strav, BIL1_pirsp, BIL1_chrvi, o3395_versp, o5687_versp, o649_versp, BIL1_glovi, BIL2_glovi, BIL3_glovi, and BIL4_glovi. Amino acid residues are indicated in standard single-letter code. X—any amino acid; X(1-100)—any amino acid sequence composed of 1 to 100 amino acid residues.
FIG. 2b is a sequence diagram depicting an amino acid sequence motif (SEQ ID NO: 106) exclusively defining amino acid sequences of a subset of Type B BIL domains, including: 4825_rhosp, BIL2_rhosp, 00588_rhoca, 02710_rhoca, 01524_rhoca, 01523_rhoca, 00126_rhoca, 01216_rhoca, 00949_rhoca, 01374_rhoca, 00459_rhoca, 00460_rhoca, 00746_rhoca, 03530_rhoca, 00199_rhoca, BIL3_magma, BIL4_magma, BIL1_brusu, BIL1_unknwn, BIL2_unknwn, 06786_metex, BIL1_silpo, BIL2_silpo, BIL3_silpo, BIL4_silpo, BIL5_silpo, BIL6_silpo, BIL7_silpo, BIL8_silpo, BIL9_silpo, BIL10_silpo, BIL11_silpo, BIL12_silpo, BIL13_silpo, BIL14_silpo, BIL15_silpo, BIL16_silpo, BIL1_rhile, and II0519_brume. Amino acid residues are indicated in standard single-letter code. X—any amino acid; X(1-100)—any amino acid sequence composed of 1 to 100 amino acid residues.
FIGS. 3a-z are block-logo diagrams depicting conserved amino acids of Hint-like motifs of Type A BIL domains (FIGS. 3a-g) and Type B BIL domains (FIGS. 3h-o) and of homologous Hint motifs of Hog proteins (FIGS. 3p-t) and inteins (FIGS. 3u-z). Unique BIL domain motifs are underlined with hatched lines. Motifs are ordered left to right in the amino to carboxy terminal positions along the protein sequences. Similar motifs are vertically aligned. The motifs are shown as sequence logos in which the heights of amino acid letter designations are proportional to their degree of conservation in each position. Protein splicing active site residues of intein Hint domains are indicated by asterisks. Motifs were identified and are displayed as previously described (Pietrokovski S., 1998. Protein Sci. 7, 64). The BIL domain motifs shown include sequences described in FIGS. 1a-b and Tables 1-2. The intein and hedgehog Hint domain sequences were obtained from previously published sources (Aspock G., 1999. Genome Res. 9, 909; Pietrokovski S., 2001. Trends Genet. 17, 465). The amino acid residue position shown forming the carboxy terminal end of the motif depicted in FIG. 3z actually represents the amino acid residue forming the amino terminal end of the carboxy terminal extein. Only intein and hedgehog motifs common to Hint domains are shown.
FIGS. 4a-b are dendrograms depicting phylogenetic relationships of Type A and Type B BIL domains, respectively.
FIGS. 5a-b are autoradiographs depicting SDS-PAGE separation of in-vitro translated, [35S]-methionine-labeled, protein products of the MBP-BIL-CBD expression constructs pC2C-PsyBIL, and pC2C-RspBIL2 (FIGS. 5a and 5b, respectively, “MBC” lane). Translation of control constructs pC2C (“MC” lane) was used as a positive control and no DNA template (“-” lane) were used as a negative control. Molecular weights were estimated using translation products of pBESTluc as reference standards. In FIG. 5a, molecular weights (“mw” lane) were further estimated using unlabeled protein markers (dotted lines). In FIG. 5b [MBP-CBD]′ and [MBP]′ indicate splicing and amino terminal cleavage products derived from MBP-RspBIL2-CBD. These fragments migrated at molecular weights greater than those the corresponding control fragments due to their containing residual BIL2_rhosp-flanking amino acid residues as a result of the pC2C-RspBIL2 cloning scheme described under Materials and Methods. Expected molecular weights of identified fragments are indicated in parentheses. The expected molecular weights of MBP-PsyBIL-CBD and of its carboxy terminal cleavage product MBP-PsyBIL, and its MBP-CBD splicing product were 66.3 and 59.1, and 50.6 kDa, respectively.
FIG. 5c is a diagram of the amino acid sequence of FhaB_psesy (SEQ ID NO: 107) depicting the putative protein splicing motifs (underlined) and catalytic residues (double-underlined) responsible for auto-cleavage/-splicing activity by this Type A BIL domain.
FIGS. 6a-b are photographs depicting SDS-PAGE analysis of autocatalytically processed protein products overexpressed in-vivo in E. coli transformed with the MBP-PsyBIL-CBD (“MBC” lane) expression construct pC2C-PsyBIL. FIG. 6a depicts Coomassie Blue staining of chitin (lane 2) or amylose (lane 3) affinity column separated protein. Lane 4 is a control showing protein from E. coli transformed with pC2C to overexpress MBP-CBD (“MC” lane) chimeric protein. Fragments corresponding to the MBP-PsyBIL carboxy terminal auto-cleavage product and the MBP-CBD auto-splicing product of the chimera are indicated. FIG. 6b is an autoradiograph depicting Western immunoblotting analysis of separated protein following purification on chitin beads. Both chitin purified samples from E. coli transformed with pC2C-PsyBIL or control plasmid pC2C to overexpress MBP-PsyBIL-CBD or MBP-CBD chimeric proteins, respectively, were separated in duplicate lanes and blotted onto a single nitrocellulose membrane. The membrane was cut in half and each sample was reacted in duplicate with either anti-MBP (anti-M) or anti-CBD (anti-C) antibodies. Both anti-M and anti-C antibodies reacted with the protein band corresponding to the mass of the MBP-CBD product. Protein bands corresponding to MBP-PsyBIL and MBP products, that appear following purification on chitin beads result from non specific binding by excess amounts of overexpressed protein. Expected molecular weights of identified fragments are indicated in parentheses. The expected molecular weights of MBP-PsyBIL-CBD and of its carboxy terminal cleavage product MBP-PsyBIL, and its MBP-CBD splicing product were 66.3 and 59.1, and 50.6 kDa, respectively.
FIGS. 6c-d are data plots depicting MALDI mass spectra of MBP-CBD ligation product (FIG. 6c) and MBP-PsyBIL carboxy terminal cleavage product (FIG. 6d) electroeluted from SDS-PAGE gels. The expected molecular weights of MBP-PsyBIL-CBD and of its carboxy terminal cleavage product MBP-PsyBIL, and its MBP-CBD splicing product were 66.3 and 59.1, and 50.6 kDa, respectively.
FIG. 7 is an amino acid sequence diagram depicting positioning of peptide sequences identified by MALDI peptide mass mapping analysis within the amino acid sequence of the MBP-CBD splicing product (SEQ ID NO: 108). Twenty-five tryptic peptide masses (underlined) were assigned to the amino acid sequence of the MBP-CBD protein, corresponding to 49% coverage of the MBP-CBD sequence. Lettering in non-bold/italic font indicates the amino acid sequence of the MBP tag, and the amino acid sequence of the CBD tag (amino acids 394-461) is indicated in bold+italic font. The peptide corresponding to amino acids 388-396 contains the BIL domain splice site between amino acids Ser393 and Thr394.
FIG. 8 is an amino acid sequence diagram depicting MALDI peptide mapping of the 59.3 kDa MBP-PsyBIL carboxy terminal cleavage product (SEQ ID NO: 109). Underlined sequences correspond to peptides detected by MALDI. Lettering in non-bold/italic font indicates the amino acid sequence of the MBP tag and that in bold/italic font indicates the amino acid sequence of the PsyBIL domain. The carboxy terminal end of the protein, asparagine N541 represents the carboxy terminal of the PsyBIL domain. The expected molecular weights of MBP-PsyBIL-CBD and of its carboxy terminal cleavage product MBP-PsyBIL, and its MBP-CBD splicing product were 66.3 and 59.1, and 50.6 kDa, respectively.
FIG. 9 is a schematic diagram depicting functions of BIL domains. Hint domains are shown as dark gray horseshoes with their flanks as ovals. Proteins are depicted with amino termini positioned on the left.
FIGS. 10a-c are electrophoretic analyses depicting C-terminal auto-cleavage by MBP-RspBIL2a-CBD chimera. FIG. 10a is a photograph of a Coomassie blue stained electrophoretic separation of in-vivo expressed MBP-RspBIL2a-CBD chimera affinity purified on amylose depicting C-terminal cleavage activity (“MB” product). FIG. 10b is a photograph of a Western immunoblotting analysis depicting C-terminal cleavage activity (“MB” product) using anti-MBP antibodies (anti-M). FIG. 10c is an autoradiograph of an SDS-PAGE separation of the in-vitro translated, [35S]-methionine-labeled chimera depicting C-terminal cleavage activity (“MB” product). M-MBP specific fragment, MB-MBP-BIL specific fragment, MBC-intact chimera.
FIGS. 11a-b are electrophoretic analyses depicting N-terminal auto-cleavage by in-vivo expressed MBP-4825rhosp-CBD chimera. FIG. 11a is a photograph of a Coomassie blue stained electrophoretic separation of chimera protein products depicting N-terminal cleavage activity of protein products affinity purified on chitin (“BC” product) and affinity purified on amylose (“M” product). FIG. 11b is a photograph of a Western immunoblotting analysis depicting N-terminal cleavage activity using anti-CBD antibody as a probe (“BC” product) and anti-MBP antibody as a probe (“M” product). BC-BIL-CBD specific fragment, M-MBP specific fragment, MB-MBP-BIL specific fragment, MBC-intact chimera.
FIGS. 12a-c are electrophoretic analyses depicting auto-processing and by in-vivo expressed MBP-BIL4_cloth-CBD chimera. FIG. 12a is a photograph of a Western immunoblotting analysis of amylose (lane “A”) or chitin (lane “C”) purified protein products depicting auto-splicing activity using anti-CBD antibody as a probe (“MC” product). FIG. 12b is a photograph of a Western immunoblotting analysis of amylose (lane “A”) or chitin (lane “C”) purified protein products depicting auto-splicing activity (“MC” product). FIG. 12b also shows carboxy terminal auto-cleavage of protein products affinity purified via amylose based affinity chromatography (lane “A”, “MB” product). FIG. 12c is a photograph of a Coomassie blue stained electrophoretic separation of protein products isolated via amylose based (lane “A”) or chitin based (lane “C”) affinity chromatography depicting auto-splicing activity (“MC” species). FIG. 12c also shows carboxy terminal auto-cleavage of protein products affinity purified via amylose based affinity chromatography (lane “A”, “MB” species). Note the very small amounts of the uncleaved precursor (lane “C”, “MBC” species) suggesting very efficient autoprocessing activity by this chimera. M-MBP specific fragment, MB-MBP-BIL specific fragment, MBC-intact chimera.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is of chimeric autoprocessing polypeptides, polynucleotides encoding such polypeptides, and uses of such polypeptides and polynucleotides for reversibly binding proteins to specific substrates, reversibly binding specific substrates to each other, and auto-splicing amino acid sequences. Specifically, the present invention can be used to purify proteins, to affinity-select display phages, and to post-translationally ligate proteins together.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Autoprocessing polypeptides, polypeptides having the capacity to post-translationally auto-cleave and/or auto-splice, can be used to greatly facilitate various industrially and scientifically important biochemical procedures, as described above. For example, such autoprocessing polypeptides can be used in applications which involve reversible binding of proteins to specific substrates, such as protein purification, and reversible binding of specific substrates to each other, such as affinity-selection of display phages.
Various chimeric autoprocessing polypeptides and methods of using such have been described in the prior art (reviewed in: Perler and Adam, 2000. Curr Opin Biotechnol. 11, 377-83; Paulus H., 2000. Annu Rev Biochem. 69, 447).
However, all such prior art chimeric autoprocessing polypeptides suffer from various drawbacks. As described above, these drawbacks include suboptimal activity, poor stability, insolubility, requirement for strong auxiliary nucleophiles causing undesirable modifications at carboxy termini of cleaved amino terminal fragments, and undesirable amino terminal cleavages.
Thus, all prior art approaches have failed to provide optimal chimeric autoprocessing polypeptides for use in protein engineering.
While reducing the present invention to practice it was uncovered that the chimeric polypeptides of the present invention display efficient auto-cleavage, including auto-cleavage resulting in auto-splicing.
The chimeric polypeptides of the present invention comprise novel autoprocessing domains characterized by unique amino acid sequences, unique host protein/organism type origins, and unique natural biological capacities. Thus, the chimeric polypeptides of the present invention are highly novel and significantly enlarge and enhance the prior art spectrum of available types of chimeric autoprocessing polypeptides and their possible applications.
Thus, according to one aspect of the present invention, there are provided chimeric polypeptides having efficient auto-cleavage activity and comprising an autoprocessing segment having an amino acid sequence set forth by SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106.
Preferably, the amino acid sequence of the autoprocessing segment is set forth by SEQ ID NO: 12, 31, 76, or 77.
As used herein, the phrase “auto-cleavage activity” refers to cleavage of a polypeptide of the present invention in a region adjacent to the autoprocessing segment. Auto-cleavage occurs following exposure of the polypeptide of the present invention to suitable conditions in the absence of any other protein. Suitable auto-cleavage conditions are described hereinbelow.
Depending on the purpose, application and configuration, the polypeptides of the present invention may display different types of auto-cleavage activity.
Preferably, the auto-cleavage activity of the polypeptides of the present invention results in removal of a segment of the polypeptide adjacent to the amino terminal end or the carboxy terminal end of the autoprocessing segment.
Preferably, the segment of the polypeptide adjacent to the autoprocessing segment is an amino terminal segment or a carboxy terminal segment of the polypeptide.
According to one embodiment, the polypeptides of the present invention may comprise the autoprocessing domain as an amino terminal segment thereof. In this configuration, the polypeptides of the present invention may display removal of the segment thereof adjacent to the carboxy terminal end of the autoprocessing segment, i.e., the segment removed is the carboxy terminal segment of the polypeptide.
According to further embodiments, the polypeptides of the present invention may comprise the autoprocessing domain as a carboxy terminal segment thereof. In this configuration, the polypeptides of the present invention may display removal of the segment thereof adjacent to the amino terminal end of the autoprocessing segment, i.e., the segment removed is the amino terminal segment of the polypeptide.
According to yet further embodiments, the polypeptides of the present invention may comprise the autoprocessing domain as an internal segment of the polypeptide. In this configuration, the polypeptides of the present invention may display one or more of the following: removal of a segment adjacent to the amino terminal end of the autoprocessing segment, (i.e., the amino terminal segment of the polypeptide); removal of a segment adjacent to the carboxy terminal end thereof (i.e., the carboxy terminal segment of the polypeptide); removal of both the carboxy and amino terminal segments.
In some cases, removal of both segments may result in subsequent covalent fusion between the removed segments and, as such, auto-splicing of the polypeptide.
As used herein, the term “auto-splicing” refers to covalent bond formation between the amino acid residue forming the carboxy terminal end of a segment of the polypeptide adjacent to the amino terminal end of the autoprocessing domain and the amino acid residue forming the amino terminal end of a segment of the polypeptide adjacent to the carboxy terminal end of the autoprocessing domain.
As is illustrated in the Examples section which follows, the polypeptides of the present invention demonstrate such auto-splicing activity.
The polypeptides of the present invention may be advantageously used to post-translationally ligate essentially any protein to essentially any other protein via formation of a covalent bond between amino acid residues forming complementary terminal ends thereof. This may be effected by using a polypeptide of the present invention comprising the proteins to be ligated in the configuration described hereinabove enabling auto-splicing to yield the desired ligation product.
Preferably, the covalent bond is a peptide bond. Alternately, as described above, the covalent bond may be an ester bond, such as the ester bond formed during auto-cleavage of prior art autoprocessing polypeptides.
Depending on the application and purpose, auto-cleavage of the polypeptide may be specifically induced under suitable conditions, preferably a specific temperature, pH, or concentration of dithiothreitol (DTT).
Preferably, the temperature is in the range of 33 to 41° C., more preferably the temperature is in the range of 34 to 40° C., more preferably the temperature is in the range of 35 to 39° C., more preferably the temperature is in the range of 36 to 38° C., more preferably the temperature is in the range of 36.5 to 37.5° C., and most preferably the temperature is 37.0° C.
Preferably, the pH is in the range of pH 7.8 to 8.2, more preferably the pH is in the range of pH 7.9 to 8.1, and most preferably the pH is 8.0.
Preferably, the concentration of dithiothreitol is in the range of 0.1-20 millimolar, more preferably the concentration of dithiothreitol is in the range of 0.2-10 millimolar, more preferably the concentration of dithiothreitol is in the range of 0.5-5 millimolar, and most preferably the concentration of dithiothreitol is in the range of 1.0-2.0 millimolar.
Without being bound to a paradigm, the present inventors are of the opinion that auto-cleavage activity, in addition to being governed by the amino acid sequence of the autoprocessing segment, is also influenced by the amino acid sequence of the segments adjacent to the autoprocessing segment.
For example, the present inventors are of the opinion that in configurations in which the polypeptide comprises a carboxy terminal segment adjacent to the carboxy terminal end of the autoprocessing segment, the efficiency of cleavage may be enhanced if such a carboxy terminal segment includes at its amino terminal end, an amino acid residue comprising a nucleophilic group such as a sulfhydryl group, or more preferably a hydroxyl group.
As such, in cases wherein the polypeptide of the present invention includes a carboxy terminal segment adjacent to the autoprocessing segment, the amino acid residue forming the amino terminal end of such a carboxy terminal segment is preferably cysteine, serine, or more preferably threonine. As is illustrated in the Examples section below, polypeptides of the present invention having such a carboxy terminal segment display auto-cleavage of the carboxy terminal segment, including auto-cleavage resulting in auto-splicing.
In configurations wherein the polypeptide of the present invention includes an amino terminal segment adjacent to the autoprocessing segment, the amino acid residue forming the carboxy terminal end of such an amino terminal segment is preferably serine. As is illustrated in the Examples section below, polypeptides of the present invention having such an amino terminal segment display auto-cleavage of the amino terminal segment, including auto-cleavage resulting in auto-splicing.
It is recognized in the art that certain prior art autoprocessing segments lack the capacity to auto-cleave the bond attaching a given terminal end of the autoprocessing segment to particular flanking amino acid residues (Perler and Adam, 2000. Curr Opin Biotechnol. 11, 377-83).
In particular, certain prior art autoprocessing amino acid sequences lack the capacity to auto-cleave the bond attaching their amino terminal end to a flanking serine residue. As is illustrated in the Examples section which follows, and in sharp contrast to polypeptides containing such prior art autoprocessing amino acid sequences, the polypeptides of the present invention possess the capacity to auto-cleave such a bond.
Specific amino acid sequences of autoprocessing domains comprised in the polypeptides of the present invention may be obtained by referring to Table 1, FIGS. 1a-b, and FIGS. 2a-b of the Examples section below. Table 1 provides database coordinates which can be used to retrieve the nucleic acid sequences encoding such amino acid sequences. Such amino acid sequences may easily be determined from such nucleic acid sequences by the ordinarily skilled artisan using a suitable nucleic acid-to-amino acid sequence translation software, such as, for example translation software made publicly available by the National Center for Biotechnology Information (NCBI) or the European Molecular Biology Laboratory (EMBL) on the World Wide Web (WWW). FIGS. 1a-b provide specific amino acid sequences of various autoprocessing domains comprised in the polypeptides of the present invention. FIGS. 2a-b provide amino acid sequence motifs which define autoprocessing domains comprised in the polypeptides of the present invention.
For example, as is shown in the Examples section which follows, polypeptides of the present invention comprising autoprocessing segments FhaB_psesy (SEQ ID NO: 12), BIL4_cloth (SEQ ID NO: 31), 4825_rhosp (SEQ ID NO: 76) or BIL2_rhosp (SEQ ID NO: 77) display auto-cleavage activity, including auto-cleavage activity resulting in auto-splicing.
The polypeptides of the present invention may further comprise at least one affinity tag capable of specifically binding a substrate. As is further described hereinbelow, the affinity tag type depends on the intended use of the polypeptide.
As used herein, the phrase “affinity tag” refers to any moiety (preferably a peptide or polypeptide moiety) which is capable of specifically binding a substrate.
While the substrate can be essentially any substance or particle which can be specifically bound by the affinity tag, the substrate is preferably a molecule, a compound, a virus, or a cell.
The polypeptides of the present invention may comprise essentially any affinity tag.
Examples of peptide/polypeptide affinity tags include streptavidin, His-tags, strep-tags, epitope tags, maltose-binding proteins, and chitin-binding domains.
His-tags (histidine tags) consist of a chain of 2 to 10, most preferably 6, contiguous histidine amino acid residues. His-tags have the capacity to specifically bind substrates including nickel. Ample guidance regarding tagging polypeptides with His-tags is available in the literature of the art (for example, refer to: Sheibani N. 1999. Prep Biochem Biotechnol. 29:77). Purification of molecules comprising histidine tags is routinely effected using nickel-based automatic affinity column purification techniques. A suitable capture ligand for histidine-tagged molecules is the anti histidine tag single chain antibody 3D5 (Kaufmann, M. et al., 2002. J Mol. Biol. 318. 135-47).
Examples of epitope tags include an 11-mer Herpes simplex virus glycoprotein D peptide, and an 11-mer N-terminal bacteriophage t7 peptide, being commercially known as HSVTag and t7Tag, respectively (Novagen, Madison, Wis., USA), and 10- or 9-amino acid c-myc or Hemophilus influenza hemagglutinin (HA) peptides, which are recognized by the variable regions of monoclonal antibodies 9E10 and 12Ca5, respectively.
Strep-tags are peptides having the capacity to specifically bind streptavidin. Ample guidance regarding the use of strep-tags is provided in the literature of the art (see, for example: Schmidt, T G M. and Skerra, A. 1993. Protein Eng. 6, 109; Schmidt T G M. et al., 1996. Journal of Molecular Biology 255, 753-766; Skerra A. and Schmidt T G M., 1999. Biomolecular Engineering 16, 79-86; Sano T. and Cantor C R. 2000. Methods Enzymol. 326, 305-11; Sano T. et al., 1998. Journal of Chromatography B 715, 85-91).
Preferably, the affinity tag is a maltose-binding domain or a chitin-binding domain.
Preferably, the maltose-binding domain is malE-encoded maltose-binding protein (MBP). Ample guidance regarding the use of maltose-binding protein as an affinity tag is provided in the Examples section which follows and in the literature of the art (see, for example: Guan M. et al., 2002. Protein Expr Purif. 26, 229-34; Cattoli F. and Sarti G C., 2002. Biotechnol Prog. 18, 94-100).
In cases where the affinity tag is a maltose-binding protein, the substrate is preferably amylose, a specific ligand of such an affinity tag. Alternately, the substrate may be maltose, also a specific ligand of such an affinity tag.
As is shown in the Examples section below, polypeptides of the present invention comprising maltose-binding protein (MBP) can specifically bind a support including amylose.
Preferably, the chitin-binding domain is B. circulans cbd-encoded chitin binding domain (CBD). Ample guidance regarding the use of chitin-binding domain as an affinity tag is provided in the Examples section which follows and in the literature of the art (see, for example: Humphries H E. et al., 2002. Protein Expr Purif. 26, 243-8; Chong S. et al., 1997. Gene 192, 271-81).
In cases where the affinity tag is cbd-encoded chitin binding domain (CBD), the substrate is preferably chitin, a specific ligand of such an affinity tag.
As is illustrated in the Examples section which follows, polypeptides of the present invention comprising cbd-encoded chitin-binding domain can specifically bind a support including chitin.
The polypeptides of the present invention can be generated using chemical synthesis approaches or preferably recombinant techniques.
While reducing the present invention to practice, nucleic acid sequences encoding polypeptides having putative autoprocessing segments were identified in nucleic acid sequence databases (see Examples section below for further detail). The sequences were analyzed and the autoprocessing segment encoding regions were identified and used to generate polynucleotides encoding the polypeptides of the present invention.
Thus, according to another aspect of the present invention there is provided a polynucleotide sequence which encodes the auto-cleavable polypeptide of the present invention.
The polynucleotides of the present invention can be assembled from genomic, and/or complementary sequences.
As used herein, the phrase “complementary sequence” refers to a polynucleotide having a nucleic acid sequence resulting from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such sequences can be subsequently amplified in-vivo or in-vitro using a DNA dependent DNA polymerase.
As used herein, the phrase “genomic sequence” refers to a polynucleotide derived from a chromosome which thus reflects a contiguous portion of a chromosome.
In the case of a polynucleotide of the present invention encoding an autoprocessing domain expressed in a prokaryotic organism, the nucleic acid sequence encoding the autoprocessing domain may be conveniently generated via polymerase chain reaction (PCR) amplification using genomic DNA of the prokaryotic organism as a template.
Alternately, in the case of a polynucleotide of the present invention encoding an autoprocessing domain expressed in a eukaryotic organism, the nucleic acid sequence encoding the autoprocessing domain may be conveniently generated via PCR amplification using a cDNA library derived from the organism as a template.
Suitable oligonucleotide primers for PCR amplifying nucleic acid sequences encoding specific autoprocessing domains comprised in polypeptides of the present invention can be designed using the nucleic acid sequences identified by the database coordinates provided in Table 1 of the Examples section which follows.
For example, as is illustrated in the Examples section below, oligonucleotide primers suitable for PCR amplifying nucleic acid sequences encoding the autoprocessing domains FhaB_psesy, or BIL2_rhosp were derived from nucleic acid sequences retrieved using the relevant database coordinates provided in Table 1 of the Examples section below. The nucleic acid sequences of such primers are set forth by SEQ ID NOs: 4-5, or 6-7, respectively. Ample guidance for determining suitable reaction conditions for amplifying nucleic acid sequences encoding the aforementioned autoprocessing domains is provided in the Examples section which follows. PCR amplification of nucleic acid sequences is a commonly performed procedure and suitable primers and reaction conditions for a broad range of such procedures can generally be routinely determined by one of ordinary skill in the art, for example via suitable software, such as, for example, OLIGO 4.0 (National Biosciences, Plymouth, Minn.).
It will be appreciated that since autoprocessing activity is a characteristic of the amino acid sequence, one of ordinary skill in the art may alternatively use the amino acid sequences provided herein as a template for designing nucleic acid sequences which encode such amino acid sequences, and which take into consideration parameters such as codon usage which may increase the efficiency of expression of such sequences in specific organisms.
As described above, the polypeptides of the present invention may further comprise at least one affinity tag. In order to produce polypeptides of the present invention comprising affinity tags, coding nucleotides are formed comprising nucleic acid sequences encoding such affinity tags.
As described hereinabove, methods of generating nucleic acid sequences encoding affinity tags are well known to one of ordinary skill in the art. For example, nucleic acid sequences encoding affinity tags can be advantageously generated by PCR amplification of nucleic acid sequences encoding such affinity tags.
For example, as described and demonstrated in the Examples section which follows, suitable oligonucleotide primers for amplification of nucleic acid sequences encoding B. circulans cbd-encoded chitin-binding domain are set forth in SEQ ID NOs: 1-2.
Alternately, autoprocessing domains may be conveniently cloned into nucleic acid constructs configured for expressing fusion proteins comprising an affinity tag fused to a polypeptide encoded by a nucleic acid insert cloned into such a construct.
As is shown in the Examples section below, polynucleotides of the present invention comprising nucleic acid sequences encoding the affinity tag maltose-binding protein were assembled by cloning a nucleic acid sequence encoding an autoprocessing domain of the polypeptides of the present invention into the expression construct pMALC2 (New England Biolabs) which is designed to express fusion proteins comprising maltose-binding protein fused to a polypeptide encoded by a cloned insert.
Such nucleic acid constructs can be advantageously used to insert and/or express a chimeric polynucleotide within a host cell.
Thus, according to yet another aspect of the present invention there is provided a nucleic acid construct comprising a polynucleotide of the present invention.
The nucleic acid constructs of the present invention preferably comprise suitable promoter sequences so as to enable efficient expression of the polynucleotide of the present invention in an expression system.
Expression of the polynucleotides of the present invention may be conveniently controlled using an inducible promoter. A suitable inducible promoter is an isopropyl beta-D-thiogalactoside (IPTG)-inducible promoter, such as a T7 promoter. As described in the Examples section which follows, a T7 promoter can be used to drive IPTG-inducible expression of a polynucleotide of the present invention in a suitable cell-free expression system or in a cellular expression system.
IPTG-induced expression of polynucleotides under the regulatory control of T7 promoters is widely practiced in the art by the ordinarily skilled practitioner and ample guidance regarding the use of such promoters is available in the literature of the art (see, for example, Sambrook et al., infra).
The nucleic acid constructs of the present invention can be used to produce the polypeptides of the present invention in a suitable expression system.
Thus, according to still another aspect of the present invention there is provided a method of generating a polypeptide of the present invention.
The method is effected by generating a chimeric amino acid sequence including an autoprocessing segment of the present invention. Preferably the chimeric amino acid sequence is generated by expressing a polynucleotide of the present invention in an expression system suitable for generating the chimeric amino acid sequence from the chimeric polynucleotide.
The nucleic acid constructs of the present invention can be used to express the polypeptides of the present invention in various expression systems, including any cellular or cell-free expression systems suitable for expressing recombinant proteins such as the polypeptides of the present invention.
When used to express the polypeptides of the present invention in a cell-free expression system, the constructs of the present invention may be advantageously expressed in any suitable in-vitro transcription/translation system.
Numerous in-vitro transcription/translation systems are commercially available for expressing recombinant proteins such as the polypeptides of the present invention.
For example, a suitable cell-free expression system for expressing nucleic acid constructs of the present invention is an E. coli S30 extract expression system, as described and as demonstrated in the Examples section below.
Numerous cellular expression systems, including yeast, bacterial, insect, and mammalian cells can be employed to express the nucleic acid constructs of the present invention.
As described and illustrated in the Examples section which follows, the polypeptides of the present invention may be advantageously expressed in E. coli by transforming E. coli with the nucleic acid constructs.
Transformation of E. coli with nucleic acid constructs is a routine procedure widely practiced in the art (see, for example, Sambrook et al., infra).
For example, for expression of the polypeptides of the present invention using the nucleic acid constructs of the present invention in E. coli, competent cells capable of DNA uptake may be prepared from cells harvested in exponential growth phase and rendered competent via the widely practiced CaCl2 method. Addition of MgCl2 or RbCl to the transformation reaction medium may be employed to increase transformation efficiency. Alternative transformation methods include methods such as electroporation or host cell protoplast transformation.
As described hereinabove, the capacities of the polypeptides of the present invention to specifically bind substrates and to auto-cleave can be advantageously used in various practical applications involving reversible binding of substrates, such as protein purification.
Thus, according to a further aspect of the present invention there is provided a method of purifying a protein.
The method is effected by generating a polypeptide of the present invention comprising an autoprocessing segment being terminally attached to, or flanked by, an amino acid sequence of the protein, immobilizing the polypeptide to a support, and subjecting the immobilized polypeptide to suitable conditions for enabling auto-cleavage resulting in removal of the protein from the polypeptide.
According to a preferred embodiment, the polypeptide of the present invention is configured such that one terminal end of the autoprocessing segment is adjacent to a terminal segment of the polypeptide being the protein to be purified and the other terminal end of the autoprocessing segment is adjacent to a terminal segment of the polypeptide comprising an affinity tag.
According to this embodiment, the polypeptide of the present invention is preferably immobilized via a specific binding of the affinity tag to a specific ligand thereof included in the support. Optionally, in cases where auto-cleavage further results in detachment of the autoprocessing segment from the terminal segment of the polypeptide comprising the affinity tag, the method may advantageously further comprise the step of separating the protein from the autoprocessing segment so as to further facilitate purification of the protein. Such separation may be effected as described further hereinbelow.
According to another embodiment, the polypeptide of the present invention consists of a chimera comprising the autoprocessing segment fused to the protein to be purified.
According to this embodiment, the polypeptide of the present invention is preferably immobilized via a specific binding of the autoprocessing segment to a specific ligand of the autoprocessing segment included in the support.
According to yet another embodiment, the polypeptide of the present invention consists of an autoprocessing segment being flanked at its amino terminal end with an amino terminal segment of the amino acid sequence of the protein to be purified, and being flanked at its carboxy terminal end with the carboxy terminal segment of the amino acid sequence of the protein to be purified complementing the amino terminal segment of the amino acid sequence of the protein to be purified.
According to this embodiment, the polypeptide of the present invention is preferably immobilized via a specific binding of the autoprocessing segment to a specific ligand of the autoprocessing segment included in the support, and auto-cleavage results in auto-splicing of the complementary amino and carboxy terminal segments of the protein to be purified to thereby release the protein.
In embodiments in which the polypeptide of the present invention is immobilized via the autoprocessing segment, the specific ligand included in the support is preferably an antibody or antibody fragment capable of specifically binding the autoprocessing segment.
Purification of a protein according to the method of the present invention may be advantageously effected via standard affinity chromatography techniques. For example, a suitable support for immobilizing a polypeptide of the present invention may be an affinity resin coupled to a specific ligand of the polypeptide of the present invention packed in a standard affinity purification column. Following subjecting of the support-bound polypeptide of the present invention to conditions suitable for auto-cleavage thereof, the highly purified protein released from the support-bound autoprocessing segment may be conveniently recovered as a flow-through fraction eluted from the column.
In the case described hereinabove, wherein auto-cleavage further results in detachment of the autoprocessing segment from the support-bound segment of the polypeptide of the present invention, separating the protein from the autoprocessing segment may be effected analogously to the method described hereinabove by using such standard affinity chromatography techniques wherein the affinity resin includes a specific ligand of the autoprocessing segment.
Alternately, various methods suitable for separating such mixtures of polypeptides may be practiced by the ordinarily skilled artisan. Such techniques include, for example high-performance liquid chromatography (HPLC), size-exclusion chromatography, and similar methodologies.
Ample guidance regarding chromatographic isolation of proteins is widely available in the literature of the art (see, for example: Wilchek M. and Chaiken I., 2000. Methods Mol Biol 147, 1-6; Jack G W., 1994. Mol Biotechnol 1, 59-86; Narayanan S R., 1994. Journal of Chromatography A 658, 237-258; Nisnevitch M. and Firer M A., 2001. J Biochem Biophys Methods 49, 467-80; Janson J C. and Kristiansen T. in Packings and Stationary Phases in Chromatography Techniques. Unger K K. (ed.), Marcel Dekker, New York, pp. 747 (1990); Clonis Y D: HPLC of Macromolecules: A Practical Approach, IRL Press, Oxford, pp. 157 (1989); Nilsson J. et al., 1997. Protein Expr Purif. 11, 1-16).
As described in the Examples section which follows, amylose affinity ligand based column chromatography of a polypeptide of the present invention comprising the autoprocessing domain BIL2_rhosp flanked at its amino terminal end with an amino terminal segment including the affinity tag MBP, and flanked at its carboxy terminal end with the amino acid sequence of a protein to be purified resulted in column-retention of a segment of the polypeptide of the present invention lacking the amino acid sequence of the protein to be purified (FIG. 10a), thereby demonstrating the utility of the method of the present invention for purifying proteins.
As described in the Examples section which follows, chitin affinity ligand based column chromatography of a polypeptide of the present invention comprising the autoprocessing domain 4825_rhosp flanked at its carboxy terminal end with an carboxy terminal segment including the affinity tag CBD, and flanked at its amino terminal end with the amino acid sequence of a protein to be purified resulted in column-retention of a segment of the polypeptide of the present invention lacking the amino acid sequence of the protein to be purified (FIG. 11b), thereby demonstrating the utility of the method of the present invention for purifying proteins.
According to the teachings of the present invention, the polypeptides of the present invention may include affinity tags flanking the autoprocessing segment.
Thus, according to yet a further aspect of the present invention there is provided a method of reversibly attaching a first substrate to a second substrate.
The first and second substrates may be the same or may be different.
The method is effected using a polypeptide of the present invention in which the autoprocessing segment is flanked by a first amino acid sequence capable of binding the first substrate and a second amino acid sequence capable of binding the second substrate, such that auto-cleavage releases the first amino acid sequence from the second amino acid sequence. Exposing the first substrate and the second substrate to the polypeptide of the present invention generates a complex including the first substrate attached via the polypeptide to the second substrate. Following generation thereof, the complex is subjected to suitable conditions for auto-cleavage, thereby detaching the first substrate from the second substrate.
Complex generation may be effected in various ways, depending on the application and purpose. For example, complex generation may be effected wherein neither, one or both substrates is included in, or consists of, a support specifically binding an affinity tag comprised in the polypeptide of the present invention.
According to a preferred embodiment, the method is used for reversibly attaching a first substrate being a protein displayed by a bacteriophage to a second substrate specifically binding the phage-displayed protein, which substrate being included in a support.
According to this embodiment, the method is effected using a polypeptide of the present invention comprising a first amino acid sequence having the capacity to specifically bind the substrate and a second amino acid sequences having the capacity to bind the phage-displayed protein.
The method according to this aspect of the present invention may be advantageously employed with phage-display libraries for selecting bacteriophages displaying a protein having high affinity to a specific substrate. This may be effected by exposing a phage display library to a support including a substrate being a target molecule to which a high affinity ligand is desired. Elements of the phage display library not being bound with high affinity to the support may be washed and recovery of phages specifically binding the substrate with high affinity via a displayed protein capable of specifically binding the target molecule with high affinity may be conveniently recovered by subjecting the support-bound phages to conditions suitable for auto-cleavage of the polypeptide of the present invention so as to effect the detachment thereof from the support.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.
Example 1
Bacterial-Intein Like (BIL) Domains
Novel Auto-Cleaving/Auto-Splicing Protein Domains
Autoprocessing polypeptides capable of auto-cleavage have been shown to be uniquely useful in a wide range of protein engineering applications, for example for protein purification without the requirement for proteases. However, all prior art autoprocessing polypeptides suffer from various drawbacks, including suboptimal activity, stability, solubility, and requirement for auxiliary molecules causing undesirable protein modifications. In order to enlarge and enhance the current repertoire of autoprocessing polypeptides, the present inventors have identified, generated and demonstrated the functionality of novel autoprocessing polypeptides, as follows.
Materials and Methods:
In order to identify novel auto-cleaving/-splicing proteins, databases storing genomic sequences of various organisms, including bacterial pathogens were searched for open reading frames (ORFs) coding for protein sequences containing Hint domains. Following identification of protein sequences containing Hint domains, such protein sequences were cloned and tested for auto-cleaving/-splicing activity, as described below.
Data sources: BILs were identified in bacterial genomes by searching the following databases: National Center for Biotechnology Information (NCBI) sequence databases for Brucella melitensis (B. melitensis) 16M, Streptomyces coelicolor (S. coelicolor) A3(2), Neisseria meningitidis (N. meningitidis) MC58, N. meningitidis Z2491, Pseudomonas fluorescens (P. fluorescens) PfO-1, Leptospira interrogans (L. interrogans) 56601, Streptomyces avermitilis (S. avermitilis) MA-468, Pirellula species 1, and Chromobacterium violaceum (C. violaceum) ATCC 12472 sequences; Integrated Genomics (http://www.integratedgenomics.com) sequence databases for Rhodobacter capsulatus (R. capsulatus) SB 1003 genomic sequences (Haselkorn et al., 2001. Photosynthesis Research 70, 43-52) and Methylobacterium extorquens (M. extorquens) AM1, Joint Genome Institute (http://wwwjgi.doe.gov) sequence databases for Rhodobacter sphaeroides (R. sphaeroides) 2.4.1 (Mackenzie et al., 2001. Proc Natl Acad Sci USA. 99, 2275-2280), Magnetospirillum magnetotacticum (M. magnetotacticum) MS-1, Clostridium thermocellum (C. thermocellum) ATCC 27405, and Thermobifida fusca (T. fusca) YX genomic sequences; The Institute for Genomic Research (http://www.tigr.org) sequence databases for Pseudomonas syringae (P. syringae) DC3000 (Fouts et al., 2002. Proc Natl Acad Sci USA 99, 2275-2280), Silicibacter pomeroyi (S. pomeroyi) DSS-3, Gemmata obscuriglobus (G. obscuriglobus) UQM 2246, Myxococcus xanthus (M. xanthus) DK1622, and Verrucomicrobium spinosum (V. spinosum) DSM 4136 sequences; The Sanger Institute (http://www.sanger.ac.uk) sequence databases for Neisseria meningitidis (N. meningitidis) FAM18, P. fluorescens SBW25, and Rhizobium leguminosarum (R. leguminosarum) bv. viciae 3841 sequences; University of Oklahoma, Advanced Center for Genome Technology (http://www.genome.ou.edu) sequence databases for Neisseria gonorrhoeae (N. gonorrhoeae) FA1090 genomic sequences; Baylor College of Medicine Human Genome Sequencing Center (http://www.hgsc.bcm.tmc.edu) sequence databases for Mannheimia haemolytica (M. haemolytica) PHL213 genomic sequences; and Kazusa DNA Research Institute database (Japan; http://www.kazusa.orjp) for Gloeobacter violaceus (G. violaceus) PCC 7421 sequences.
BIL domain nomenclature: BIL domains were named using the format “a_b” where “b” is an abbreviation of the bacterial species of origin and where “a” is either host protein name (e.g., “FhaB” or “MafB”); an arbitrary BIL# designation; an Integratedgenomics Database (http://ergo.integratedgenomics.com/R_capsulatus.html) number for R. capsulatus; a Computational Biology Program at ORNL (http://genome.ornl.gov/microbial/rsph) analysis code for R. sphaeroides; or a gene number for B. melitensis strain 16M and N. meningitidis strains MC58 and Z2491. Available gene identifier accession numbers and further information relevant to identified BIL domains are provided in Table 1, below.
Computational sequence analysis: The BLAST software package of the NCBI was used for sequence-to-sequence searches (Altschul, S F. et al., 1997. Nucleic Acids Res. 25, 3389) of BIL domains with BIL domains and with intein sequences, and the BLIMPS software was employed for block-to-sequence searches (Henikoff S. et al., 1995. Gene 163, GC17). Multiple block sequence alignments were constructed using BLOCKMAKER (Henikoff S. et al., 1995. Gene 163, GC17) and MACAW (Schuler G D. et al., 1991. Structure, Function and Genetics 9, 180) software, as previously described (Pietrokovski S., 1998. Protein Science 7, 64). BIL domains were aligned with other BIL domains having higher scores than with intein sequences and alignments of BIL domains with each other was across their whole, or almost whole, lengths (results not shown). This is also a practical way to classify BIL domains as such. Phylogenetic analysis was performed using the PHYLIP software package (Felsenstein J., 1989. Cladistics 5, 164) version 3.55.
Generation of BIL domain phylogeny dendrograms: BIL domain phylogeny dendrograms were computed from DNA multiple sequence alignment of 49 mostly complete BIL domains aligned across 201 positions, coding for 67 amino acids which could be confidently aligned across BIL domains. Nodes with bootstrap values below 440/1000 were collapsed, and bootstrap values above 800/1000 are shown. Bootstrap values of the nodes grouping all A-type and B-type BILs are 441 and 519, respectively. The D. melanogaster Hedgehog Hint domain (Porter J A., et al., 1996. Cell 86, 21) was used as an outgroup to root the tree. The dendrogram was calculated using the DNADIST program (version 3.5) of the PHYLIP software package (Felsenstein J., 1989. Cladistics 5, 164). Results were verified against those obtained using CLUSTALW software (Thompson J D. et al., 1994. Nucl Acid Res. 22, 4673) and from protein multiple sequence alignments obtained using PHYLIP, PROTDIST, and CLUSTALW software.
BIL Functional Activity Assays:
In order to analyze the capacity of BIL domains to auto-cleave/auto-splice flanking sequences, genetic sequences encoding BIL domains and portions of flanking sequences were cloned for expression as chimeric proteins tagged at their amino terminal ends with the malE gene-encoded maltose-binding protein (MBP) affinity tag, and at the carboxy terminal end with the B. circulans cbd gene-encoded chitin-binding domain (CBD) affinity tag. These chimeras were expressed in an in-vitro transcription/translation system, or overexpressed in-vivo in E. coli, and resulting protein products were analyzed for evidence of BIL domain-mediated autoprocessing activity.
Such chimeras were cloned using BIL domain genetic sequences encoding:
(i) the Type A BIL domain FhaB_psesy (Table 1, FIG. 1a) and its downstream-flanking threonine residue to generate the chimera “MBP-PsyBIL-CBD”; and
(ii) the Type B BIL domain BIL2_rhosp (Table 1, FIG. 1b) including 32 amino terminal-flanking and 11 carboxy terminal-flanking amino acids to generate the chimera “MBP-RspBIL2-CBD”.
Constructs: B. circulans cbd gene sequences encoding CBD were cloned by PCR from expression vector pTYB2 (New England Biolabs, Beverly, Mass.) using the primers 5′-AAATGTCGACTGCGGTGGCCTGACC-3′ (SEQ ID NO: 1) and 5′-TGTCGTATTGCTTCCTTTCGGGCTT-3′ (SEQ ID NO: 2), and inserted, including the upstream linker 5′-TGCGGTGGCCTGACCGGTCTGAACTCAGGCCTC-3′ (SEQ ID NO: 3), into the SalI/PstI linearized, isopropyl beta-D-thiogalactoside (IPTG)-inducible MBP-fusion protein expression construct pMALC2 to generate the MBP-CBD fusion protein expression construct pC2C. Construct pC2C was used in functional assays as a positive control for expression of MBP-CBD.
For MBP-PsyBIL-CBD expression, genetic sequences encoding PsyBIL and flanking sequences were PCR amplified from P. syringae DC3000 strain genomic DNA (kindly provided by Dr. G. Sessa, Tel-Aviv University) using the primers 5′-AAAAGGATCCTGCTTTGCGGCCGGAACGA-3′ (SEQ ID NO: 4) and 5′-AAAATCTAGAGGTATTATGCACCCATGTCTTG-3′ (SEQ ID NO: 5), and cloned in BamHI/XbaI linearized pC2C, between the malE MBP-encoding sequences and the CBD expressing cbd sequences to generate the MBP-PsyBIL-CBD expression construct pC2C-PsyBIL.
For MBP-RspBIL2-CBD expression, genetic sequences encoding RspBIL2 and flanking sequences were PCR-amplified from R. sphaeroides 2.4.1 strain genomic DNA (supplied by Dr. Steven L. Porter, Department of Biochemistry, University of Oxford) using the primers 5′-GAATTCGGTGATTCATCCTTGGGGCGA-3′ (SEQ ID NO: 6) and 5′-TCTAGAAAAACACGGCAAGGGCGAGCGG-3′ (SEQ ID NO: 7), and cloned in EcoRI/XbaI linearized pC2C, between the MBP-encoding malE sequences and the CBD-encoding cbd sequences to generate the MBP-RspBIL2-CBD expression construct pC2C-RspBIL2.
The MBP-BIL4Cloth-CBD Expression Construct pC2C-BIL4Cloth.
Polymerase chain reactions were performed using a Biometra thermal cycler in a 50 μl reaction mixture containing Taq polymerase buffer (Sigma, St. Louis, Mich.), 1 μl Taq DNA polymerase, 200 mM dNTP, 10 mM of each primer and 100 ng genomic DNA
The chimeras were constructed such that carboxy terminal or amino terminal cleavage thereof was expected to generate [MBP-BIL+CBD] or [MBP+BIL-CBD] specific protein products, respectively. The MBP- or CBD-containing protein products were expected to vary in size according to the size of the BIL domain flanking sequences included in the BIL sequences cloned in the chimeric proteins. Auto-splicing by the chimeras was expected to generate MBP-CBD protein products having a molecular weight varying according to the size of the BIL domain flanking sequences included in the cloned BIL segment.
Chimeras were expressed and analyzed for evidence of BIL processing activity, as described below.
In-vitro BIL protein expression and activity assays: In-vitro transcription-translation of MBP-BIL-CBD and MBP-CBD chimeric proteins was achieved using chimera expression constructs pC2C-RspBIL2 and pC2C-RspBIL2a, and expression construct pC2C as DNA templates, respectively, using E. coli S30 extract for circular DNA system (Promega Kit #L1030, Promega, Madison Wis.). Reactions were carried out according to the manufacturer's instructions, using a reaction containing 0.25 mM [35S]-methionine, 220 nmol of expression construct DNA as template, 1-2 mM dithiothreitol, and having a pH of about 8.0. Reactions were incubated at 37° C. for 90-120 minutes. Prior to electrophoresis of expressed protein, 5 μl or 10 μl aliquots of reaction mixtures were mixed with four volumes of acetone in order to remove polyethylene glycol. Acetone precipitation was followed by centrifugation at 12,000×g for 5 minutes. The supernatant was discarded and the protein-containing pellet was mixed with gel loading buffer to give a final concentration of 0.06 M Tris-Cl, 2% SDS, 10% (v/v) glycerol, and 0.01% bromophenol blue. Proteins were separated via 7.5% or 10% SDS-PAGE, and the separated proteins were visualized using a phosphor-imaging screen. Phosphor-imaging signals were quantified using NIH IMAGE 1.62 software. Product quantities were derived from values of three independent experiments averaged for each sample together with their standard deviation of the means. The molar percentage of each product was calculated.
The expected molecular weights of MBP-RspBIL2-CBD, and of its splicing product MBP-CBD, its carboxy terminal cleavage product MBP-RspBIL2, and its MBP-containing amino terminal cleavage product are 68.0, 55.1, 60.5 and 46.7 kDa, respectively.
The expected molecular weights of control vector pC2C-expressed MBP-CBD and of its MBP portion were 50.3 and 43.0 kDa, respectively.
In-vivo Type A BIL domain protein expression, purification and activity assay: Competent TB1 E. coli cells (NEB, Beverly, Mass.) were transformed with constructs for expression of MBP-BIL-CBD chimeras. Transformants were plated on LB agar supplemented with ampicillin (100 μg/ml). Single colonies were used to inoculate 3 ml aliquots of LB medium supplemented with ampicillin (100 μg/ml). Following incubation at 37° C. for 16 hours with shaking, 1 ml of culture was used to inoculate a 2 liter flask containing 500 ml of LB supplemented with ampicillin (100 μg/ml). Incubation was continued at 37° C. with shaking until the optical density at 600 nm was 0.6, at which point IPTG was added to a final concentration of 0.3 mM. After further incubation for 3 hours, cells were harvested by centrifugation at 5,000×g for 20 minutes, re-suspended in solution containing 20 mM Tris (pH 7.4), 200 mM NaCl, and protease inhibitor cocktail (Sigma, St. Louis, Mich.), and lysed by sonication. Lysates were centrifuged at 17,000×g for 20 minutes to remove cell debris, and supernatants were harvested for subsequent analyses. Proteins were then affinity purified with either chitin (NEB, Beverly, Mass.) or amylose beads (NEB, Beverly, Mass.) which bind to the CBD or MBP affinity tags included in the chimeric protein. Elution of protein from beads prior to electrophoresis was performed by mixing the protein-bound beads with SDS-PAGE sample loading buffer.
Western immunoblotting assays and protein staining: Products generated by expression of MBP-BIL-CBD chimeras were separated by SDS-PAGE. Briefly, protein samples were mixed with protein loading buffer to give a final concentration of 0.06 M Tris-Cl, 2% SDS, 10% (v/v) glycerol, 0.1 M dithiothreitol, and 0.01% bromophenol blue. All samples were boiled for 3 minutes prior to electrophoresis. Separated proteins were analyzed by Western immunoblotting using either monoclonal mouse anti-MBP (Novus Biologicals, Inc. Littleton, Colo.) antibody for identification of the MBP tag, or polyclonal rabbit anti-CBD (NEB, Beverly, Mass.) for identification of the CBD tag. Secondary antibodies used were HRP conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.). Relative apparent molecular weights were calculated using TriChromoRanger (Pierce, Rockford Ill.) prestained markers.
Electrophoretic gels containing separated proteins were fixed in 40% methanol/7% acetic acid and stained with PhastGel Blue R stain (Pharmacia Biotech AB, Sweden). Gels were destained in 40% methanol/7% acetic acid and then in deionized water, and visualized protein bands were excised and electroeluted for MALDI mass spectroscopy (MS) analysis.
Electroelution of protein: Protein was electroeluted from gels at 150 volts for 2 hours in GeBAflex tubes (Gene Bio Application Ltd., Israel) using elution buffer containing 0.025% SDS, Tris and Tricine (pH 8.5). Following electroelution SDS was removed from electroeluted protein using cold TCA:acetone precipitation in the presence of 0.5% sodium deoxycholate (NaDOC; T. Mehlman and A. Shainskaya, unpublished).
In-gel proteolysis: Protein bands from PhastGel Blue R stained gels were destained using multiple washes in 50% acetonitrile in 50 mM ammonium bicarbonate. Destained protein bands were subsequently reduced, alkylated and in-gel proteolysed using either bovine trypsin (sequencing grade, Roche Diagnostics, Germany) or chymotrypsin, (Boehringer Mannheim, Germany) by incubation with 12.5 ng/μl protease in 50 mM ammonium bicarbonate at 37° C., as previously described (Shevchenko et al., 1996. Analytical Chemistry 68, 850). Extracted peptide solutions were dried for subsequent MALDI-MS analysis.
Mass Spectrometry: Intact molecular mass measurement and peptide mass mapping were performed using a Bruker Reflex III MALDI time-of-flight (TOF) mass spectrometer (Bruker, Bremen, Germany) equipped with SCOUT source, delayed ion extraction, reflector and a 337 nm nitrogen laser. Each mass spectrum was generated using data accumulated from 200 laser shots. Both external and nearby calibrations for proteins were performed using BSA and myoglobin (Sigma). For peptide mapping, internal calibration with molecular ions of regularly occurring matrix ions and peptides derived from trypsin was additionally performed to consolidate further peptide assignment.
Intact molecular weight measurements by MALDI MS: Gel electroeluted proteins were further purified by cold acetone precipitation. The dried extract from one lane of the gel was re-dissolved in 0.5 ml of 80% formic acid and immediately diluted with water to yield a solution containing 20% formic acid, and 50% of this solution was applied to a target plate.
Peptide mass mapping by MALDI mass spectrometry: Aliquots of one tenth of the extracted peptide mixture volume, dissolved in 0.1% TFA or formic acid/isopropanol/water (1:3:2), were used for MALDI-MS using the fast evaporation or dry droplet method. Matrix surfaces of α-cyano-4-hydroxycinnamic acid (4-HCCA) or 2,5-dihydroxybenzoic acid (DHB) were utilized for the fast evaporation (Jensen O N. et al., 1996. Rapid Commun in Mass Spectrom. 10, 1371; Vorm O. et al., 1994. Analyt Chem. 66, 3281) or dry droplet method (Kussmann K. et al., 1997. J Mass Spectrom. 32, 593), respectively.
Experimental Results:
Identification of two novel bacterial intein-like domains containing Hint-like motifs: Searches of sequence databases of diverse bacterial species for Hint-like motif-containing putative ORFs identified open reading frames coding for proteins comprising two related types of novel intein-like protein domains termed by the present inventors Type A and Type B bacterial intein-like (BIL) domains. Novel type A and B BIL domain sequences identified are shown aligned in FIGS. 1a (SEQ ID NOs: 8-62) and 1b (SEQ ID NOs: 63-104), respectively. The bioinformatic sources used to identify these BIL domains are shown in Table 1.
An amino acid sequence motif (SEQ ID NO: 105) was identified (FIG. 2a) which exclusively defines a subset of Type A BIL domains, including domains: 39—9_thefus, SCP1.201_strco, 3875—87_magma, B0372+_neimeB, B0655+_neimeB, A2115_neime, MafB1_neimeC, BIL2_neimeC, BIL4_neimeC, BIL5_neimeC, BIL6_neimeC, MafB1_neigo, BIL2_neigo, BIL3_neigo, MafB2_neigo, BIL5_neigo, BIL6_neigo, FhaB_psesy, FhaB_manha, FhaB1_psefl-PfO-1, FhaB1_-psefl-SBW25, BIL6_cloth, BIL5_cloth, BIL2_cloth, BIL4_cloth, BIL1_cloth, BIL8_cloth, BIL9_cloth, BIL1_gemob, BIL2_gemob, 0709_lepin, 3725_lepin, o1078_myxxa, o1070_myxxa, BIL1_strav, BIL2_strav, BIL3_strav, BIL1_pirsp, BIL1_chrvi, o3395_versp, o5687_versp, o649_versp, BIL1_glovi, BIL2_glovi, BIL3_glovi, and BIL4_glovi.
An amino acid sequence motif (SEQ ID NO: 106) was identified (FIG. 2b) which exclusively defines a subset of Type. B BIL domains, including: 4825_rhosp, BIL2_rhosp, 00588_rhoca, 02710_rhoca, 01524_rhoca, 01523_rhoca, 00126_rhoca, 01216_rhoca, 00949_rhoca, 01374_rhoca, 00459_rhoca, 00460_rhoca, 00746_rhoca, 03530_rhoca, 00199_rhoca, BIL3_magma, BIL4_magma, BIL1_brusu, BIL1_unknwn, BIL2_unknwn, 06786_metex, BIL1_silpo, BIL2_silpo, BIL3_silpo, BIL4_silpo, BIL5_silpo, BIL6_silpo, BIL7_silpo, BIL8_silpo, BIL9_silpo, BIL10_silpo, BIL11_silpo, BIL12_silpo, BIL13_silpo, BIL14_silpo, BIL15_silpo, BIL16_silpo, BIL1_rhile, and II0519_brume.
This new type of domain appears in non-conserved regions of hyper-variable proteins. Thus, these domains are distinct from the Hint domains of inteins and Hog-proteins by the species and proteins in which they appear. An analysis of amino acid residue conservation within Hint-like motifs of BIL domains and within homologous Hint domain motifs of Hog proteins and inteins is shown in (FIGS. 3a-z). Examination of BLAST sequence alignments (Altschul, S F. et al., 1997. Nucleic Acids Result. 25, 3389) of BIL domains with BIL domains and with intein sequences showed that BIL domains aligned with each other with higher scores than with intein sequences across their whole, or almost whole, lengths (results not shown). Therefore, BIL domains were found to be distinct from inteins by their global sequence features.







TABLE 1







Databases used to identify BIL domains.












BIL
BIL domain






Type
name*
Source**
Date***
Contig/Entry
Coordinates





A
BIL1_cloth
NCBI

23022619
—


A
BIL2_cloth
NCBI

28N′aa + 23020813 +
—






59aa + 23020812


A
BIL3_cloth
NCBI

23022239 + 14N′aa
—


A
BIL4_cloth
NCBI

23020817 + 5N′amino
311-445






acid/gi|23020817


A
BIL5_cloth
NCBI

23020815 + 13N′aa
—


A
BIL6_cloth
NCBI

23022237
—


A
BIL7_cloth
NCBI

23022587 + 7N′aa
—


A
BIL9_cloth
NCBI

23022893 + 59aa +
—






23022892


A
BIL10_cloth
NCBI

22262017
34594-34986


A
BIL11_cloth
NCBI

22262176
416-728


A
3875_87_magma
NCBI

21614488
76532-75165


A
FhaB_manha
BCM
 4 Oct. 2001
C78-C85
11046-20977


A
BIL2_neigo
OU-
26 Sep. 2000
AE004969
1563413-1564129




ACGT


A
BIL3_neigo
OU-
26 Sep. 2000
AE004969
1565033-1565809




ACGT


A
BIL5_neigo
OU-
26 Sep. 2000
AE004969
1351509-1350766




ACGT


A
BIL6_neigo
OU-
26 Sep. 2000
AE004969
1349978-1349310




ACGT


A
MafB1_neigo
OU-
26 Sep. 2000
AE004969
1560214-1561941




ACGT


A
MafB2_neigo
OU-
26 Sep. 2000
AE004969
1355876-1354062




ACGT


A
B0369+_neimeB
NCBI

7225591 + 34 N′ aa
—


A
B0372+_neimeB
NCBI

7225594 + 11 N′ aa
—


A
B0655+_neimeB
NCBI

7225882 + 14 N′ aa
—


A
BIL2_neimeC
Sanger
15 May 2002
NmC
1836857-1837573


A
BIL3_neimeC
Sanger
15 May 2002
NmC
1838418-1838981


A
BIL4_neimeC
Sanger
15 May 2002
NmC
1839771-1840439


A
BIL5_neimeC
Sanger
15 May 2002
NmC
627204-627920


A
BIL6_neimeC
Sanger
15 May 2002
NmC
628395-629102


A
MafB1_neimeC
Sanger
15 May 2002
NmC
1833717-1835480


A
FhaB1_psefl-
NCBI-

205922-575
  3-6313



PfO-1
TIGR


A
FhaB1_psefl-
Sanger
 2 Sep. 2002
Pflu552a01
41728-29387



SBW25


A
FhaB_psesy
TIGR
30 Aug. 2002
5668
5148986-5149429


A
SCP1.201_strco
NCBI

13620683 + 32 N′ aa


A
39_9_thefus
JGI
 1 Nov 2000
39
13655-15508


A
BIL1_gemob
TIGR
23 Sep. 2002
14
  32-20392


A
BIL2_gemob
TIGR
14 Feb. 2003
354
16692-11734


A
0709_lepin
NCBI
10 Nov. 2002
24213409
—


A
3725_lepin
NCBI
10 Nov. 2002
24216424
—


A
3719_lepin
NCBI
10 Nov. 2002
24197710
—


A
o665_myxxa
TIGR
23 Apr. 2003
168
495-3704


A
o1078 myxxa
TIGR
23 Apr. 2003
157
103477-106710


A
o1070_myxxa
TIGR
23 Apr. 2003
168
495-3704


A
BIL1_strav
NCBI
20 Jul. 2003
29826740
—


A
BIL2_strav
NCBI
20 Jul. 2003
29826826
—


A
BIL3_strav
NCBI
20 Jul. 2003
29831835
—


A
BIL1_pirsp
NCBI
20 Jul. 2003
32470666
—


A
BIL1_chrvi
NCBI
07 Sep. 2003
34104178
—


A
BIL1_glovi
Kazusa
04 Sep. 2003
gll0211
—


A
BIL2_glovi
Kazusa
04 Sep. 2003
gll0207
—


A
BIL3_glovi
Kazusa
04 Sep. 2003
gll0213
—


A
BIL4_glovi
Kazusa
04 Sep. 2003
gll0212
—


A
BIL5_glovi
Kazusa
04 Sep. 2003
gll0205
—


A
BIL6_glovi
Kazusa
04 Sep. 2003
gll0208
—


A
BIL7_glovi
Kazusa
04 Sep. 2003
gsl3615
—


A
o649_versp
TIGR
02 Sep. 2003
65738
  3-1949


A
o5687_versp
TIGR
02 Sep. 2003
65921
18348-1288


A
o3395_versp
TIGR
02 Sep. 2003
65925
56853-46669


B
II0519_brume¶
NCBI

17988864


B
BIL2_magma
NCBI

21613062
 922-1590


B
BIL3_magma
NCBI

21614112
2449-1475


B
BIL4_magma
NCBI

21614173
2216-3187


B
BIL5_magma
NCBI

21612572
 3-338


B
BIL6_magma
NCBI

21613847
2033-1774


B
06786_metex
IG
June 2002
1507
6076-7113


B
00126_rhoca
IG
December 2001
2G06-2D11
114767-113670


B
00199_rhoca
IG
December 2001
2G06-2D11
178648-177806


B
00459_rhoca
IG
December 2001
2G06-2D11
434469-435083


B
00460_rhoca
IG
December 2001
2G06-2D11
435094-436191


B
00746_rhoca
IG
December 2001
2D10-2D06
2243-3079


B
00949_rhoca
IG
December 2001
2A12-2D05
325707-326651


B
01216_rhoca
IG
December 2001
2A12-2D05
222590-223555


B
01374_rhoca
IG
December 2001
2A12-2D05
148470-149462


B
01523_rhoca
IG
December 2001
1A01-1C09
279638-280444


B
01524_rhoca
IG
December 2001
1A01-1C09
280569-281423


B
02710_rhoca
IG
December 2001
1D09-1F02
197288-199588


B
03530_rhoca
IG
December 2001
1A01-1C09
521700-521173


B
4825_rhosp
JGI
26 Mar. 2001
184
67785-67165


B
BIL2_rhosp
JGI
26 Mar. 2001
177
 9673-10194


B
BIL1_silpo
TIGR
18 Jun. 2002
50
10679-14440


B
BIL2_silpo
TIGR
18 Jun. 2002
50
  1-2688


B
BIL3_silpo
TIGR
18 Jun. 2002
4
10260-12356


B
BIL4_silpo
TIGR
18 Jun. 2002
290
18579-19640


B
BIL5_silpo
TIGR
18 Jun. 2002
11
3224-2217


B
BIL6_silpo
TIGR
18 Jun. 2002
199
13687-15303


B
BIL7_silpo
TIGR
18 Jun. 2002
199
15104-16399


B
BIL8_silpo
TIGR
18 Jun. 2002
32
1329-2600


B
BIL9_silpo
TIGR
18 Jun. 2002
32
28672-26588


B
BIL10_silpo
TIGR
18 Jun. 2002
60
19338-18265


B
BIL11_silpo
TIGR
18 Jun. 2002
89
10027-11181


B
BIL12_silpo
TIGR
18 Jun. 2002
126
 9020-10066


B
BIL13_silpo
TIGR
18 Jun. 2002
110
13640-14440


B
BIL14_silpo
TIGR
18 Jun. 2002
125
 9769-10353


B
BIL15_silpo
TIGR
18 Jun. 2002
129
7727-8257


B
BIL16_silpo
TIGR
18 Jun. 2002
195
7271-8050


B
Bil1_rhile
Sanger
14 Jul. 2003
RHIZ10E3Cb12.slk
—


B
BIL1_unknwn
TIGR
13 Mar. 2001
14712
1124-51 


B
BIL2_unknwn
TIGR
13 Mar. 2001
12703
  2-1369





*the bacterial species origin of the BIL domains is identified by the bacterial species code following the underscore in the BIL name (refer to Table 2 below for the code-species correspondance).


**The sources are named as follows JGI—Joint Genome Institute (http://www.jgi.doe.gov), NCBI—(http://www.ncbi.nlm.nih.gov), Sanger—The Sanger Institute (http://www.sanger.ac.uk), OU-ACGT—University of Oklahoma, Advanced Center for Genome Technology (http://www.genome.ou.edu), TIGR—The Institute for Genomic Research (http://www.tigr.org), BCM—Baylor College of Medicine Human Genome Sequencing Center (http://www.hgsc.bcm.tmc.edu), IG—IntegratedGenomics (http://www.integratedgenomics.com), and Kazusa—Kazusa DNA Research Institute database (Japan; http://www.kazusa.or.jp).


***Dates refer to the data release dates used. The NCBI entries were extended as noted. Coordinates of the BIL host protein ORF are given for nucleotide contigs/entries. The positions of BILs within these ORFs are provided in Figures 1a-b.


¶An identical sequence was identified in Brucella suis






Conserved motifs in BIL domains, Hog proteins and inteins: Alignments of conserved motifs in Type A BIL domains, Type B BIL domains, Hog proteins, and inteins are shown in FIGS. 3a-g, 3h-o, 3p-t, and 3u-z, respectively. Type A BIL domains were found to share 7 Hint-like consensus sequence motifs (FIGS. 3a-d and 3f-g) and one novel motif (FIG. 3e) having no known Hog or intein counterpart.
Almost all Type A BIL domains were found to comprise apparent functional protein splicing active sites corresponding to those present in inteins (marked by asterisks in FIGS. 3u, 3w and 3z), and several are also flanked at their carboxy terminal ends with serine or threonine amino acid residues, similarly to the carboxy terminal ends of inteins.
Type A BIL domains were found to contain an invariant His-Asn amino acid residue pair adjacent to the carboxy terminal end thereof (FIG. 3g), similarly to the His-Asn amino acid residue pair typically forming the carboxy terminal ends of inteins (FIG. 3z, positions 7-8). Conservation of the Type A BIL domain carboxy terminal end with that of inteins suggests that, similarly to inteins, Type A BILs undergo cyclization of the Asn residue forming the carboxy terminal end thereof. However, the residue at the carboxy terminal end of Type A BIL domains (FIG. 3g, position 8), which corresponds to the residue forming the amino terminal end of polypeptide segments flanking the carboxy terminal ends of inteins which is always Cys, Ser or Thr (FIG. 3z, position 9), is not conserved, since only a few Type A BIL domains have a serine or threonine residue in that position. Other Type A BIL domains have aspartate, glutamate, asparagine, tyrosine or alanine residues in that position, which are not found in any intein.
Type B BIL domains were also found to share 6 Hint-like consensus sequence motifs (FIGS. 3h-i and 3l-o) and two novel motifs (FIGS. 3j-k) having no known Hog protein or intein Hint domain counterpart. Type B BIL domain carboxy terminal ends were found to have a conserved position comprising Cys, Ser or Thr residues (FIG. 3o, position 6), potentially corresponding to the carboxy terminal flanking position of inteins (FIG. 3z, position 9), however this carboxy terminal residue of Type B BIL domains (FIG. 3o, position 7) is not preceded by the His-Asn motif typically found in inteins (FIG. 3z, positions 7-8) and in Type A BIL domains (FIG. 3g, positions 6-7). The —SH/—OH groups on the side chains of the aforementioned Cys, Ser or Thr residues in intein host proteins have been found to be essential for ligation of the intein carboxy and amino flanks in the protein splicing reaction (Xu M Q. and Perler F B., 1996. EMBO J. 15, 5146).
In Type A BIL domains whose carboxy terminal ends are not flanked by Thr or Ser residues, Asn cyclization may nevertheless occur without trans-esterification by the flanking residue. Alternately, trans-esterification may occur by the mildly nucleophilic residues found in this position. In the first case the BIL domain would be cleaved from a segment flanking its carboxy terminal end, and in the second case protein splicing would occur. Since Type B BIL domains do not have any conserved Asn or Gln residue at their carboxy terminal end, cleavage of this end could then proceed by a mechanism different from the Asn and Gln cyclizations of inteins (Paulus H., 2000. Annu Rev Biochem. 69, 447).
Key amino acid residues corresponding to protein splicing active sites (marked by asterisks in FIGS. 3u, 3w, and 3z-position 9) were found to be conserved in Type B BIL domains.
Both Type A and Type B BIL domains were found to be distinct from inteins in having additional unique sequence motifs, in not being integrated in highly conserved sites of essential proteins, and in not comprising endonuclease domains.
Phylogenetic distribution of BIL domains: The phylogenetic distribution of the BIL domains identified is shown in Table 2. BIL domains were identified in 3 evolutionarily distant bacterial types—alpha, beta and gamma proteobacteria (gram-negative bacteria), actinobacteria (high GC gram-positive bacteria), and Bacillus/Clostridium group bacteria (low GC gram-positive bacteria).
Both presence and genomic distribution were found to be variable, even in closely related species and strains. For example, 1, 3 and 6 ORFs encoding BIL domains were identified, respectively, in N. meningitidis strains whose genomes have been completely or almost completely sequenced; 2 and 14 ORFs encoding BIL domains were identified in 2 different Rhodobacter species; and while one such ORF was identified in P. syringae, none were found in P. aeruginosa and P. putida. 
BIL domains and inteins were found to coexist in certain species. For example, the genome of M. magnetotacticum was found to comprise ORFs encoding both Type A and Type B BIL domains, and that of T. fusca was found to comprise ORFs encoding both BIL domains and inteins.
The variability observed in the number of BIL domain ORFs in different species is probably due to gene duplications. As shown in a dendrogram demonstrating phylogenetic relationships of Type A BIL domains (FIG. 4a), all BIL domains derived from Neisseria species cluster together, and BIL domains from different species sub-cluster as well, implying that all Neisseria BIL domains arose by duplication from a single ancestor and that some are paralogs within different species. The latter is corroborated by the apparent duplication of some gene loci containing BIL domains in these species (not shown). Clustering of BIL domains from the same species was also observed in C. thermocellum (FIG. 4a) and M. Magnetotacticum (FIG. 4b).
BIL domain host proteins: BIL domains were identified in putative ORFs coding for a few hundred to a few thousand amino acids. Several BIL domains were found to be flanked by domains present in secreted bacterial proteins. In P. syringae and M. haemolytica, BIL domains were identified near the carboxy terminal end of FhaB-like ORFs. FhaB is a very large Bordetella gene coding for a secreted filamentous hemagglutinin protein, which functions as an adhesin important for B. pertussis virulence (Smith A M. et al., 2001. FEMS Microbiol Rev. 25, 309). Three of the R. capsulatus BIL domain-containing ORFs include RTX repeats—calcium binding repeats found in various secreted bacterial proteins, including many toxins (Coote J G., 1992. FEMS Microbiol Rev. 8, 137). In N. meningitidis and N. gonorrhoeae, BIL domains were identified in MafB proteins. These are part of multiple adhesin family possibly involved in glycolipid adhesion to cells (Naumann et al., 1999. Curr Opin Microbiol 2, 62-70; Paruchuri et al., 1990. Proc Natl Acad Sci U S A. 87, 333-7). Three other Neisseria BIL domains were found to have an HNH nuclease domain in amino acid sequences flanking their carboxy terminal ends. HNH domains are found in various DNase and endonuclease proteins including secreted toxins (Belfort M. and Roberts R J., 1997. Nucleic Acids Res. 25, 3379; James R. et al., 1996. Microbiology 142, 1569). A domain present in the amino acid sequence flanking the carboxy terminal end of a BIL domain in the gram-positive bacterium T. fusca is also found in a short, conserved Salmonella ORF (GenBank accession NP—454902) and in an amino acid sequence flanking the carboxy terminal end of a N. meningitidis FhaB/hemolysin protein (gene NMA0688). Both of these proteins are from gram-negative bacteria and are likely to be secreted.







TABLE 2







Phylogenetic distribution of BIL domains identified.















No. of BIL






domains






identified





BIL
in


Taxonomic group
Species
Species code
type
organism





alpha proteobacteria
Rhodobacter capsulatus SB1003
rhoca
B
14*


alpha proteobacteria
Rhodobacter sphaeroides 2.4.1
rhosp
B
 2*


alpha proteobacteria
Silicibacter pomeroyi DSS-3
silpo
B
16*


alpha proteobacteria
Brucella melitensis 16M/Brucella suis
brume
B
 1


alpha proteobacteria
Magnetospirillum magnetotacticum MS-1
magma
A
 1


alpha proteobacteria


B
 5*


alpha proteobacteria
Methylobacterium extorquens AM1
metex
B
 1*


alpha proteobacteria
Rhizobium leguminosarum bv. viciae 3841
rhile
B
 1


beta proteobacteria
Neisseria meningitidis Z2491
neimeA
A
 1


beta proteobacteria
Neisseria meningitidis MC58
neimeB
A
 3


beta proteobacteria
Neisseria meningitidis FAM18
neimeC
A
 6*


beta proteobacteria
Neisseria gonorrhoeae FA1090
neigo
A
 6


beta proteobacteria
Chromobacterium violaceum ATCC 12472
chrvi
A
 1


gamma
Pseudomonas syringae DC3000
psesy
A
 1*


proteobacteria


gamma
Pseudomonas fluorescens PfO-1
psefl-PfO-1
A
 1*


proteobacteria


gamma
Pseudomonas fluorescens PfSBW25
psefl-SBW25
A
 1*


proteobacteria


gamma
Mannheimia haemolytica PHL213
manha
A
 1*


proteobacteria


delta proteobacteria
Myxococcus xanthus DK1622
myxxa
A
 3*


spirochaetes
Leptospira interrogans 56601
lepin
A
 3


actinobacteria
Streptomyces coelicolor A3(2)
strco
A
 1


actinobacteria
Streptomyces avermitilis MA-468
strav
A
 3


actinobacteria
Thermobifida fusca YX
thefu
A
 1*


Bacillus/Clostridium
Clostridium thermocellum ATCC 27405
cloth
A
10*


group


planctomycetes
Pirellula species 1
pirsp
A
 1


planctomycetes
Gemmata obscuriglobus UQM 2246
gemob
A
 2*


cyanobacteria
Gloeobacter violaceus PCC 7421
glovi
A
 7


verrucomicrobium
Verrucomicrobium spinosum DSM 4136
versp
A
 3*


unknown
Unknown

B
 2*





*Genome not fully sequenced - total number of BILs may be greater.






BIL Domain-Mediated Auto-Cleavage/Auto-Splicing Activity:
Type A BIL domain-mediated auto-cleavage/auto-splicing activity in an in-vitro transcription/translation system: Electrophoretic analysis (FIG. 5a) showed that protein products generated following in-vitro transcription/translation of the MBP-PsyBIL-CBD expression construct pC2C-PsyBIL displayed molecular weights corresponding to the uncleaved precursor MBP-PsyBIL-CBD, the splicing product MBP-CBD, and the carboxy terminal cleavage product MBP-PsyBIL. Two additional protein products displayed molecular weights of 43 and 45 kDa. Control reactions using the MBP-CBD expression construct pC2C as a transcription template produced two protein products, one corresponding in weight to MBP-CBD and the other to the MBP portion thereof. The latter may be observed in chimeric proteins having MBP as an amino terminal tag (refer to: NEB instruction manual “pMAL protein fusion and purification system”, Catalog #E8000S). The 43 kDa protein product may represent a premature transcription or translation stop side product unrelated to BIL domain-mediated activity. Appearance of the 45 kDa band, not seen in the control reaction and slightly larger than the expected weight of MBP, may be due to an additional termination point introduced in the BIL domain. As radioactive methionine was used to label the reaction products, and as, unlike the MBP and BIL domains, the CBD domain lacks a methionine residue, its isolated product cannot be visualized according to the protocol employed.
The relative amounts of the MBP-PsyBIL-CBD, MBP-PsyBIL and MBP-CBD protein products were found to be 15%, 57% and 28%, respectively.
These results therefore demonstrated the capacity of Type A BIL domains to auto-cleave and auto-splice flanking sequences.
Type B BIL domain-mediated auto-cleavage and auto-splicing activity in an in-vitro transcription/translation system: Electrophoretic analysis (FIG. 5b) showed that protein products generated following in-vitro translation of the MBP-RspBIL2-CBD expression construct pC2C-RspBIL2 included proteins with sizes corresponding to the unprocessed MBP-RspBIL2-CBD precursor, the carboxy terminal cleavage product MBP-RspBIL2, the MBP-containing amino terminal cleavage product, and the splicing product MBP-CBD. Also apparent was a 43 kDa MBP-containing fragment which also appeared in the control reaction, as described above.
These results therefore demonstrated the capacity of Type B BIL domains to auto-cleave and auto-splice flanking sequences. The putative protein splicing motifs and catalytic residues of the FhaB_psesy amino acid sequence responsible for the observed autoprocessing activity are shown in FIG. 5c (SEQ ID NO: 107).
Recombinant BIL domains expressed in-vivo in E. coli display auto-cleavage and auto-splicing activity—mass spectrometric confirmation of BIL-mediated autoprocessing activity: In order to examine the possibility of industrially producing functional recombinant Type A BIL domain, pC2C-PsyBIL was overexpressed in-vivo in E. coli, and the recombinant protein products were analyzed for BIL domain-mediated autoprocessing activity.
Similarly to the in-vitro results described above, purified protein from E. coli transformed with pC2C-PsyBIL was found to include a protein product having a molecular weight corresponding to the MBP-PsyBIL carboxy terminal cleavage product, as well as a protein product having a molecular weight corresponding to the MBP-CBD auto-splicing product, as determined by both Coomassie Blue staining and Western immunoblotting analysis of SDS-PAGE separated proteins (FIGS. 6a and 6b, respectively). The main product was again observed to be MBP-PsyBIL protein, as displayed by comparing the quantity thereof produced to that of the MBP-CBD protein product when both were purified using amylose beads (FIG. 6a, lane 3).
The identities of the PsyBIL reaction products, the MBP-CBD and MBP-BIL protein products were also confirmed by mass spectrometry analysis (FIGS. 6c and 6d, respectively). The measured mass of the MBP-CBD protein (50,602.07 Da) was found to be in close agreement to the expected mass of the unmodified protein (50,266.39 Da). The measured and expected masses of MBP-BIL protein were also found to be in close agreement: 59,332.79 and 59,070.11 Da, respectively. A prominent peak with a mass of 43,303 Da, was also observed in MALDI spectra of electroeluted 50 kDa MBP-CBD protein. Such cross-contamination can be observed in gel purified protein bands (A. Shainskaya, unpublished). Reactivity of the 43 kDa band with anti MBP tag antibody (FIG. 6b) indicates that this band is a truncated product.
Peptide mass mapping of the 50.1 and 59.3 kDa protein products proteins by MALDI analysis (Tables 3 and 4, respectively) confirmed their assigned identity as MBP-CBD (FIG. 7; SEQ ID NO: 108) and MBP-BIL (FIG. 8; SEQ ID NO: 109), respectively, in particular by identifying the splice junction of the MBP-CBD protein (peptide position 388-396; FIG. 7; Table 3) and the carboxy terminal end of the MBP-BIL protein (peptide position 535-541; FIG. 8; Table 4) with accuracies of 27 and 100 ppm, respectively.
Thus, following extensive experimentation, the splicing junction and cleavage points were found to precisely correspond to those predicted from the sequence similarity of the BIL and intein domains, thereby unambiguously demonstrating BIL domain autoprocessing capacity.







TABLE 3







MALDI identification of peptides of the 50.1 kDa


MBP-CBD splicing product of MBP-PsyBIL-CBD.













[M + H]+
[M + H]+
Mass



Peptide
calculated
measured
accuracy



position*
mass (Da)
mass (Da)
(ppm)
















1-2
 278.1538
278.1460
28



3-7
 563.2677
563.2599
13



 8-16
1057.604
1057.596
7



 8-26
2047.363
2047.35
6



27-35
1064.532
1064.586
50



28-35
 936.442
936.491
52



28-30
 423.2244
423.215
22



90-99
1267.647
1267.6
37



129-138
1201.522
1201.602
66



129-141
1571.732
1571.823
57



191-201
1188.642
1188.711
58



172-180
1129.55 
1129.57
17



253-274
2137.972
2138.147
81



279-296
2095.812
2096.030
104



297-306
1010.472
1010.612
138



328-345
2109.02 
2109.017
1



356-387
3461.516
3461.33
53



364-387
2576.53 
2576.41
46



388-396
983.48
983.55
71



397-435
3986.34*
3986.13
52



397-438
4380.80*
4379.90
205



439-461
2634.93*
2634.72
79







*mass corresponds to peptide with an alkylated cysteine residue






These results therefore fully and clearly demonstrate the capacity of BIL domains to auto-cleave and auto-splice flanking sequences, similarly to inteins. The presently described BIL domains therefore represent a novel and highly useful class of autoprocessing proteins which can be harnessed for manipulating and modifying proteins, for example as depicted in FIG. 9. These results furthermore demonstrate the suitability of utilizing genetically transformed host cells, such as E. coli, to industrially express chimeric proteins which comprise functional BIL domains.







TABLE 4







MALDI-identification of carboxy terminal peptides


of the 59.3 kDa MBP-PsyBIL carboxy terminal


cleavage product of MBP-PsyBIL-CBD.











[M + H]+
[M + H]+



Peptide
calculated mass
measured mass
Mass accuracy


position
(Da)
(Da)
(ppm)













535-541
897.4947
897.47
27


537-541
656.3156
656.25
100









Discussion: BIL domains are present in several hyper-variable bacterial proteins, such as FhaB adhesins and MafB proteins of Neisseria strains. Their immediate flanks are the most variable portions of the proteins and they themselves are not always present in these proteins, even in closely related strains of the same species. Some, and perhaps all, proteins with BIL domains seem to be secreted proteins. BIL domains might enhance the variability of secreted proteins by their protein splicing and cleavage activity as detailed below.
As described above, the amino terminal ends of BIL domains, and of Hint domains of inteins and Hog proteins are very similar (FIGS. 3a-z). Thus all these domains probably form labile ester bonds on their amino terminal ends. In proteins with BIL domains these ester bonds could be attacked by various nucleophilic molecules, including peptides, proteins and small reactive compounds, such as glutathione or cysteine. Such reactions would ligate the attacking nucleophiles to a carboxy terminal position of the host protein and release the BIL domain and the host protein region downstream to it. This is analogous to Hedgehog protein maturation where the Hint domain mediates the attachment of a cholesterol molecule to the cleaved Hedge domain. In adhesins with BIL domains this putative ligation might serve to covalently attach the bacteria to its adhesion target. Additionally, released BIL and carboxy terminal domains could have a function of their own. For example, in pathogenic bacteria that have such proteins, the released domains could serve as decoys to the immune system.
In Neisseria strains, sequences encoding BIL domains appear as either as short open reading frames downstream of MafB genes and in the carboxy terminal ends of these proteins upstream of a variable domain. This suggests that, at least in such Neisseria strains, BIL domains function as cassettes which can be fused to genes by genetic rearrangement to promote the variability of the encoded proteins. Other microevolutionary processes in Neisseria and Ralstonia solanacearum, a plant pathogen bacterium with a wide host range, are known to generate different carboxy terminal ends for surface-exposed and virulence proteins (Parkhill et al., 2000. Nature, 404, 502-506; Salanoubat et al., 2002. Nature 415, 497-502).
Not all species with BIL domains are pathogens and many pathogenic bacteria with fully sequenced genomes do not have BIL domains. BIL domains might be used in different processes not connected with pathogenicity. For example, BIL domain activity might be one way for bacteria to attach to diverse surfaces.
In summary, two novel types of Hint domain-containing proteins, BIL Types A and B, were identified. Both types have the active site sequence features of the Hint domains but also possess sequence features that distinguish them from the known Hint domains and from each other. BIL domains appear in different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and P. syringae. These domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous hemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL domain sequences suggests that they were positively selected for in different lineages. Type A and Type B BIL domains were cloned and shown to display auto-cleavage and auto-splicing of flanking polypeptide sequences in an in-vitro transcription/translation system, as well as when overexpressed in E. coli, thereby indicating the capacity of BIL domains to autocatalyze post-translational modifications of host proteins.
Conclusion: The above-described experimental results demonstrate the capacity of the autoprocessing polypeptides of the present invention to efficiently auto-cleave and auto-splice flanking sequences. The presently described experimental results furthermore demonstrate the feasibility of utilizing genetically transformed host cells, such as E. coli, for efficient industrial production of such autoprocessing polypeptides. Thus, the autoprocessing polypeptides and the polynucleotides encoding such polypeptides of the present invention significantly expand and enhance the available repertoire of available autoprocessing polypeptides having utility in numerous commercially important protein engineering applications, such as protein purification, affinity selection of display phages and post-translational protein ligation.
Example 2
Supplementary Evidence Demonstrating C-Terminal In-Vitro and In-Vivo Autocleavage by Chimeric Protein Including the Type B BIL Domain BIL2_rhosp
Materials and Methods:
In order to analyze the capacity of Type B BIL domain BIL2_rhosp (Table 1, FIG. 1b) to display autoprocessing activity, genetic sequences encoding this BIL domain including one flanking amino acid residue at each terminus were cloned for expression as a chimeric protein tagged at its amino terminal end with the malE gene-encoded maltose-binding protein (MBP) affinity tag, and at the carboxy terminal end with the B. circulans cbd gene-encoded chitin-binding domain (CBD) affinity tag. The resultant “MBP-RspBIL2a-CBD” chimera was expressed in-vivo and in-vitro and resulting protein products were analyzed for evidence of BIL domain-mediated autoprocessing activity according to methods described in Example 1 above.
Experimental Results:
The in-vivo expressed MBP-RspBIL2a-CBD chimera was shown to display C-terminal auto-cleavage activity via amylose-based affinity purification, electrophoretic separation, and Coomassie blue staining of the electrophoretically separated proteins (FIG. 10a). The in-vivo expressed MBP-RspBIL2a-CBD chimera was also shown to display C-terminal cleavage via Western immunoblotting analysis of SDS-PAGE separated proteins (FIG. 10b). The in-vitro expressed MBP-RspBIL2a-CBD chimera was shown to display C-terminal cleavage following [35S]-methionine-labeling and autoradiography of electrophoretically separated protein (FIG. 10c). Evidence from intact mass mass-spectrometry of the MBP-BIL specific carboxy terminal cleavage product indicates that the C-terminal end of this product is located 6-11 amino acid residues from the predicted carboxy terminal end of the BIL domain towards the N-terminus. Evidence for the MBP-RspBIL2a identity of the cleavage product was obtained from Western Blot, and affinity column analysis. The presence of the protein chaperone DnaK was detected during affinity purification of the BIL products. This chaperone may bind the BIL domain, and may also be involved in its activity.
Conclusion: There is ample evidence that a chimeric protein which comprises the type B BIL domain BIL2_rhosp has the capacity to efficiently display carboxy terminal auto-cleavage activity. Hence, such a BIL domain can therefore be advantageously exploited in applications benefiting from an auto-cleaving chimeric protein.
Example 3
N-Terminal Autocleavage of In-Vivo Expressed Chimeric Polypeptide Comprising the Type B BIL Domain 4825_rhosp
Materials and Methods:
In order to analyze the capacity of Type B BIL domain 4825_rhosp (Table 1, FIG. 1b) to display autoprocessing activity, genetic sequences encoding this BIL domain with 14 amino terminal-flanking and 51 carboxy terminal-flanking amino acids were cloned for expression as a chimeric protein tagged at its amino terminal end with the malE gene-encoded maltose-binding protein (MBP) affinity tag, and at the carboxy terminal end with the B. circulans cbd gene-encoded chitin-binding domain (CBD) affinity tag. The resultant “MBP-4825rhosp-CBD” chimera was expressed in-vivo and resulting protein products were analyzed for evidence of BIL domain-mediated autoprocessing activity according to methods described in Example 1 above.
Experimental Results:
The in-vivo expressed MBP-4825rhosp-CBD chimera was shown to display N-terminal auto-cleavage activity via chitin or amylose based affinity purification of expressed protein and electrophoretic separation, and Coomassie blue staining of electrophoretically separated protein (FIG. 11a). The chimera was also shown to display N-terminal cleavage via Western immunoblotting analysis of SDS-PAGE separated proteins using anti-CBD and anti-MBP antibodies (FIG. 11b). The cleavage site was found to be located exactly between the predicted N-terminal residue of the BIL domain and the amino terminal flanking sequence as demonstrated by amino terminal end sequencing. Large amounts of the chaperone GroEL were detected during the purification of the protein products when overexpressing the MBP-4825rhosp-CBD chimera the whereas no GroEL was detected when overexpressing control chimera lacking the BIL domain.
Conclusion: A chimeric protein which comprises the type B BIL domain 4825_rhosp has the capacity to efficiently display N-terminal auto-cleavage activity. Hence, such a BIL domain can therefore be advantageously exploited in applications benefiting from an auto-cleaving chimeric protein.
Example 4
Auto-Splicing and C-Terminal Auto-Cleavage of In-Vivo Expressed Chimeric Protein Including the Type A BIL Domain BIL4_cloth
Materials and Methods:
In order to analyze the capacity of Type A BIL domain BIL4_cloth (Table 1, FIG. 1a) to display autoprocessing activity, genetic sequences encoding this BIL domain were cloned, including one flanking amino acid residue of the host protein at the C-terminal end, for expression as a chimeric protein tagged at its amino terminal end with the malE gene-encoded maltose-binding protein (MBP) affinity tag, and at the carboxy terminal end with the B. circulans cbd gene-encoded chitin-binding domain (CBD) affinity tag. The resultant “MBP-BIL4cloth-CBD” chimera was expressed in-vivo and resulting protein products were analyzed for evidence of BIL domain-mediated autoprocessing activity according to methods described in Example 1 above.
Experimental Results:
The in-vivo expressed MBP-BIL4cloth-CBD chimera was shown to display auto-splicing activity (MBP-CBD specific fragment) as shown by Western immunoblotting assay using anti CBD and anti MBP antibody probes (FIGS. 12a and 12b, respectively), and as shown by Coomassie blue staining of electrophoretically separated protein products isolated via amylose based or chitin based affinity chromatography depicting auto-splicing activity. The identity of the auto-splicing product was verified via mass-spectrometry peptide mapping (data not shown). Carboxy terminal auto-cleavage activity (MBP-BIL domain specific fragment) was also detected by Western immunoblotting assay using an anti MBP antibody probe (FIG. 12b) and by Coomassie blue staining of electrophoretically separated protein products isolated via amylose based affinity chromatography (FIG. 12c).
Conclusion: A chimeric protein which comprises A type a BIL domain, such as the type A BIL domain BIL4_cloth, has the capacity to efficiently display auto-splicing and carboxy terminal auto-cleaving activity. Hence, such a BIL domain can therefore be advantageously exploited in applications benefiting from an auto-splicing and/or carboxy terminal auto-cleaving chimeric protein.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications and sequences identified by their accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, patent application or sequence identified by their accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

## Claims

1. A chimeric polypeptide comprising an autoprocessing segment having the amino acid sequence set forth by SEQ ID NO: 31.

2. The chimeric polypeptide of claim 1, wherein a carboxy terminal end of said autoprocessing segment is attached to a first polypeptide segment and further wherein said autoprocessing results in removal of said first polypeptide segment.

3. The chimeric polypeptide of claim 1, wherein said autoprocessing segment is flanked by a first and a second polypeptide segments and further wherein said autoprocessing results in splicing of said first and said second polypeptide segments.

4. The chimeric polypeptide of claim 2, wherein said segment of the polypeptide adjacent to said carboxy terminal end of said autoprocessing segment includes an amino acid residue comprising a nucleophilic group at an amino terminal end thereof.

5. The chimeric polypeptide of claim 4, wherein said nucleophilic group is a hydroxyl group.

6. The chimeric polypeptide of claim 4, wherein said amino acid residue is a threonine residue.

7. The chimeric polypeptide of claim 2, wherein a segment of the chimeric polypeptide adjacent to an amino terminal end of said autoprocessing segment includes a serine amino acid residue at a carboxy terminal end thereof.

8. The chimeric polypeptide of claim 1, wherein said autoprocessing results in auto-splicing.

9. The chimeric polypeptide of claim 8, wherein said auto-splicing is auto-splicing of segments of the polypeptide flanking said autoprocessing segment.

10. The chimeric polypeptide of claim 1, wherein the chimeric polypeptide is active in said autoprocessing under a condition selected from the group consisting of a temperature selected from a range of 33° C. to 41° C., a pH selected from a range of pH 7.8 to pH 8.2, and a concentration of dithiothreitol selected from a range of 0.1 mM to 20 mM.

11. The chimeric polypeptide of claim 1, further comprising an affinity tag capable of specifically binding a molecule.

12. The chimeric polypeptide of claim 11, wherein said affinity tag is a maltose-binding domain or a chitin-binding domain.

13. The chimeric polypeptide of claim 11, wherein said molecule is displayed on a virus or a cell.

14. The chimeric polypeptide of claim 11, wherein said molecule is amylose or chitin.

15. The chimeric polypeptide of claim 13, wherein said virus is a bacteriophage.