PEGylated tetanus neurotoxin and the treatment of hypotonia

Abstract

The present invention relates to a composition comprising: a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT) and a second tetanus neurotoxin (TeNT), wherein the PEG-TeNT comprises a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG). The present invention also relates to various PEG-TeNTs. The present invention also relates to a method of treating hypotonia using the composition or various PEG-TeNTs and a kit comprising the composition or various PEG-TeNTs. In one embodiment, the hypotonia is obstructive sleep apnea.

Description

Technical Field

[0001] The present invention relates to a composition comprising a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) and a second TeNT. The present invention also relates to a method of treating hypotonia using said composition. Background Art

[0002] Tetanus neurotoxin (TeNT) is produced by Clostridium tetani. TeNT acts on the spinal cord and blocks the release of γ-aminobutyric acid (GABA) and glycine, which are inhibitory neurotransmitters, at spinal inhibitory interneurons. Thus, TeNT causes spastic paralysis. There are no multiple serotypes of TeNT.

[0003] The use of the biological properties of TeNT or at least TeNT fragments for therapeutic purposes has been proposed. However, long-term treatment with protein therapeutics often leads to a targeted immune response. Since only one serotype of TeNT is known, serotype switching cannot be selected for TeNT-based therapies to avoid immunity. In addition, many populations have been vaccinated against TeNT, thus precluding TeNT-based therapies.

[0004] US 2002 / 0197278 A1 discloses the use of polyethylene glycolylated botulinum toxin for treating inappropriate muscle contraction disorders. US 2002 / 0197278 A1 also proposes the use of polyethylene glycolylated TeNT for treating inappropriate muscle contraction disorders such as migrainous headache or strabismus. However, as described above, TeNT causes muscle contraction, thus precluding its use, whether polyethylene glycolylated or not, for treating inappropriate muscle contraction disorders.

[0005] Wan et al., Process Biochemistry (2017) 52:183-191 discloses the effect of polyethylene glycolylation on the anti-PEG immune response generated by the administration of polyethylene glycolylated proteins, but does not utilize its findings for any treatment.

[0006] WO 2016 / 001762 A1 discloses the use of polyethylene glycolylated TeNT fragment c(c) for increasing muscle mass. When TeNT is digested by papain, fragment c (50 kDa) is produced, and this fragment c corresponds to 451 amino acids at the C-terminus of the TeNT heavy chain. Fragment c retains the binding, internalization, and trans-synaptic transport capabilities of undigested TeNT, but does not disrupt any neuronal processes and is thus non-toxic.

[0007] There is a need for a TeNT-based treatment that avoids pre-existing anti-TeNT immune responses in subjects who have been immunized with tetanus toxoid.

[0008] It should be understood that if any prior art publication is cited herein, such citation does not constitute an admission that the publication forms part of the common general knowledge in the art in Australia or any other country. Summary of the Invention

[0009] The inventors of the present application have recognized that due to the adaptive immune system, the utilization of TeNT has not been fully realized. When a protein therapeutic agent is administered, the adaptive immune system generates an antibody response, thereby reducing the efficacy of the protein therapeutic agent. As a result of vaccination, the adaptive immune response may be intentional, as demonstrated by many populations that have been immunized against TeNT. Alternatively, the adaptive immune response may be unintentional, resulting from repeated exposure to the protein therapeutic agent.

[0010] The inventors of the present application have generated a family of modified TeNTs and treatment regimens that address these problems. Specifically, the present invention provides a family of pegylated TeNTs (PEG-TeNTs) that each evade the immune system and a graded treatment regimen, wherein different alternative PEG-TeNTs are used for treatment when the efficacy of a previously administered PEG-TeNT decreases or when the immunological characteristics of the patient preclude the use of another PEG-TeNT.

[0011] The first aspect provides a tetanus neurotoxin (TeNT) or a fragment thereof that comprises one or more surface serine→cysteine amino acid substitutions relative to SEQ ID NO:1.

[0012] In one embodiment of the first aspect, the substituted cysteine is conjugated to polyethylene glycol (PEG).

[0013] The second aspect provides a TeNT or a fragment thereof that, relative to SEQ ID NO:1, comprises: R1225K; R1225E; W1288A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; R1225del; W1288del; or R1225del and W1288del; E270A; Y374A; E270A and Y374; G270del; Y374del; or a combination thereof, wherein the TeNT or fragment is inactive.

[0014] The third aspect provides a composition comprising: (i) a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT) that comprises a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT, wherein the first PEG-TeNT is not conjugated to the second TeNT.

[0015] The fourth aspect provides a method for treating hypotonia, the method comprising administering to a subject: (i) a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT) that comprises a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT.

[0016] The fourth aspect also provides the use of a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT) that comprises a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG) in the manufacture of a medicament for treating hypotonia in a subject to whom a second TeNT has been administered.

[0017] The fourth aspect also provides the use of a second tetanus neurotoxin (TeNT) in the manufacture of a medicament for treating hypotonia in a subject who has received a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT), the first polyethylene glycolylated tetanus neurotoxin comprising a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

[0018] The fourth aspect also provides the use in the manufacture of a medicament for treating hypotonia of: (i) a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT) that comprises a tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT.

[0019] The fourth aspect also provides a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG), which is used in a method for treating hypotonia in a subject administered a second TeNT.

[0020] The fourth aspect also provides a second tetanus neurotoxin (TeNT) which is used in a method for treating hypotonia in a subject administered a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT), wherein the first polyethylene glycolylated tetanus neurotoxin comprises tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG).

[0021] The fourth aspect also provides: (i) a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT; the composition being used in a method for treating hypotonia.

[0022] In an embodiment of the fourth aspect, the composition may comprise the first PEG-TeNT and the second TeNT.

[0023] The fourth aspect also provides a composition comprising: (i) a first polyethylene glycolylated tetanus neurotoxin (PEG-TeNT) comprising tetanus neurotoxin (TeNT) conjugated to polyethylene glycol (PEG); and (ii) a second TeNT, the composition being used in a method for treating hypotonia.

[0024] In one embodiment, the first PEG-TeNT or the second TeNT comprises polyethylene glycolylated TeNT light chain (LC), polyethylene glycolylated TeNT heavy chain (HC), polyethylene glycolylated TeNT heavy chain (HC) and polyethylene glycolylated TeNT light chain (LC), or polyethylene glycolylated TeNT fragment c (c). In one embodiment, the first PEG-TeNT or the second TeNT comprises PEG-TeNT-LC-HC.

[0025] In another embodiment, the first PEG-TeNT or the second TeNT is PEG-TeNT-HC comprising polyethylene glycolylated HC. In this embodiment, LC is not polyethylene glycolylated. In another embodiment, the first PEG-TeNT or the second TeNT is PEG-TeNT-LC-c comprising polyethylene glycolylated LC and polyethylene glycolylated c. In this embodiment, HN is not polyethylene glycolylated.

[0026] In another embodiment, the first PEG-TeNT is PEG-TeNT-HC, and the second TeNT is PEG-TeNT-LC-c.

[0027] In one embodiment, the second TeNT comprises inactivated TeNT. Relative to SEQ ID NO:1, the inactivated TeNT may comprise: R1225K; R1225E; W1288A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; R1225del; W1288del; R1225del or W1288del; E270A; Y374A; E270A and Y374A; G270del; Y374del; or a combination thereof. In one embodiment, the second TeNT comprises inactivated TeNT, and the inactivated TeNT comprises R1225E and W1288A.

[0028] In one embodiment, the subject is administered: PEG-TeNT (PEG-TeNT-c) comprising pegylated c until efficacy decreases; then a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c until efficacy decreases; then PEG-TeNT (PEG-TeNT-LC-HC) comprising pegylated LC and pegylated HC.

[0029] In one embodiment, treatment comprises administering to the subject: PEG-TeNT (PEG-TeNT-c) comprising pegylated c; then a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c; then PEG-TeNT (PEG-TeNT-LC-HC) comprising pegylated LC and pegylated HC to determine the immunological profile of the anti-TeNT antibodies of the subject and to determine an effective composition of PEG-TeNT based on that profile.

[0030] In one embodiment, the hypotonia is obstructive sleep apnea.

[0031] In another embodiment, the composition is a therapeutic composition. In another embodiment, the composition is a cosmetic composition.

[0032] Also disclosed is a method for preparing a protein (LC-HN) consisting of TeNT LC and TeNT HN, the method comprising expressing a nucleic acid molecule encoding an amino acid sequence comprising SEQ ID NO:6 in a host cell.

[0033] The fifth aspect provides a polyethylene glycolylated tetanus neurotoxin (PEG-TeNT), which comprises a tetanus neurotoxin (TeNT) conjugated with polyethylene glycol (PEG).

[0034] In one embodiment, the TeNT light chain (LC) is polyethylene glycolylated, the TeNT heavy chain (HC) is polyethylene glycolylated, or the TeNT fragment c (c) is polyethylene glycolylated. In one embodiment, LC is polyethylene glycolylated and HC is polyethylene glycolylated (PEG-TeNT-LC-HC), or LC is polyethylene glycolylated and c is polyethylene glycolylated (PEG-TeNT-LC-c).

[0035] In another embodiment, PEG is conjugated to a lysine residue of TeNT. In another embodiment, PEG is conjugated to a cysteine residue of TeNT. In one embodiment, PEG is conjugated to a cysteine residue of TeNT, wherein the cysteine residue is native or a substitution of a serine residue relative to SEQ ID NO:1.

[0036] In one embodiment, PEG has a molecular weight of about 2 kDa, about 5 kDa, about 10 kDa, or about 20 kDa or about 30 kDa.

[0037] The sixth aspect provides a method for treating hypotonia, the method comprising administering to a subject the PEG-TeNT of the fifth aspect, wherein the PEG-TeNT is not conjugated to a second TeNT.

[0038] The sixth aspect also provides the use of the PEG-TeNT of the fifth aspect in the manufacture of a medicament for treating hypotonia, wherein the PEG-TeNT is not conjugated to a second TeNT.

[0039] Alternatively, the sixth aspect provides the PEG-TeNT of the fifth aspect for use in a method for treating hypotonia, the method comprising administering the PEG-TeNT to a subject, wherein the PEG-TeNT is not conjugated to a second TeNT.

[0040] In one embodiment, a first PEG-TeNT (PEG-TeNT-HC) comprising polyethylene glycolylated HC and a second PEG-TeNT (PEG-TeNT-LC-c) comprising polyethylene glycolylated LC and polyethylene glycolylated c are administered to the subject.

[0041] In another embodiment, the subject is administered: PEG-TeNT-c comprising a polyethylene glycolylated c; and / or a first PEG-TeNT (PEG-TeNT-HC) comprising a polyethylene glycolylated HC and a second PEG-TeNT (PEG-TeNT-LC-c) comprising a polyethylene glycolylated LC-c; and / or a PEG-TeNT (PEG-TeNT-LC-HC) comprising a polyethylene glycolylated HC and a polyethylene glycolylated LC.

[0042] In one embodiment, the treatment comprises administering to the subject: PEG-TeNT-c comprising a polyethylene glycolylated c until the efficacy decreases. Thereafter, the treatment may comprise administering to the subject: PEG-TeNT-HC and PEG-TeNT-LC-c until the efficacy decreases. Thereafter, the treatment may comprise administering to the subject: a PEG-TeNT (PEG-TeNT-LC-HC) comprising a polyethylene glycolylated LC and a polyethylene glycolylated HC.

[0043] In one embodiment, the treatment comprises administering to the subject: PEG-TeNT-c comprising a polyethylene glycolylated c until the efficacy decreases; then administering PEG-TeNT-HC and PEG-TeNT-LC-c until the efficacy decreases; then administering a PEG-TeNT (PEG-TeNT-LC-HC) comprising a polyethylene glycolylated LC and a polyethylene glycolylated HC.

[0044] In one embodiment, the treatment comprises administering to the subject: PEG-TeNT-c comprising a polyethylene glycolylated c, which comprises PEG having a molecular weight of about 5 kDa, until the efficacy decreases; then administering PEG-TeNT-c comprising a polyethylene glycolylated c, which comprises PEG having a molecular weight of about 10 kDa, until the efficacy decreases; then administering PEG-TeNT-c comprising a polyethylene glycolylated c, which comprises PEG having a molecular weight of about 20 kDa, until the efficacy decreases. Thereafter, the treatment may comprise administering to the subject: PEG-TeNT-HC and PEG-TeNT-LC-c, either or both of which comprise PEG having a molecular weight of 5 kDa, until the efficacy decreases; then administering PEG-TeNT-HC and PEG-TeNT-LC-c, either or both of which comprise PEG having a molecular weight of 10 kDa, until the efficacy decreases; then administering PEG-TeNT-HC and PEG-TeNT-LC-c, either or both of which comprise PEG having a molecular weight of 20 kDa, until the efficacy decreases. Thereafter, the treatment may comprise administering to the subject: PEG-TeNT (PEG-TeNT-LC-HC) comprising a polyethylene glycolylated LC and a polyethylene glycolylated HC, either or both of which comprise PEG having a molecular weight of 5 kDa, until the efficacy decreases; then administering PEG-TeNT (PEG-TeNT-LC-HC) comprising a polyethylene glycolylated LC and a polyethylene glycolylated HC, either or both of which comprise PEG having a molecular weight of 10 kDa, until the efficacy decreases; then administering PEG-TeNT (PEG-TeNT-LC-HC) comprising a polyethylene glycolylated LC and a polyethylene glycolylated HC, either or both of which comprise PEG having a molecular weight of 20 kDa.

[0045] In one embodiment, the treatment comprises administering to the subject: PEG-TeNT-c comprising a polyethylene glycolylated c; then administering a composition comprising PEG-TeNT-HC and PEG-TeNT-LC-c; then administering PEG-TeNT (PEG-TeNT-LC-HC) comprising a polyethylene glycolylated LC and a polyethylene glycolylated HC to determine the immunological profile of the anti-TeNT antibodies of the subject and to determine an effective composition of PEG-TeNT based on that profile.

[0046] In another embodiment, treatment with TeNT also includes administering inactivated TeNT. Relative to SEQ ID NO:1, the inactivated TeNT can include: R1225K; R1225E; W1228A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225E and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; R1225del; W1288del; R1225del or W1288del. In one embodiment, the second TeNT includes inactivated TeNT, and the inactivated TeNT includes R1225E and W1288A; E270A; Y374A; E270A and Y374A; G270del; Y374del; or a combination thereof.

[0047] In one embodiment, the hypotonia is obstructive sleep apnea.

[0048] The seventh aspect provides a kit, which includes the TeNT of the first aspect, the composition of the second aspect, or the PEG-TeNT of the fifth aspect.

[0049] In one embodiment, the composition or PEG-TeNT is used according to the methods of the third aspect or the sixth aspect, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a schematic diagram of an exemplary PEG-TeNT of the present invention: A is PEG-TeNT-c; B is PEG-TeNT-HC; C is PEG-TeNT-LC-c; D is PEG-TeNT-LC-HC, where the TeNT is complete and active, that is, TeNT includes two chains, and polyethylene glycolation exists in specific regions, namely c, HC, LC-c or LC-HC.

[0051] Figure 2 is the amino acid sequence (SEQ ID NO:1) of the mature TeNT containing 1314 amino acids.

[0052] Figure 3 is the nucleic acid sequence (SEQ ID NO:2) of the vector pRSET-TeNT encoding TeNT.

[0053] Figure 4It is a figure of the vector pRSET-TeNT encoding TeNT. TeNT is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0054] Figure 5 is the amino acid sequence of the HC (SEQ ID NO:3) containing amino acids 457 to 1314 of SEQ ID NO:1.

[0055] Figure 6 is the amino acid sequence of the c (SEQ ID NO:4) containing amino acids 864 to 1314 of SEQ ID NO:1.

[0056] Figure 7 is the nucleic acid sequence (SEQ ID NO:5) of the vector pRSET-TeNT-c encoding c.

[0057] Figure 8 It is a figure of the vector pRSET-TeNT-c encoding c. c is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0058] Figure 9 is the amino acid sequence of the LC-HN (SEQ ID NO:6) containing amino acids 1 to 863 of SEQ ID NO:1.

[0059] Figure 10 is the nucleic acid sequence (SEQ ID NO:7) of the vector pRSET-TeNT-LC-HN encoding LC-HN.

[0060] Figure 11 It is a figure of the vector pRSET-TeNT-LC-HN encoding LC-HN. LC-HN is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0061] Figure 12 is the amino acid sequence of the non-functional c (SEQ ID NO:8) containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1.

[0062] Figure 13 is the nucleic acid sequence (SEQ ID NO:9) of the vector pRSET-TeNT-c encoding the non-functional c of Figure 12 (SEQ ID NO:8) containing the amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1.

[0063] Figure 14It is a diagram of vector pRSET-TeNT-c encoding non-functional c (SEQ ID NO:8) of Figure 12 containing amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1. The c containing amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1 is expressed with an N-terminal His6 tag. This nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0064] Figure 15 is the amino acid sequence (SEQ ID NO:10) of non-functional TeNT containing amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1.

[0065] Figure 16 is the nucleic acid sequence (SEQ ID NO:11) of vector pRSET-TeNT encoding non-functional TeNT of Figure 15 containing amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1.

[0066] Figure 17 It is a diagram of vector pRSET-TeNT encoding non-functional TeNT (SEQ ID NO:10) of Figure 15 containing amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1. The TeNT containing amino acid substitutions W1288A and R1225E relative to SEQ ID NO:1 is expressed with an N-terminal His6 tag. This nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0067] Figure 18 is the amino acid sequence (SEQ ID NO:12) of mature TeNT with 1314 amino acids, which contains amino acid substitutions S81C, S120C, S144C, S248C, S335C, S428C, S600C, S963C, S1041C, S1155C, and S1187C of surface serine to cysteine relative to SEQ ID NO:1.

[0068] Figure 19 is the nucleic acid sequence (SEQ ID NO:13) of vector pRSET-TeNT encoding mature TeNT (SEQ ID NO:12) with surface serine substituted by cysteine of Figure 18.

[0069] Figure 20 isFigure of vector pRSET-TeNT (SEQ ID NO:13) of Figure 19 encoding mature TeNT (SEQ ID NO:12) with surface serine replaced by cysteine in Figure 18. TeNT with surface serine replaced by cysteine is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0070] Figure 21 is the amino acid sequence of mature TeNT with 1314 amino acids (SEQ ID NO:14), which, relative to SEQ ID NO:1, contains amino acid substitutions of surface serine→cysteine, S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C in the LC region and the c region.

[0071] Figure 22 is the nucleic acid sequence of vector pRSET-TeNT encoding mature TeNT (SEQ ID NO:14) with surface serine replaced by cysteine in Figure 21 (SEQ ID NO:15).

[0072] Figure 23 Figure of vector pRSET-TeNT (SEQ ID NO:15) of Figure 22 encoding mature TeNT (SEQ ID NO:14) with surface serine replaced by cysteine in Figure 21. TeNT with surface serine replaced by cysteine is expressed with an N-terminal His6 tag. The nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0073] Figure 24 is the amino acid sequence of TeNT (SEQ ID NO:16) containing amino acid substitutions of HC surface serine→cysteine, S600C, S963C, S1041C, S1155C, and S1187C relative to SEQ ID NO:1.

[0074] Figure 25 is the nucleic acid sequence of vector pRSET-TeNT encoding mature TeNT (SEQ ID NO:16) with surface serine replaced by cysteine in Figure 24 (SEQ ID NO:17).

[0075] Figure 26It is a figure of vector pRSET-TeNT (SEQ ID NO:17) in Figure 25 encoding TeNT (SEQ ID NO:16) with surface serine substituted by cysteine in Figure 24. TeNT with surface serine substituted by cysteine is expressed with an N-terminal His6 tag. This nucleic acid is inserted into the MCS of the pRSET-A vector and expressed under the control of the T7 promoter.

[0076] Figure 27 It is a 3D protein structure model of TeNT derived from crystallographic data stored in the Protein Data Bank (accession ID PDB: 5N0B), on which the epitopes recognized by major human antibody clonotypes (identified by da Silva Antunes et al. (2017) and Palermo et al. (2017)) are located using Discovery Studio. Surface serine residues in or near the identified epitopes are selected to be mutated to cysteine for subsequent PEGylation.

[0077] Figure 28 It includes two photos of SDS-PAGE analysis of PEG-TeNT, where PEG-TeNT contains PEG with increasing molecular weights, and (A) is detected using Coomassie blue and (B) is detected by Western blot using a polyclonal anti-TeNT antibody. (B) shows that the immunogenicity is proportional to the PEG molecular weight. The left margin has a molecular weight marker in kDa: lane 1; TeNT lane 2; 2 kDa PEG-TeNT-HC-LC lane 3; 5 kDa PEG-TeNT-LC-HC lane 4; 10 kDa PEG-TeNT-LC-HC lane 5; 20 kDa PEG-TeNT-LC-HC lane 6.

[0078] Figure 29 packageFour line graphs representing competitive ELISA assays. Four PEG-TeNTs and four PEG-TeNT-LC-c serine mutants each containing PEGs of different molecular weights (2 kDa, 5 kDa, 10 kDa, and 20 kDa) were assayed for TeNT using a polyclonal anti-TeNT antibody. (A) TeNT was adsorbed onto the ELISA plate and then probed with a polyclonal anti-TeNT antibody pre-incubated with each of the four PEG-TeNT antigens (2 kDa, 5 kDa, 10 kDa, and 20 kDa) at four concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL). (B) Each of the PEG-TeNTs (2 kDa, 5 kDa, 10 kDa, and 20 kDa) was adsorbed onto a separate ELISA plate and then probed with a polyclonal anti-TeNT antibody pre-incubated with each of the TeNT antigens at four concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL). (C) The TeNT-LC-c serine mutant was adsorbed onto the ELISA plate and then probed with a polyclonal anti-TeNT antibody pre-incubated with each of the four PEG-TeNT-LC-c serine mutant antigens (2 kDa, 5 kDa, 10 kDa, and 20 kDa) at four concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL). (D) Each of the PEG-TeNT-LC-c serine mutants (2 kDa, 5 kDa, 10 kDa, and 20 kDa) was adsorbed onto a separate ELISA plate and then probed with a polyclonal anti-TeNT antibody pre-incubated with each of the TeNT-LC-c serine mutant antigens at four concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL).

[0079] Figure 30 package Four photographs showing local limb twitching in the hindlimbs of female C57BL / 6 mice that received an injection of PEG-TeNT-LC-HC are presented. A and B show local limb twitching in mice that had not undergone the experiment. C shows no limb twitching after injection of 100 units of TeNT in mice vaccinated with tetanus toxoid vaccine, while D shows persistent limb twitching after injection of 80 units of PEG-TeNT LC-HC 20 kDa in mice vaccinated with tetanus toxoid vaccine.

[0080] Figure 31Include two dot plots showing the clinical tetanus levels exhibited in the hind limbs of female C57BL / 6 mice after injection of a defined number of units of TeNT, PEG-TeNT-LC-HC 20 kDa, TeNT-LC-c serine mutant, PEG-TeNT-LC-c serine mutant 2 kDa, or PEG-TeNT-LC-c serine mutant 20 kDa. A) Mice were injected with TeNT or PEG-TeNT-LC-HC 20 kDa. B) Mice were injected with TeNT, TeNT-LC-c serine mutant, PEG-TeNT-LC-c serine mutant 2 kDa, or PEG-TeNT-LC-c serine mutant 20 kDa. 1 unit is the minimum amount of toxin required to produce stage IV convulsions in 24 hours in naïve mice.

[0081] Figure 32 Include two line plots showing the progression of clinical convulsions in mice vaccinated with tetanus toxoid vaccine. A shows the progression of convulsions after injection of 4000 units of TeNT in the presence or absence of inactivated TeNT bait. B shows the progression of convulsions after injection of 40 units of TeNT-PEG 20 kDa in the presence or absence of inactivated TeNT bait. In both cases, the presence of the bait significantly promoted the progression.

[0082] Figure 33 Is a box plot showing the effect of increasing the dose of tetanus toxin on the respiratory disturbance index (RDI) of English bulldogs treated according to Example 23. The black bars represent the median RDI of six studies conducted at each tetanus toxin dose. The shaded boxes represent the interquartile range and the brackets represent the minimum and maximum RDI samples at each dose. Compared to the RDI after administration of placebo, after administration of 10 IU / kg, increasing the tetanus toxin was associated with a decrease in RDI (P = 0.043; Wilcoxon Signed Ranks test). The medians of placebo and 10 IU / kg are additionally indicated by ○ and *, respectively. Detailed Description

[0083] The present invention relates to TeNT and PEG-TeNT molecules that evade immunity ( Figure 1 ), their compositions, and their therapeutic and cosmetic uses. In one embodiment, the present invention relates to treating hypotonia, optionally obstructive sleep apnea.

[0084] This text describes the use of specifically modified TeNT and compositions of TeNT and a decoy (inactive TeNT) for treating hypotonia in subjects with a protective immune response against tetanus toxoid. To achieve this goal, active TeNT is modified by adding PEG, introducing specific mutations, or a combination thereof, for delivering a bioactive compound capable of increasing muscle tone in tetanus-immunized patients.

[0085] The activity can be demonstrated by administering a unit-determined dose of the modified toxin or formulation, where TeNT will not exhibit activity in vaccinated subjects at the same unit dose. Based on the analysis of the three-dimensional structure of TeNT, introducing specific surface mutations to direct the attachment of PEG molecules allows masking specific TeNT epitopes known to be targeted by the protective antibody response in vaccinated subjects. The combination of pegylation, site-directed mutagenesis, and decoy molecule formulation greatly enhances the molecule's effect of increasing muscle tone in a vaccinated mammalian model relative to an equivalent unit of TeNT administered.

[0086] US 2002 / 0197278 discloses a series of pegylated botulinum toxins for treating inappropriate muscle contraction disorders and indicates that TeNT can be used as an alternative to botulinum toxin. However, TeNT cannot be used to treat muscle contraction. Additionally, since the disclosed method does not include site-directed masking of epitopes, the so-called invention of US 2002 / 0197278 appears to be unworkable, and to the knowledge of the inventors of the present application, the three-dimensional structure and epitope identification required for deliberately masking TeNT epitopes were unknown at the priority date of US 2002 / 0197278.

[0087] Wan et al. addressed the effect of pegylation on the anti-PEG immune response generated by administering pegylated proteins but did not utilize their findings for any treatment. Although Wan et al. disclosed that pegylated tetanus toxoid exhibits reduced immunogenicity relative to non-pegylated tetanus toxoid, Wan et al. did not propose a therapeutically relevant molecule or formulation. Additionally, the pegylation of tetanus toxoid is not relevant to the modification of active TeNT because tetanus toxoid is an inactive TeNT used for vaccination, which can be produced by formaldehyde cross-linking of TeNT. That is, pegylated or non-pegylated tetanus toxoid does not possess the combined enzymatic, binding, and translocation activities of active TeNT.

[0088] WO 2016 / 001762 A1 only relates to the TeNT c fragment, which is a molecule with no specific activity other than binding neurotransmitters and entering neurons.

[0089] Accordingly, there is a need for a TeNT-based therapy that avoids pre-existing anti-TeNT immunity in subjects vaccinated with tetanus toxoid. The solution to this problem is disclosed herein and is provided in part by a masked active and therapeutically relevant polyethylene glycolylated TeNT.

[0090] Tetanus neurotoxin (TeNT)

[0091] TeNT is approximately 150 kDa and is expressed by the tetX gene. A codon-optimized nucleic acid sequence corresponding to the coding region of tetX but lacking the start methionine codon is provided in the vector sequence of Figure 3 (SEQ ID NO:2). TeNT is expressed as a protein that is post-translationally cleaved, i.e., first the start methionine is removed and then it is cleaved into two parts: a 50 kDa light chain (LC or A chain) derived from the N-terminus of the uncleaved protein and a 100 kDa heavy chain (HC or B chain) derived from the C-terminus of the uncleaved protein. These two chains are linked by an interchain disulfide bond, which is crucial for neurotoxicity. The 1314 amino acid sequence of mature TeNT is provided in Figure 2 (SEQ ID NO:1).

[0092] The LC has zinc endopeptidase activity and attacks the vesicle-associated membrane protein (VAMP) required for vesicle-membrane fusion, thereby preventing the release of neurotransmitters.

[0093] After digestion with papain, the HC can be cleaved into two domains, each 50 kDa: an N-terminal translocation domain named HN; and a C-terminal ganglioside (membrane) binding domain named fragment c (c). TeNT lacking c is referred to herein as LC-HN.

[0094] c bears two polysialic acid ganglioside binding sites and binds to polysialic acid gangliosides (GD2, GD1b, and GT1b) on the neuronal membrane. Thus, c mediates the binding of TeNT to the presynaptic membrane of the surrounding motor axons and helps TeNT move across this membrane into the neuron.

[0095] Amino acid sequences:

[0096] The amino acid sequence of TeNT lacking the start methionine is provided in Figure 2 (SEQ ID NO:1);

[0097] The amino acid sequence of the HC is provided in Figure 5 (SEQ ID NO:3);

[0098] The amino acid sequence of c is provided in Figure 6 (SEQ ID NO:4); and

[0099] The amino acid sequence of LC-HN is provided in Figure 9 (SEQ ID NO:6).

[0100] The codon-optimized nucleic acid sequences and vector maps of vectors encoding the following:

[0101] c is provided in Figure 7 (SEQ ID NO:5) and Figure 8 in; and

[0102] LC-HN is provided in Figure 10 (SEQ ID NO:7) and Figure 11 in.

[0103] As used herein, "TeNT" is used to refer to the complete TeNT molecule composed of a heavy chain and a light chain. Subdomains and fragments are referred to herein by their abbreviations: light chain "LC"; heavy chain "HC"; heavy chain N-terminal domain "HN"; heavy chain fragment c "c"; light chain plus heavy chain N-terminal domain "LC-HN" (i.e., the TeNT molecule lacking c). In the case where any subdomain or fragment is pegylated, the prefix PEG is used: PEG-LC; PEG-HC; PEG-HN; PEG-c; PEG-LC-HN. In a complete TeNT molecule containing a pegylated fragment or subdomain, the prefix PEG is used and the pegylated subdomain or fragment is indicated: PEG-TeNT-LC; PEG-TeNT-HC; PEG-TeNT-LC-HC; PEG-TeNT-HN; PEG-TeNT-c; PEG-TeNT-LC-c; PEG-TeNT-LC-HN, etc.

[0104] It should be understood that since subdomains and fragments have specific functions, subdomains and fragments are not interchangeable for the complete TeNT.

[0105] The TeNT disclosed herein can be active or inactive. Active TeNT has the same biological activity as native TeNT. Inactive TeNT lacks one or more activities of native TeNT. In one embodiment, inactive TeNT does not block the release of inhibitory neurotransmitters. The inactive TeNT of the present disclosure can also be referred to as a "decoy". Inactive TeNT includes tetanus toxoid. In one embodiment, the inactive TeNT is the inactive TeNT disclosed herein.

[0106] In one embodiment, two or more TeNTs can be conjugated.

[0107] In another embodiment, TeNT is not conjugated. In one embodiment of the compositions, methods, and uses disclosed herein, the first PEG-TeNT is not conjugated to the second TeNT.

[0108] The immune-evasive PEG-TeNTs disclosed herein and depicted in Figure 1 include:

[0109] TeNT with a pegylated fragment c (PEG-TeNT-c) ( Figure 1 A);

[0110] TeNT with a pegylated heavy chain (PEG-TeNT-HC) ( Figure 1 B);

[0111] TeNT with a pegylated light chain and a pegylated fragment c (PEG-TeNT-LC-c) ( Figure 1 C); and

[0112] Fully pegylated TeNT (PEG-TeNT-LC-HC) ( Figure 1 D).

[0113] PEG-TeNT-c advantageously evades pre-existing immune responses of the adaptive immune system in vaccinated subjects. In one embodiment, PEG-TeNT-c provides a first level of therapy to be used until its efficacy decreases.

[0114] PEG-TeNT-HC and PEG-TeNT-LC-c advantageously evade pre-existing immune responses of the adaptive immune system in vaccinated subjects and also evade immune responses of the adaptive immune system triggered by repeated exposure to PEG-TeNT-c.

[0115] In one embodiment, PEG-TeNT-HC and PEG-TeNT-LC-c together provide a second level of therapy to be used until their efficacy decreases.

[0116] PEG-TeNT-LC-HC advantageously evades pre-existing immune responses of the adaptive immune system in vaccinated subjects and also evades immune responses of the adaptive immune system triggered by repeated exposure to PEG-TeNT-c and exposure to PEG-TeNT-HC plus PEG-TeNT-LC-c.

[0117] In one embodiment, PEG-TeNT-LC-HC provides a third level of therapy to be used until its efficacy decreases.

[0118] Also disclosed are TeNT with a polyethylene glycolylated light chain (PEG-TeNT-LC), TeNT with a polyethylene glycolylated LC and HN (PEG-TeNT-LC-HN), and TeNT with a polyethylene glycolylated HN (PEG-TeNT-HN).

[0119] Those skilled in the art will appreciate that the specific combinations of PEG-TeNT and the order of using those PEG-TeNT combinations for treatment can be varied.

[0120] Polyethylene glycol (PEG)

[0121] PEG can be conjugated, for example, to the following amino acid residues in TeNT: lysine (e.g., amino-polyethylene glycolylation), cysteine (e.g., thiol-polyethylene glycolylation and bridging polyethylene glycolylation), histidine, arginine, aspartic acid, asparagine (e.g., N-glycosyl-polyethylene glycolylation), glutamic acid, glutamine (e.g., transglutaminase-mediated polyethylene glycolylation), serine (e.g., O-glycosyl-polyethylene glycolylation), threonine (e.g., O-glycosyl-polyethylene glycolylation), or tyrosine residues. Examples of polyethylene glycolylation also include N-terminal polyethylene glycolylation and C-terminal polyethylene glycolylation.

[0122] Polyethylene glycolylation can be achieved by reacting PEG with a hydroxyl-reactive functional group, such as an anhydride, acyl chloride, chloroformate, and carbonate. Alternatively, polyethylene glycolylation can be achieved with functional groups such as aldehydes, esters, and amides.

[0123] PEG can be linear or branched.

[0124] PEG can be a modified PEG, such as poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA).

[0125] Polyethylene glycolylation can be site-specific polyethylene glycolylation.

[0126] In one embodiment, surface serine residues of TeNT or a TeNT fragment are mutated to surface cysteine residues to facilitate directed PEG conjugation at immunogenic epitopes. In this context, mutation is synonymous with substitution, e.g., serine is substituted with cysteine. Such mutations or substitutions include any combination of one or more of the following: S81C; S120C; S144C; S248C; S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C.

[0127] Functional groups for heterobifunctional PEG include maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS ester.

[0128] In one embodiment, PEG is conjugated to TeNT using carbodiimide-EDC and sulfo-NHS with carboxyl-amine crosslinking.

[0129] The present invention also contemplates PEG-TeNTs comprising PEGs of different molecular weights conjugated to different subdomains or fragments of TeNT.

[0130] For example, PEG can be conjugated or linked to the TeNT of the present disclosure for 2 to 6 hours at 4°C to 25°C. In one embodiment, PEG is conjugated to TeNT for 6 hours at room temperature.

[0131] Indications

[0132] As used herein, "hypotonia" refers to any condition including involuntary muscle weakness that can be treated by inhibiting inhibitory neurotransmitters such as GABA or glycine. Thus, "hypotonia" includes decreased muscle tone secondary to a decrease in nervous system drive or other etiologies and disorders of decreased muscle tone, strength, or nervous system drive or insufficiency. Thus, in one embodiment, hypotonia can be neurogenic hypotonia.

[0133] Hypotonia conditions that can be treated with the PEG-TeNT, compositions, or methods according to the present disclosure include obstructive sleep apnea, apnea, snoring, ptosis, Horner's syndrome, muscle atrophy, impaired muscle nerve function, amyotrophic lateral sclerosis (ALS), motor neuron disease, any myopathy, multiple sclerosis, myasthenia gravis, decreased facial muscle tone, optional ectropion, flaccid paralysis or weakness of any skeletal or smooth muscle caused by any etiology, respiratory muscle weakness caused by any etiology, including post-ventilator weakness; muscle weakness caused by trauma or poor posture caused by muscle relaxation, pelvic floor muscle relaxation or weakness, or nasal or upper airway relaxation.

[0134] Other conditions that can be treated with the PEG-TeNT, compositions, or methods according to the present disclosure include muscle atrophy, muscular dystrophy, or decreased muscle mass.

[0135] In cases where the condition to be treated according to the present disclosure is not a hypotonia condition, increasing muscle tone by treating with the TeNT of the present disclosure can alleviate the symptoms of the condition.

[0136] Cosmetic applications of PEG-TeNT can include tightening the abdominal muscles, tightening the chest muscles, tightening the gluteus maximus muscles, tightening skeletal muscles, or treating facial sagging caused by muscle relaxation.

[0137] Smooth muscle, skeletal muscle, tissues or organs that can be treated with the PEG-TeNT, composition or method according to the present disclosure include the upper esophagus, esophageal wall, esophageal sphincter, lower esophageal sphincter, anal sphincter, bladder, bladder sphincter, vaginal sphincter, pyloric sphincter, sphincter of Oddi, ileocecal sphincter, pelvic floor muscles, vaginal wall muscles, prostate, submandibular gland, parotid gland, sublingual gland, minor salivary glands of the oral mucosa, vocal cords, facial muscles, masticatory muscles, scalp muscles, chest muscles, back muscles, upper limb muscles, forearm muscles, lower limb muscles, hand muscles, foot muscles, gastric wall muscles, colonic wall muscles, neck muscles, pharyngeal dilator muscles, masseter muscle, medial pterygoid muscle, lateral pterygoid muscle, geniohyoid muscle, genioglossus muscle, tensor veli palatini muscle, levator veli palatini muscle, stylopharyngeus muscle, styloglossus muscle, mylohyoid muscle, stylohyoid muscle, hyoglossus muscle, digastric muscle, sternocleidomastoid muscle, trapezius muscle, temporal muscle, cricopharyngeal muscle, uterine muscle and cervix, gastric innervation, nasal mucosa, lung mucosa, skin, thymus, bone, coronary artery, pulmonary smooth muscle and myocardium.

[0138] Compositions and Administration

[0139] The compositions of the present disclosure can be therapeutic compositions or cosmetic compositions. That is, the compositions can be used for therapeutic or cosmetic purposes.

[0140] As used herein, the terms "therapeutic composition" or "cosmetic composition" refer to compositions that contain TeNT that inhibits or treats hypotonia in a subject as described herein. The compositions have been formulated for administration to a subject. In one embodiment, the compositions are sterile. In one embodiment, the compositions are pyrogen-free. The compositions can contain a pharmaceutically acceptable carrier. Preferably, the compositions are manufactured in accordance with Good Laboratory Practice (GLP) or Good Manufacturing Practice (GMP).

[0141] The TeNT of the present disclosure can be administered at a dose of up to 10 mg / kg or higher. The TeNT of the present disclosure can be administered at the following doses: about 1 fg / kg, about 5 fg / kg, about 10 fg / kg, about 50 fg / kg, about 100 fg / kg, about 500 fg / kg, about 1 pg / kg, about 5 pg / kg, about 10 pg / kg, about 50 pg / kg, about 100 pg / kg, about 500 pg / kg, about 1 ng / kg, about 2 ng / kg, about 3 ng / kg, about 4 ng / kg, about 5 ng / kg, about 6 ng / kg, about 7 ng / kg, about 8 ng / kg, about 9 ng / kg, about 10 ng / kg, about 11 ng / kg, about 12 ng / kg, about 13 ng / kg, about 14 ng / kg, about 15 ng / kg, about 16 ng / kg, about 17 ng / kg, about 18 ng / kg, about 19 ng / kg, about 20 ng / kg, about 30 ng / kg, about 40 ng / kg, about 50 ng / kg, about 60 ng / kg, about 70 ng / kg, about 80 ng / kg, about 90 ng / kg, about 100 ng / kg, about 200 ng / kg, about 300 ng / kg, about 400 ng / kg, about 500 ng / kg, about 600 ng / kg, about 700 ng / kg, about 800 ng / kg, about 900 ng / kg, about 1 μg / kg, about 5 μg / kg, about 10 μg / kg, about 50 μg / kg, about 100 μg / kg, about 500 μg / kg, about 1 mg / kg or about 10 mg / kg. The TeNT of the present disclosure can be administered within any range of any of the doses listed above.

[0142] The TeNT of the present disclosure can be administered at a dose of up to 1000 IU / kg or higher. The TeNT of the present disclosure can be administered at the following doses: about 0.1 IU / kg, about 0.2 IU / kg, about 0.3 IU / kg, about 0.4 IU / kg, about 0.5 IU / kg, about 0.6 IU / kg, about 0.7 IU / kg, about 0.8 IU / kg, about 0.9 IU / kg, about 1 IU / kg, about 2 IU / kg, about 3 IU / kg, about 4 IU / kg, about 5 IU / kg, about 6 IU / kg, about 7 IU / kg, about 8 IU / kg, about 9 IU / kg, about 10 IU / kg, about 11 IU / kg, about 12 IU / kg, about 13 IU / kg, about 14 IU / kg, about 15 IU / kg, about 16 IU / kg, about 17 IU / kg, about 18 IU / kg, about 19 IU / kg, about 20 IU / kg, about 30 IU / kg, about 40 IU / kg, about 50 IU / kg, about 60 IU / kg, about 70 IU / kg, about 80 IU / kg, about 90 IU / kg, about 100 IU / kg, about 200 IU / kg, about 300 IU / kg, about 400 IU / kg, about 500 IU / kg, about 600 IU / kg, about 700 IU / kg, about 800 IU / kg, about 900 IU / kg, about 1000 IU / kg. The TeNT of the present disclosure can be administered within any range of any of the doses listed above.

[0143] In a composition comprising two PEG-TeNTs, such as a composition comprising a first PEG-TeNT (PEG-TeNT-HC) in which TeNT-HC is polyethylene glycolated and a second PEG-TeNT (PEG-TeNT-LC-c) in which TeNT-LC is polyethylene glycolated and TeNT-c is polyethylene glycolated, the ratio of the first PEG-TeNT to the second PEG-TeNT can vary. For example, the ratio of the first PEG-TeNT to the second PEG-TeNT can be about 1000:1, about 500:1, about 100:1, about 50:1, about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:50, about 1:100, about 1:500 or about 1:1000.

[0144] The composition can comprise any combination of TeNT and is not limited to the combination of PEG-TeNT-HC and PEG-TeNT-LC-c. The composition can comprise: PEG-TeNT-c and PEG-TeNT-HC; PEG-TeNT-c and PEG-TeNT-LC-c; PEG-TeNT-c and PEG-TeNT-LC-HC; PEG-TeNT-HC and PEG-TeNT-LC-HC; and PEG-TeNT-LC-c and PEG-TeNT-LC-HC. Also disclosed is a composition comprising: PEG-TeNT-c, PEG-TeNT-HC and PEG-TeNT-LC-c; PEG-TeNT-c, PEG-TeNT-HC and PEG-TeNT-LC-HC; PEG-TeNT-c, PEG-TeNT-LC-c and PEG-TeNT-LC-HC; PEG-TeNT-HC, PEG-TeNT-LC-c and PEG-TeNT-LC-HC; and PEG-TeNT-c, PEG-TeNT-HC, PEG-TeNT-LC-c and PEG-TeNT-LC-HC. In the composition, any TeNT can be replaced, and any composition can also comprise PEG-TeNT-LC, PEG-TeNT-LC-HN and / or PEG-TeNT-HN.

[0145] In one embodiment, the composition further comprises inactivated TeNT, which serves as a decoy for anti-TeNT antibodies generated by prior exposure to TeNT, such as by vaccination. Relative to SEQ ID NO:1, the inactivated TeNT can comprise: R1225K; R1225E; W1228A; W1288Y; W1288F; W1288L; R1225K and W1288A; R1225K and W1288Y; R1225E and W1288Y; R1225K and W1288F; R1225E and W1288F; R1225K and W1288L; R1225E and W1288L; R1225del; W1288del; or R1225del and W1288del; E270A; Y374A; E270A and Y374A; E270del; Y374del or a combination thereof. In one embodiment, the second TeNT comprises inactivated TeNT, which comprises R1225E, W1288A, E270A and Y374A.

[0146] In a composition comprising at least one PEG-TeNT and a decoy TeNT, the decoy TeNT will be in molar excess relative to the PEG-TeNT. For example, the ratio of decoy TeNT to PEG-TeNT can be about 10 6 :1; 10 5 :1, 10 4 :1, 1000:1, about 500:1, about 400:1, about 300:1, about 200:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1 or about 1:1.

[0147] The PEG-TeNT of the present disclosure can be administered once, twice, or three times a week; once, twice, or three times a month; once, twice, or three times a quarter; once, twice, or three times every 6 months; or once, twice, or three times a year.

[0148] PEG-TeNT can be administered in a single dose, divided doses, or multiple doses. In the case where muscles are present in pairs, PEG-TeNT can be administered unilaterally to one muscle of the pair or bilaterally to both muscles of the pair.

[0149] As an alternative to a combination of two or more PEG-TeNTs of the present disclosure, the two or more PEG-TeNTs can be administered in combination sequentially or simultaneously.

[0150] PEG-TeNT can be administered locally to a subject by any suitable method, such as by injection, surgical implantation, topical application, or intranasal administration. In one embodiment, TeNT is administered intramuscularly by injection into the affected muscle.

[0151] PEG-TeNT will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the specific type of hypotonia being treated, the specific subject being treated, the clinical condition of the subject, the site of administration, the method of administration, the timing of administration, and other factors known to a physician, including a dentist. The therapeutically effective amount of PEG-TeNT to be administered will depend on these considerations.

[0152] Pharmaceutically acceptable carriers include water, buffered water, saline solutions such as physiological saline or balanced saline solutions such as Hank's balanced solution or Earle's balanced solution, glycine, and hyaluronic acid.

[0153] The composition can be formulated for intramuscular administration. A composition for intramuscular administration can comprise a pharmaceutically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Examples of suitable aqueous and non-aqueous carriers, solvents, diluents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol), carboxymethylcellulose and mixtures thereof, vegetable oils (such as olive oil), injectable organic esters (such as ethyl oleate).

[0154] The composition can comprise penetration enhancers to facilitate their TeNT delivery. Penetration enhancers can include fatty acids such as oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, reclinate, glyceryl monooleate, glyceryl dilaurate, caprylic acid, arachidonic acid, 1-monodecanoyl glycerol, monoglycerides and diglycerides and physiologically acceptable salts thereof.

[0155] The composition can further include chelating agents such as ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (such as sodium salicylate, 5-methoxysalicylate, homovanillate).

[0156] An article and / or kit is also provided, which includes a container containing PEG-TeNT or a composition comprising PEG-TeNT. The container can be a bottle, vial or syringe containing PEG-TeNT or the composition (optionally in unit dosage form). For example, the PEG-TeNT or the composition can be in the form of an injectable solution in a disposable container, optionally a syringe. The article and / or kit can further comprise printed instructions and / or labels, etc., indicating the treatment of a subject according to the methods disclosed herein.

[0157] The term "therapeutically effective amount" refers to the amount of PEG-TeNT that is effective in treating hypotonia in a subject.

[0158] The terms "treat", "treating" or "treatment" refer to therapeutic treatment and prophylactic or preventive measures, wherein the aim is to prevent, reduce or improve hypotonia in a subject or to slow (mitigate) the progression of hypotonia in the subject. Subjects in need of treatment include subjects who already have hypotonia and subjects who are to prevent or improve hypotonia.

[0159] The terms "preventing", "prevention", "preventive", or "prophylactic" refer to avoiding the occurrence of hypotonia or impeding, defending against, or preventing the occurrence of hypotonia. Subjects in need of prevention may be predisposed to hypotonia.

[0160] The terms "ameliorate" or "amelioration" refer to alleviating, reducing, or eliminating hypotonia.

[0161] Hypotonia can be quantified. Hypotonia can be quantified on a semi - quantitative scale, such as 0 to 5, where 0 represents absence, 1 to 4 represent discernible increases in severity, and 5 represents the maximum severity. Alternatively, hypotonia can be quantified as a binary event, i.e., present or absent (0 or 1). Other semi - quantitative scales will be obvious to those skilled in the art. In another embodiment, hypotonia can be quantified on a quantitative scale, such as using a dynamometer.

[0162] Any quantification of hypotonia can be compared to a control, such as a healthy control subject not receiving PEG - TeNT, an affected control subject receiving treatment for hypotonia but not treated with PEG - TeNT, or a population.

[0163] Treatment of hypotonia by administration of PEG - TeNT can result in a reduction of hypotonia by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0164] As used herein, the term "subject" can refer to a mammal. The mammal can be a primate, particularly a human, or can be a farm animal, a zoo animal, or a companion animal. Although the PEG - TeNT, compositions, and methods disclosed herein are particularly contemplated for use in the medical treatment of humans, they are also applicable to veterinary treatment, including treatment of farm animals such as horses, cattle, and sheep; companion animals such as dogs and cats; or zoo animals such as felines, canines, bovines, and ungulates.

[0165] Unless otherwise defined in this specification, 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 by reference to the published texts.

[0166] It should be noted that the term "a / an" refers to one or more. For example, "a TeNT" should be understood to represent one or more TeNTs. Thus, the terms "a / an", "one or more" and "at least one" are used interchangeably herein.

[0167] In the appended claims and the description of the present invention, unless the context requires otherwise due to the language of expression or necessary implication, the word "comprise" or variants such as "comprises" or "comprising" are used in an inclusive sense, that is, specifying the presence of the stated features, but not excluding the presence or addition of further features in various embodiments of the present invention.

[0168] As used herein, the term "about" encompasses a range of values that vary from the magnitude of a given quantity by ±25%. In other embodiments, the term "about" encompasses a range of values that vary from the magnitude of a given quantity by ±20%, ±15%, ±10%, ±5%, ±4%, ±3%, ±2% or ±1%. For example, in one embodiment, "about 3 grams" represents a value from 2.7 grams to 3.3 grams (i.e., 3 grams ±10%), etc.

[0169] Similarly, the time or duration of an event can vary by at least 25%. For example, although a specific event may be disclosed as lasting one day in one embodiment, the event can last more than one day or less than one day. For example, "one day" can include a time period from about 18 hours to about 30 hours. In other embodiments, the time period can vary by ±20%, ±15%, ±10%, ±5%, ±4%, ±3%, ±2% or ±1% of the time period.

[0170] Examples

[0171] Example 1: Preparation of PEG-TeNT-c ( Figure 1 A)

[0172] In this example, the surface serine residues of TeNT-c were mutated to surface cysteine residues to facilitate site-directed PEG conjugation at immunogenic epitopes to generate Figure 1 molecules of A. The mutations will be: S963C, S1041C, S1155C and S1187C.

[0173] The gene of TeNT with surface serine to cysteine substitutions S963C, S1041C, S1155C and S1187C will be synthesized by a commercial provider (such as Integrated DNA Technologies). The gene will be subcloned into the pRSET-A expression vector by restriction enzyme digestion to add a 6× histidine tag from the vector to the N-terminus of the mutant protein.

[0174] TeNT containing S963C, S1041C, S1155C and S1187C will be expressed, pegylated and purified according to Example 5 to produce PEG-TeNT-c.

[0175] Example 2: Preparation of PEG-TeNT-HC( Figure 1 B)

[0176] In this example, the surface serine residues of TeNT-HC were mutated to surface cysteine residues to facilitate site-directed PEG conjugation at immunogenic epitopes, resulting in Figure 1 molecules of B. The mutations will be: S600C, S963C, S1041C, S1155C and S1187C (Figure 24, SEQ ID NO:16).

[0177] The gene of TeNT with surface serine to cysteine substitutions S600C, S963C, S1041C, S1155C and S1187C will be synthesized by a commercial provider (such as Integrated DNA Technologies). The gene will be subcloned into the pRSET-A expression vector (Figure 25, SEQ ID NO:17, Figure 26 ) to add a 6× histidine tag from the vector to the N-terminus of the mutant protein.

[0178] TeNT containing S600C, S963C, S1041C, S1155C and S1187C will be expressed, pegylated and purified according to Example 5 to produce PEG-TeNT-HC.

[0179] Example 3: Preparation of PEG-TeNT-LC-c( Figure 1 C)

[0180] In this example, the surface serine residues of LC and c were mutated to surface cysteine residues to facilitate site-directed PEG conjugation at immunogenic epitopes, resulting in Figure 1Molecules of C. The mutations are: S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C (Figure 21, SEQ ID NO:14).

[0181] Genes of TeNT with surface serine to cysteine substitutions S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C in LC and c were synthesized by a commercial provider (Integrated DNA Technologies). The gene was subcloned into the pRSET-A expression vector by restriction enzyme digestion (Figure 22, SEQ ID NO:15, Figure 23 ) to add a 6× histidine tag from the vector to the N-terminus of the mutant protein.

[0182] TeNT containing S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C was expressed, pegylated and purified according to Example 5 to produce PEG-TeNT-LC-c.

[0183] Endotoxin removal

[0184] Endotoxin was removed from the TeNT-LC-c serine mutants according to Example 4.

[0185] Linkage of PEG to cysteine residues

[0186] 1. Combine a molar excess of PEG-maleimide with the serine mutant TeNT-LC-c (0.5 mg / mL - 2 mg / mL) in PBS at pH 6.5 - 7.5, where PEG has approximately 2 kDa, approximately 5 kDa, approximately 10 kDa or approximately 20 kDa.

[0187] 2. Perform the ligation for 6 hours at room temperature.

[0188] 3. Remove the excess PEG by size exclusion chromatography.

[0189] Trypsin digestion activation of the protein

[0190] Trypsin digestion was performed according to Example 4 to activate the TeNT-LC-c serine mutants.

[0191] Example 4: Preparation of PEG-TeNT-LC-HC( Figure 1 D)(Method 1)

[0192] Preparation of TeNT

[0193] 1. Electrotransform Escherichia coli BL21 DE3 pLysS strain with pRSET-TeNT vector (Figure 3 and Figure 4 ) and grow it overnight at 37 °C on LB agar with selective antibiotics (ampicillin and chloramphenicol).

[0194] 2. Inoculate 200 mL of pre-induction broth with 1 colony from the selective plate. The pre-induction broth at pH 7.2 - 7.4 contains: 1.2% tryptone; 2.4% yeast extract; 2% glucose; 0.4% glycerol; 17 mM KH2PO4; 72 mM K2HPO4; and selective antibiotics (ampicillin and chloramphenicol).

[0195] 3. Incubate the culture overnight at 30 °C with rapid shaking.

[0196] 4. Harvest the overnight culture by centrifugation at 4000 g for 10 minutes.

[0197] 5. Resuspend the pellet in 200 mL of expression broth (pH 7.2 - 7.4), which contains: 1.2% tryptone; 2.4% yeast extract; 0.4% glycerol; 1 mM IPTG; 17 mM KH2PO4; 72 mM K2HPO4; 100 μg / mL ampicillin; and 10 μM ZnCl2.

[0198] 6. Express the protein for 6 hours at 30 °C with rapid shaking.

[0199] 7. Harvest the cells by centrifugation at 4500 g for 15 minutes and resuspend the pellet in 30 mL of TBS (pH 8) containing 20 mM imidazole.

[0200] 8. Lyse the cells by sonication.

[0201] 9. Clarify the cell lysate by centrifugation at 4500 g for 20 minutes and filter through a 0.45 μm filter.

[0202] 10. Purify the protein by His-tag affinity chromatography using an AKTA pure 25 HPLC system (GE Healthcare).

[0203] 11. Perform buffer exchange of the purified protein with PBS using size exclusion chromatography, followed by a second stage of purification by gel filtration using an AKTA pure 25 HPLC and a Superdex 200 Increase 10 / 300 GL column.

[0204] Endotoxin removal

[0205] 1. Equilibrate a 0.5 mL endotoxin removal spin column to room temperature.

[0206] 2. Remove the bottom plug of the column, loosen the column cap, place the column in a 15 mL tube, and then centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution.

[0207] 3. Replace the bottom plug of the column, remove the column cap, add 0.2 N NaOH in 95% ethanol to the resin, replace the column cap, invert the column several times to resuspend the resin, and then incubate at room temperature for 1 to 2 hours.

[0208] 4. Remove the bottom plug of the column, loosen the column cap, place the column in a 15 mL tube, and then centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution.

[0209] 5. Replace the bottom plug of the column, remove the column cap, add endotoxin-free 2 M NaCl to the resin, replace the column cap, and invert the column several times to resuspend the resin.

[0210] 6. Remove the bottom plug of the column, loosen the column cap, place the column in a 15 mL tube, and then centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution.

[0211] 7. Replace the bottom plug of the column, remove the column cap, add endotoxin-free ultrapure water to the resin, replace the column cap, and invert the column several times to resuspend the resin.

[0212] 8. Remove the bottom plug of the column, loosen the column cap, place the column in a 15 mL tube, and centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution.

[0213] 9. Replace the bottom plug of the column, remove the column cap, add endotoxin-free phosphate buffer to the resin, replace the column cap, and invert the column several times to resuspend the resin.

[0214] 10. Remove the bottom plug of the column, loosen the column cap, place the column in a 15 mL tube, and centrifuge at 500 g for 1 minute to remove the solution from the column. Discard the solution.

[0215] 11. Rinse the column two more times with phosphate buffer and discard the eluate.

[0216] 12. Replace the bottom plug of the column, remove the column cap, apply the sample to the resin, replace the column cap, and invert the column several times to resuspend the resin.

[0217] 13. Incubate the column at 4°C for at least 1 hour with inversion mixing.

[0218] 14. Remove the bottom plug of the column, loosen the column cap, place the column in an endotoxin-free 15 mL tube, and centrifuge at 500 g for 1 minute to remove the solution from the column. Retain the sample.

[0219] 15. Repeat the endotoxin removal procedure using the regenerated spin column until the endotoxin level in the sample is at an equivalent or lower level such that all doses will contain less than 5 EU units of endotoxin per kg of subject.

[0220] Preparation of PEG-TeNT-LC-HC

[0221] 1. In 500 μL total volume of PBS (pH 7.4), combine 3 μmol of purified TeNT and 0.5 mmol of mpeg-NHS(SC) (Nanocs) with one of 2 kDa, 5 kDa, 10 kDa, 20 kDa or 30 kDa PEG.

[0222] 2. Mix the sample at room temperature for 3 hours.

[0223] 3. Remove the excess PEG by size exclusion chromatography.

[0224] Trypsin digests the protein into the active form

[0225] 1. Dissolve 1 mg of the protein in 0.5 mL of digestion buffer containing 0.1 M NH4HCO3 buffer (pH 8.0) or 0.1 M Tris buffer (pH 8.5).

[0226] 2. Wash 0.10 mL to 0.25 mL of immobilized TPCK trypsin with 3 × 500 μL of digestion buffer. Separate the gel from the buffer by centrifugation after each wash.

[0227] 3. Resuspend the gel in approximately 0.2 mL of digestion buffer.

[0228] 4. Add the immobilized TPCK trypsin to the protein sample.

[0229] 5. Incubate the reaction mixture in a rapid oscillation incubator at 37 °C for 2 to 18 hours.

[0230] 6. Separate the immobilized TPCK trypsin by centrifugation.

[0231] Example 5: Preparation of PEG-TeNT-LC-HC ( Figure 1 D) (Method 2)

[0232] In this example, the surface serine residues of TeNT-LC-HC were mutated to surface cysteine residues (S→C mutants) to facilitate directed PEG conjugation at immunogenic epitopes. Relative to SEQ ID NO:1, the TeNT mutations were: S81C; S120C; S144C; S248C; S335C; S428C; S600C; S963C; S1041C; S1155C; and S1187C.

[0233] Preparation of serine mutant TeNT-LC-HC

[0234] The vector pRSET-TeNT encoding the amino acid sequence of Figure 18 (SEQ ID NO:12) containing the S→C mutation was used to electrotransform Escherichia coli strain BL21(DE3)pLysS. TeNT-LC-HC containing the S→C mutation was expressed and purified according to Example 4, with an additional treatment with 0.5 mM DTT for 15 minutes between steps 10 and 11. Figure 20)

[0235] Endotoxin removal

[0236] Endotoxin was removed from the TeNT-LC-HC serine mutant according to Example 4. Ligation of PEG to cysteine residues.

[0237] 1. A molar excess of PEG-maleimide was combined with the serine mutant TeNT-LC-HC (0.5 mg / mL - 2 mg / mL) in PBS at pH 6.5 - 7.5, where the PEG had a molecular weight of approximately 2 kDa, approximately 5 kDa, approximately 10 kDa, or approximately 20 kDa.

[0238] 2. The ligation was carried out at room temperature for 6 hours.

[0239] 3. Excess PEG was removed by size exclusion chromatography.

[0240] Trypsin digestion activation of the protein

[0241] Trypsin digestion was performed according to Example 4 to activate the TeNT-LC-HC serine mutant.

[0242] Example 6: Inactive decoy TeNT

[0243] ​In this example, a non - pegylated recombinant c with inactivating R1225E and W1288A amino acid substitutions relative to SEQ ID NO:1 in the ganglioside - binding region was generated. The inactivated c was combined with an equimolar amount of non - pegylated LC - HN to produce an inactive bait TeNT. The resulting inactive TeNT will be used as a bait for antibody - based neutralizing responses in subjects vaccinated with tetanus toxoid vaccine.

[0244] Preparation of c containing R1225E, W1288A

[0245] The gene for c with R1225E and W1288A was synthesized by a commercial provider (Integrated DNA Technologies). The gene was sub - cloned into the pRSET - A expression vector by restriction enzyme digestion to add a 6× histidine tag from the vector to the N - terminus of the mutant protein.

[0246] The Escherichia coli BL21(DE3)pLysS strain was electro - transformed with the pRSET - TeNT - c vector encoding c containing R1225E, W1288A (Figure 12, SEQ ID NO:8) (Figure 13 (SEQ ID NO:9) and Figure 14 ) and grown overnight at 37 °C on LB agar containing selective antibiotics (ampicillin, chloramphenicol). c containing R1225E, W1288A was expressed and purified according to Example 4.

[0247] Endotoxin removal

[0248] Endotoxin was removed from the purified protein according to Example 4.

[0249] Preparation of LC - HN

[0250] The Escherichia coli BL21(DE3)pLysS strain was electro - transformed with the pRSET - TeNT - LC - HN vector (Figure 10 and Figure 11 ) and grown overnight at 37 °C on LB agar containing selective antibiotics (ampicillin, chloramphenicol). LC - HN was expressed and purified according to Example 4.

[0251] Endotoxin removal

[0252] Endotoxin was removed from the purified protein according to Example 4.

[0253] Preparation of LC - HN containing E270A, Y374A

[0254] The Escherichia coli BL21(DE3)pLysS strain was electrotransformed with the pRSET-TeNT-LC-HN vector encoding LC-HN containing E270A and Y374A and grown overnight at 37 °C on LB agar containing the selectable antibiotics (ampicillin, chloramphenicol). LC-HN containing E270A and Y374A was expressed and purified according to Example 4.

[0255] Endotoxin removal

[0256] Endotoxin was removed from the purified protein according to Example 4.

[0257] Example 7: Inactive bait TeNT

[0258] The gene for TeNT containing R1225E and W1288A was synthesized by a commercial provider (Integrated DNA Technologies). The gene was subcloned into the pRSET-A expression vector by restriction enzyme digestion to add a 6× histidine tag from the vector to the N-terminus of the mutant protein (Figure 15 (SEQ ID NO:10), Figure 16 (SEQ ID NO:11) and Figure 17 )

[0259] Inactivated TeNT containing R1225E and W1288A was expressed and purified according to Example 4.

[0260] Endotoxin removal

[0261] Endotoxin was removed from the purified protein according to Example 4.

[0262] Example 8: PEG-TeNT analysis

[0263] TeNT was prepared according to Example 4 and pegylated, then analyzed by SDS-PAGE ( Figure 28 ) and detected by Western blotting using (A) Coomassie blue and (B) a polyclonal anti-TeNT antibody. (B) shows that the immunogenicity is proportional to the PEG molecular weight.

[0264] Example 9: Reduced immunogenicity of PEG-TeNT compared to TeNT

[0265] Four PEG-TeNTs each containing a different molecular weight of PEG (2 kDa, 5 kDa, 10 kDa, and 20 kDa) were prepared according to Example 4 and pegylated. Then PEG-TeNT was assayed against TeNT by competitive ELISA.

[0266] In the first assay ( Figure 29A) TeNT was adsorbed onto an ELISA plate. The adsorbed TeNT was then probed with polyclonal anti-TeNT antibodies pre-incubated with each of four PEG-TeNT antigens (2 kDa, 5 kDa, 10 kDa, and 20 kDa) at four concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL). In this assay, a higher response (OD 450 nm) indicates a greater affinity for TeNT and thus a reduced immunogenicity of the PEG-TeNT.

[0267] In a second assay ( Figure 29B ) each PEG-TeNT was adsorbed onto a separate ELISA plate. Each adsorbed PEG-TeNT (2 kDa, 5 kDa, 10 kDa, and 20 kDa) was then probed with polyclonal anti-TeNT antibodies pre-incubated with each of four concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL) of TeNT antigen. In this assay, a lower response (OD 450 nm) indicates a greater affinity for TeNT and thus a reduced immunogenicity of the PEG-TeNT.

[0268] This example demonstrates that anti-TeNT antibodies preferentially bind TeNT and that PEGylated TeNT has reduced immunogenicity relative to TeNT (i.e., non-PEGylated).

[0269] Example 10: Reduced immunogenicity of PEG-TeNT-LC-c serine mutants compared to TeNT-LC-c serine mutants

[0270] TeNT-LC-c serine mutants were prepared according to Example 5. Four samples of the TeNT-LC-c serine mutants were PEGylated according to Example 5, each sample containing PEG of a different molecular weight (2 kDa, 5 kDa, 10 kDa, and 20 kDa). The PEG-TeNT-LC-c serine mutants were then assayed for TeNT-LC-c serine mutants by competitive ELISA.

[0271] In a first assay ( Figure 29C) In this case, the TeNT-LC-c serine mutant was adsorbed onto an ELISA plate. Then the adsorbed TeNT serine mutant was probed with polyclonal anti-TeNT antibodies pre-incubated with each of four PEG-TeNT serine mutant antigens (2 kDa, 5 kDa, 10 kDa, and 20 kDa) at four concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL). In this assay, a higher response (OD 450 nm) indicates greater affinity for the TeNT serine mutant and thus a reduced immunogenicity of the PEG-TeNT serine mutant.

[0272] In a second assay ( Figure 29D ) each PEG-TeNT-LC-c serine mutant was adsorbed onto a separate ELISA plate. Then each adsorbed PEG-TeNT serine mutant (2 kDa, 5 kDa, 10 kDa, and 20 kDa) was probed with polyclonal anti-TeNT antibodies pre-incubated with each of four concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL) of TeNT serine mutant antigen. In this assay, a lower response (OD 450 nm) indicates greater affinity for the TeNT serine mutant and thus a reduced immunogenicity of the PEG-TeNT serine mutant.

[0273] This example demonstrates that anti-TeNT antibodies preferentially bind the TeNT-LC-c serine mutant and that the PEGylated TeNT-LC-c serine mutant has a reduced immunogenicity relative to the TeNT-LC-c serine mutant (i.e., non-PEGylated).

[0274] Example 11: Reduced immunogenicity of PEG-TeNT compared to TeNT

[0275] A competitive ELISA assay was performed according to Example 9, except that human sera collected from one or more subjects who had received a booster tetanus toxoid vaccination within the previous 12 months were used instead of the polyclonal antibodies. Antibodies in the sera will show greater affinity for TeNT (i.e., non-PEGylated TeNT) compared to PEG-TeNT.

[0276] Example 12: In vivo model

[0277] PEG-TeNT-LC-HC was prepared according to Example 4 by linking PEG (approx. 5 kDa, approx. 10 kDa, or approx. 20 kDa) to surface-exposed lysine residues of recombinant TeNT.

[0278] One or more units of PEG-TeNT-LC-HC in 15 μL of PBS were injected into the hind limbs of female C57BL / 6 mice. Each animal exhibited local limb twitching within 48 hours of injection( Figure 30A and Figure 30B ).

[0279] Example 13: In Vivo Model

[0280] PEG-TeNT-LC-HC with 5 kDa PEG linked to the surface lysine residues of TeNT according to Example 4 was combined with c-bait (i.e., c inactivated by R1225E and W1288A) at a molar ratio of 1:10. A 15-μL composition containing 5 ng of PEG-TeNT in PBS was injected into the hind limb muscle of female C57BL / 6 mice. Each animal exhibited local limb twitching within 48 hours, and the symptom development was indistinguishable from that of animals treated with PEG-TeNT-LC-HC in the absence of bait.

[0281] Example 14

[0282] PEG-TeNT-LC-HC containing 5 kDa, 10 kDa, or 20 kDa PEG was administered intramuscularly at 50 ng / kg - 500,000 ng / kg to the hind leg muscles of mice previously immunized with tetanus toxoid. Increased muscle contraction was observed in the injected muscle for up to 3 days, and the increased muscle contraction was greater than that observed in mice receiving the same unit of TeNT administration. Figure 31 A reports the results for PEG-TeNT-LC-HC 20 kDa.

[0283] Example 15

[0284] A composition containing TeNT-LC-HC and 10:1 or 100:1 molar excess of bait TeNT produced according to Example 6 was administered intramuscularly at 50 ng / kg - 500,000 ng / kg to the hind leg muscles of mice previously immunized with tetanus toxoid. Increased muscle contraction was observed in the injected muscle for up to 3 days, and the increased muscle contraction was greater than that observed in mice receiving TeNT administration( Figure 32 A).

[0285] Example 16

[0286] A composition containing PEG-TeNT-LC-HC with PEG of 5 kDa, 10 kDa or 20 kDa and a 10:1 or 100:1 molar excess of decoy TeNT produced according to Example 6 was intramuscularly administered at 50 ng / kg - 500,000 ng / kg to the hind leg muscles of mice previously immunized with tetanus toxoid. An increase in muscle contraction was observed in the injected muscles for up to 3 days, and the increase in muscle contraction was greater than the effect observed in mice receiving TeNT administration and greater than the effect observed in mice receiving only PEG-TeNT administration. Figure 32 B).

[0287] Example 17

[0288] PEG-TeNT-LC-HC containing 20 kDa PEG was intramuscularly administered at 0.01 ng / kg - 50,000 ng / kg to the left genioglossus muscle of human subjects previously vaccinated with tetanus toxoid vaccine. An increase in muscle contraction will be observed in the injected muscle for up to 2 weeks, and the increase in muscle contraction will be greater than the effect observed in the right genioglossus muscle of the same subject receiving only vehicle administration.

[0289] Example 18

[0290] A composition containing PEG-TeNT-LC-HC with 20 kDa PEG and a 10:1 - 1000:1 molar excess of decoy TeNT produced according to Example 6 was intramuscularly administered at 50 ng / kg to the left genioglossus muscle of human subjects previously vaccinated with tetanus toxoid vaccine. PEG-TeNT-LC-HC containing 20 kDa PEG was intramuscularly administered at 50 ng / kg to the right genioglossus muscle of the same human subject.

[0291] An increase in muscle contraction will be observed in both the left and right genioglossus muscles for up to 2 weeks, but the increase in muscle contraction will be greater in the left genioglossus muscle treated with the composition containing PEG-TeNT-LC-HC and decoy TeNT compared to PEG-TeNT-LC-HC alone.

[0292] Example 19

[0293] About 30 kg of bulldogs will be intramuscularly administered with 25 ng / kg - 50000 ng / kg of PEG-TeNT-c containing 20 kDa PEG, wherein the dose is bilaterally separated and administered to the left genioglossus muscle and the right genioglossus muscle. After administration, obstructive sleep apnea (OSA) in the animals receiving PEG-TeNT treatment will be reduced compared to the animals treated with the vehicle alone. The OSA of the bulldogs will be observed once a week and the PEG-TeNT-c dose will be repeated as needed until the efficacy decreases, as determined by the recovery of OSA comparable to that of the animals treated with the vehicle alone.

[0294] Thereafter, 25 - 50000 ng / kg of PEG-TeNT-HC or 25 - 50000 ng / kg of PEG-TeNT-LC-c, each containing 20 kDa PEG, will be administered to the bulldogs, bilaterally separated and administered to the left genioglossus muscle and the right genioglossus muscle. After administration, OSA in the animals receiving PEG-TeNT treatment will be reduced compared to the animals treated with the vehicle alone. The OSA of the bulldogs will be observed once a week and the PEG-TeNT-HC and PEG-TeNT-LC-c doses will be repeated as needed until the efficacy decreases, as determined by the recovery of OSA comparable to that of the animals treated with the vehicle alone.

[0295] Thereafter, 25 - 50000 ng / kg of PEG-TeNT-LC-HC containing 20 kDa PEG will be administered to the bulldogs, bilaterally separated and administered to the left genioglossus muscle and the right genioglossus muscle. After administration, OSA in the animals receiving PEG-TeNT treatment will be reduced compared to the animals treated with the vehicle alone. The OSA of the bulldogs will be observed once a week and the PEG-TeNT-LC-HC dose will be repeated as needed until the efficacy decreases, as determined by the recovery of OSA comparable to that of the animals treated with the vehicle alone.

[0296] Example 20

[0297] Using Discovery Studio, the TeNT epitopes recognized by the major human antibody clonotypes identified by da Silva Antunes et al. (2017) PloS One, 12(1), e0169086 and Palermo et al. (2017) Biotechnology Journal, 12(10), 1700197 were mapped onto the 3D model of TeNT derived from the crystallographic data stored in the Protein Data Bank (accession ID PDB: 5N0B). Surface serine residues in or around the identified epitopes were identified and mutated to cysteine as described in Examples 1 to 3 and Example 5 for subsequent PEGylation.

[0298] Example 21

[0299] A mixture of PEG-TeNT (branched or linear PEG of 5 kDa, 10 kDa or 20 kDa linked to surface lysine or cysteine residues) and 1 ng to 64 μg of bait TeNT in a 10 - 1000-fold molar excess was intramuscularly injected into the hind leg muscles of mice vaccinated with tetanus toxoid vaccine. Local twitching was observed for a period of several hours to several months, and this local twitching was greater than the effect observed in mice receiving native TeNT injection.

[0300] Example 22

[0301] A mixture of PEG-TeNT (branched or linear PEG of 5 kDa, 10 kDa or 20 kDa linked to surface lysine or cysteine residues) and 1 ng to 64 μg of bait TeNT in a 10 - 1000-fold molar excess was intramuscularly injected into the genioglossus muscle of humans vaccinated with tetanus toxoid vaccine. Local twitching was observed for a period of several weeks to several months, and this local twitching was greater than the effect observed in humans receiving native TeNT injection.

[0302] Example 23

[0303] TeNT at a dose of 0.001 - 10 IU / Kg was intramuscularly administered to English bulldogs with sleep apnea, where the dose was bilaterally divided and administered to the left and right genioglossus muscles. After administration, a significant reduction in obstructive sleep apnea (OSA) was observed at the highest dose. The baseline respiratory disturbance index (RDI) score was 19.9 (interquartile range 5.45), which decreased to 13.2 (interquartile range 4.45) after treatment with 10 IU / Kg TeNT, and this was determined to be significant compared to administration of placebo by the Wilcoxon signed-rank test (P = 0.043). The bulldogs were observed to maintain the reduction in RDI four months after the trial; median RDI = 13.4, P = 0.043; Wilcoxon signed-rank test. Sequence Listing <110> Snoretox Pty Ltd <120> Compositions and Methods <130> AU2018902779A <140> 2018902779 <141> 2018-07-31 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 1314 <212> PRT <213> Escherichia coli <400> 1 Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp 1 5 10 15 Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr 20 25 30 Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg 35 40 45 Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu 50 55 60 Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp 65 70 75 80 Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg 85 90 95 Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn 100 105 110 Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe Asp 115 120 125 Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Ser 130 135 140 Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly 145 150 155 160 Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg 165 170 175 Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile 180 185 190 Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val 195 200 205 Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln 210 215 220 Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly 225 230 235 240 Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys Gln 245 250 255 Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe 260 265 270 Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn 275 280 285 Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys 290 295 300 Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser 305 310 315 320 Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser Asn 325 330 335 Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser 340 345 350 Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile 355 360 365 Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile 370 375 380 Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn 385 390 395 400 Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg 405 410 415 Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val Ser 420 425 430 Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg 435 440 445 Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu 450 455 460 Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys 465 470 475 480 Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn 485 490 495 Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile 500 505 510 Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr 515 520 525 Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn 530 535 540 Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr 545 550 555 560 Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr 565 570 575 Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr 580 585 590 Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly 595 600 605 Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn 610 615 620 Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr 625 630 635 640 Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr 645 650 655 Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu 660 665 670 Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser 675 680 685 Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp 690 695 700 Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu 705 710 715 720 Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg 725 730 735 Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys 740 745 750 Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu 755 760 765 Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys 770 775 780 Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser 785 790 795 800 Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu 805 810 815 Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys 820 825 830 Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser 835 840 845 Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys 850 855 860 Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu 865 870 875 880 Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser 885 890 895 Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln 900 905 910 Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu 915 920 925 Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp 930 935 940 Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val 945 950 955 960 Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile 965 970 975 Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val 980 985 990 Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly 995 1000 1005 Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 1010 1015 1020 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp 1025 1030 1035 Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly 1040 1045 1050 Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn 1055 1060 1065 Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val 1070 1075 1080 Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys 1085 1090 1095 Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu 1100 1105 1110 Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr 1115 1120 1125 Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn 1130 1135 1140 Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn 1145 1150 1155 Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys 1160 1165 1170 Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe 1175 1180 1185 Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn 1190 1195 1200 Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn 1205 1210 1215 Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile 1220 1225 1230 Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys 1235 1240 1245 Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 1250 1255 1260 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro 1265 1270 1275 Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu 1280 1285 1290 Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp 1295 1300 1305 Glu Gly Trp Thr Asn Asp 1310 <210> 2 <211> 6816 <212> DNA <213> Escherichia coli <400> 2 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180 gatgacgata aggatcgatg gggatccgag ctcgagccga ttaccatcaa taattttcgt 240 tattcagatc cggtgaataa tgataccatc atcatgatgg aaccaccgta ctgtaaaggg 300 ctggatattt attataaagc gttcaaaatc accgaccgca tctggatcgt gccggaacgc 360 tacgaattcg gcaccaaacc ggaagatttt aatccgccga gtagtctgat cgaaggtgca 420 tcggaatact acgatccgaa ttatctgcgt actgactctg ataaagatcg ctttctgcaa 480 acgatggtaa aactgttcaa tcgtatcaaa aacaatgtag caggcgaagc cctgctggat 540 aaaatcatca acgccattcc gtatctggga aacagttatt ctctgctgga taaattcgat 600 acaaactcga actctgtgtc attcaacctg ctggaacagg acccgagcgg cgcgaccact 660 aagagtgcga tgctgactaa cctgattatt ttcggtccgg gaccggtact gaataaaaat 720 gaagttcgcg gcattgtact gcgtgtcgat aataaaaact atttcccatg tcgtgatggc 780 ttcggcagca tcatgcagat ggccttttgt ccggaatatg tgccaacttt cgataatgtg 840 attgagaaca tcacctctct gacgattggt aaaagtaaat atttccagga tccggctctg 900 ctgctgatgc atgaactgat ccatgttctg catggcctgt atggcatgca ggtttcatcc 960 cacgaaatta tcccatccaa acaggaaatt tacatgcagc atacatatcc gattagtgcc 1020 gaagaactgt tcacttttgg cggccaggat gcgaacctga tttcgattga cattaaaaac 1080 gatctgtatg aaaaaactct gaacgattat aaagcgattg ccaacaaact gtctcaggta 1140 acctcctgta acgatccgaa tattgatatt gacagttata aacaaattta tcagcagaag 1200 tatcagttcg ataaagactc taatggccag tatattgtta acgaagataa attccagatt 1260 ctgtacaata gcattatgta tggctttact gagatcgaac tgggtaaaaa atttaacatc 1320 aagactcgtc tgagctattt tagcatgaat catgatccag tgaaaatccc gaatctgctg 1380 gatgatacga tttataatga taccgaagga tttaacatcg aaagcaagga tctgaaatcc 1440 gaatataaag ggcagaacat gcgcgttaat accaatgcat ttcgcaatgt tgatggttca 1500 ggcctggtgt cgaaactgat tgggctgtgt aagaaaatca ttccaccgac aaatattcgc 1560 gaaaatctgt acaaccgtac ggcgagcctg accgatctgg ggggagaact gtgtattaaa 1620 atcaaaaatg aagatctgac cttcattgct gagaagaata gcttttccga agagccattc 1680 caggacgaaa tcgtgtctta taacaccaag aataaaccgc tgaatttcaa ctactccctg 1740 gacaaaatca ttgtggatta caacctgcag agtaaaatta ccctgccgaa tgatcgtacc 1800 accccggtga cgaaaggcat cccttacgca ccagaatata aatcaaatgc agcctcgact 1860 atcgagatcc ataatattga tgacaacact atttaccagt acctgtatgc tcagaaatct 1920 ccgacgacgc tgcagcgcat caccatgact aacagcgtgg acgatgccct gattaatagc 1980 accaaaatct actcttactt tccgtcggtg atctctaagg ttaatcaggg cgcgcaaggt 2040 atcctgtttc tgcaatgggt gcgtgatatt attgatgatt tcactaatga atctagccag 2100 aaaacgacaa ttgataaaat ttcggatgtt tccaccatcg tgccttacat cggcccagcg 2160 ctgaacatcg tgaagcaggg ttatgagggt aactttatcg gagcactgga aacgaccggc 2220 gtggttctgc tgctggaata tattccggag attactctgc cagttattgc ggctctgtcg 2280 attgcagaga gctcaacgca gaaagaaaaa attattaaga cgatcgacaa tttcctggaa 2340 aagcgctacg aaaaatggat cgaagtgtat aagctggtga aagcgaaatg gctggggacc 2400 gtgaacaccc agttccaaaa acgttcctat caaatgtatc gtagcctgga atatcaggtg 2460 gacgccatta aaaagatcat cgattacgaa tataagatct actccggtcc ggacaaagaa 2520 cagattgcgg acgaaattaa caatctgaaa aataaactgg aggaaaaagc caacaaagcg 2580 atgattaata tcaatatttt catgcgtgaa agcagccgta gcttcctggt caatcagatg 2640 attaatgaag cgaagaaaca actgctggaa tttgatacgc aatctaaaaa tattctgatg 2700 caatacatca aagccaattc taaatttatt gggatcacgg aactgaaaaa gctggaatcg 2760 aaaatcaata aagtctttag caccccgatt ccgttctcct actcgaaaaa tctggattgt 2820 tgggttgaca atgaagaaga tattgatgtt attctgaaaa agagcacgat cctgaacctg 2880 gatattaata acgatattat ctctgatatc agtggtttta attcatcagt tattacttac 2940 ccagacgctc aactggtgcc gggaatcaat gggaaagcca ttcatctggt gaataatgaa 3000 tcaagtgaag tgatcgtgca taaagcgatg gatatcgagt acaacgatat gtttaataat 3060 ttcacggtgt cgttctggct gcgtgttccg aaagtgagtg cctcccacct ggaacaatat 3120 ggaaccaacg aatactcaat cattagcagc atgaagaaac attcgctgag tattggttca 3180 ggttggagcg tttccctgaa agggaacaat ctgatctgga cactgaagga ctcagcgggc 3240 gaagtgcgtc agattacgtt tcgtgatctg ccggataaat ttaatgcata cctggctaac 3300 aaatgggtgt tcatcacaat caccaatgac cgtctgtcgt ctgcaaacct gtatattaat 3360 ggggtactga tgggctcggc agaaattaca gggctgggcg ccattcgtga agataacaat 3420 attacgctga aactggatcg ttgtaataac aataatcagt atgtgagcat tgataaattt 3480 cgtattttct gcaaagcgct gaacccgaaa gaaattgaaa aactgtatac ctcgtatctg 3540 tcaattacgt ttctgcgcga tttctgggga aacccgctgc gttacgatac ggaatactac 3600 ctgatcccgg tagccagttc tagtaaagac gttcaactga aaaatattac cgactacatg 3660 tatctgacaa acgctccatc atacacaaac ggcaaactga acatctatta ccgtcgcctg 3720 tacaatgggc tgaaattcat cattaaacgt tataccccga ataacgaaat tgattccttt 3780 gtgaagtccg gtgacttcat taagctgtat gtatcctata acaataatga acacatcgtt 3840 ggctatccga aggatggcaa tgcctttaac aacctggatc gtattctgcg tgtaggttac 3900 aacgccccgg gtattccgct gtataagaaa atggaagcag tgaaactgcg tgatctgaaa 3960 acatattccg tgcaactgaa gctgtatgat gacaaaaatg ctagcctggg tctggtaggc 4020 acgcataacg gtcagattgg aaacgatcct aatcgtgaca tcctgatcgc ctctaactgg 4080 tattttaacc acctgaaaga taaaattctg ggctgcgatt ggtattttgt ccctaccgat 4140 gaaggctgga cgaacgatta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260 gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780 gcctctcccc gcgcgttggc cgattcatta atgcag 6816 <210> 3 <211> 858 <212> PRT <213> Escherichia coli <400> 3 Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys Ile Lys Ile Lys Asn Glu 1 5 10 15 Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe Ser Glu Glu Pro Phe 20 25 30 Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn Lys Pro Leu Asn Phe 35 40 45 Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr Asn Leu Gln Ser Lys 50 55 60 Ile Thr Leu Pro Asn Asp Arg Thr Thr Pro Val Thr Lys Gly Ile Pro 65 70 75 80 Tyr Ala Pro Glu Tyr Lys Ser Asn Ala Ala Ser Thr Ile Glu Ile His 85 90 95 Asn Ile Asp Asp Asn Thr Ile Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser 100 105 110 Pro Thr Thr Leu Gln Arg Ile Thr Met Thr Asn Ser Val Asp Asp Ala 115 120 125 Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro Ser Val Ile Ser 130 135 140 Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe Leu Gln Trp Val Arg 145 150 155 160 Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys Thr Thr Ile 165 170 175 Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala 180 185 190 Leu Asn Ile Val Lys Gln Gly Tyr Glu Gly Asn Phe Ile Gly Ala Leu 195 200 205 Glu Thr Thr Gly Val Val Leu Leu Leu Glu Tyr Ile Pro Glu Ile Thr 210 215 220 Leu Pro Val Ile Ala Ala Leu Ser Ile Ala Glu Ser Ser Thr Gln Lys 225 230 235 240 Glu Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu Glu Lys Arg Tyr Glu 245 250 255 Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala Lys Trp Leu Gly Thr 260 265 270 Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln Met Tyr Arg Ser Leu 275 280 285 Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile Asp Tyr Glu Tyr Lys 290 295 300 Ile Tyr Ser Gly Pro Asp Lys Glu Gln Ile Ala Asp Glu Ile Asn Asn 305 310 315 320 Leu Lys Asn Lys Leu Glu Glu Lys Ala Asn Lys Ala Met Ile Asn Ile 325 330 335 Asn Ile Phe Met Arg Glu Ser Ser Arg Ser Phe Leu Val Asn Gln Met 340 345 350 Ile Asn Glu Ala Lys Lys Gln Leu Leu Glu Phe Asp Thr Gln Ser Lys 355 360 365 Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 370 375 380 Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn Lys Val Phe Ser Thr 385 390 395 400 Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp Cys Trp Val Asp Asn 405 410 415 Glu Glu Asp Ile Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu 420 425 430 Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser 435 440 445 Val Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys 450 455 460 Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys 465 470 475 480 Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser 485 490 495 Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr 500 505 510 Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu 515 520 525 Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile 530 535 540 Trp Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg 545 550 555 560 Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe 565 570 575 Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn 580 585 590 Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg 595 600 605 Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn 610 615 620 Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn 625 630 635 640 Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 645 650 655 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr 660 665 670 Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile 675 680 685 Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys 690 695 700 Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile 705 710 715 720 Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly 725 730 735 Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val 740 745 750 Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu 755 760 765 Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu 770 775 780 Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu 785 790 795 800 Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly 805 810 815 Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp 820 825 830 Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe 835 840 845 Val Pro Thr Asp Glu Gly Trp Thr Asn Asp 850 855 <210> 4 <211> 451 <212> PRT <213> Escherichia coli <400> 4 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 1 5 10 15 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 20 25 30 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 35 40 45 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 50 55 60 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 65 70 75 80 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 85 90 95 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 100 105 110 Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 115 120 125 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 130 135 140 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 145 150 155 160 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg 165 170 175 Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala 180 185 190 Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu 195 200 205 Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys 210 215 220 Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu 225 230 235 240 Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn 245 250 255 Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser 260 265 270 Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr 275 280 285 Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg 290 295 300 Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn 305 310 315 320 Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val 325 330 335 Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn 340 345 350 Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro 355 360 365 Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 370 375 380 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 385 390 395 400 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn 405 410 415 Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp 420 425 430 Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp 435 440 445 Thr Asn Asp 450 <210> 5 <211> 4234 <212> DNA <213> Escherichia coli <400> 5 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180 gatgacgata aggatcgatg gggatccgag ctcgagaaaa atctggattg ttgggttgac 240 aatgaagaag atattgatgt tattctgaaa aagagcacga tcctgaacct ggatattaat 300 aacgatatta tctctgatat cagtggtttt aattcatcag ttattactta cccagacgct 360 caactggtgc cgggaatcaa tgggaaagcc attcatctgg tgaataatga atcaagtgaa 420 gtgatcgtgc ataaagcgat ggatatcgag tacaacgata tgtttaataa tttcacggtg 480 tcgttctggc tgcgtgttcc gaaagtgagt gcctcccacc tggaacaata tggaaccaac 540 gaatactcaa tcattagcag catgaagaaa cattcgctga gtattggttc aggttggagc 600 gtttccctga aagggaacaa tctgatctgg acactgaagg actcagcggg cgaagtgcgt 660 cagattacgt ttcgtgatct gccggataaa tttaatgcat acctggctaa caaatgggtg 720 ttcatcacaa tcaccaatga ccgtctgtcg tctgcaaacc tgtatattaa tggggtactg 780 atgggctcgg cagaaattac agggctgggc gccattcgtg aagataacaa tattacgctg 840 aaactggatc gttgtaataa caataatcag tatgtgagca ttgataaatt tcgtattttc 900 tgcaaagcgc tgaacccgaa agaaattgaa aaactgtata cctcgtatct gtcaattacg 960 tttctgcgcg atttctgggg aaacccgctg cgttacgata cggaatacta cctgatcccg 1020 gtagccagtt ctagtaaaga cgttcaactg aaaaatatta ccgactacat gtatctgaca 1080 aacgctccat catacacaaa cggcaaactg aacatctatt accgtcgcct gtacaatggg 1140 ctgaaattca tcattaaacg ttataccccg aataacgaaa ttgattcctt tgtgaagtcc 1200 ggtgacttca ttaagctgta tgtatcctat aacaataatg aacacatcgt tggctatccg 1260 aaggatggca atgcctttaa caacctggat cgtattctgc gtgtaggtta caacgccccg 1320 ggtattccgc tgtataagaa aatggaagca gtgaaactgc gtgatctgaa aacatattcc 1380 gtgcaactga agctgtatga tgacaaaaat gctagcctgg gtctggtagg cacgcataac 1440 ggtcagattg gaaacgatcc taatcgtgac atcctgatcg cctctaactg gtattttaac 1500 cacctgaaag ataaaattct gggctgcgat tggtattttg tccctaccga tgaaggctgg 1560 acgaacgatt aagaattcga agcttgatcc ggctgctaac aaagcccgaa aggaagctga 1620 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 1680 cttgaggggt tttttgctga aaggaggaac tatatccgga tctggcgtaa tagcgaagag 1740 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 1800 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 1860 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 1920 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 1980 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 2040 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 2100 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 2160 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 2220 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa 2280 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 2340 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 2400 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 2460 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 2520 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 2580 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 2640 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 2700 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 2760 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 2820 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 2880 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 2940 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 3000 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 3060 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 3120 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 3180 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 3240 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 3300 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 3360 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 3420 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 3480 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 3540 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 3600 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 3660 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 3720 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 3780 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 3840 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 3900 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 3960 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 4020 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 4080 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 4140 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 4200 ctctccccgc gcgttggccg attcattaat gcag 4234 <210> 6 <211> 863 <212> PRT <213> Escherichia coli <400> 6 Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp 1 5 10 15 Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr 20 25 30 Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg 35 40 45 Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu 50 55 60 Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp 65 70 75 80 Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg 85 90 95 Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn 100 105 110 Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe Asp 115 120 125 Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Ser 130 135 140 Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly 145 150 155 160 Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg 165 170 175 Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile 180 185 190 Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val 195 200 205 Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln 210 215 220 Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly 225 230 235 240 Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys Gln 245 250 255 Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe 260 265 270 Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn 275 280 285 Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys 290 295 300 Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser 305 310 315 320 Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser Asn 325 330 335 Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser 340 345 350 Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile 355 360 365 Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile 370 375 380 Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn 385 390 395 400 Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg 405 410 415 Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val Ser 420 425 430 Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg 435 440 445 Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu 450 455 460 Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys 465 470 475 480 Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn 485 490 495 Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile 500 505 510 Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr 515 520 525 Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn 530 535 540 Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr 545 550 555 560 Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr 565 570 575 Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr 580 585 590 Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly 595 600 605 Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn 610 615 620 Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr 625 630 635 640 Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr 645 650 655 Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu 660 665 670 Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser 675 680 685 Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp 690 695 700 Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu 705 710 715 720 Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg 725 730 735 Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys 740 745 750 Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu 755 760 765 Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys 770 775 780 Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser 785 790 795 800 Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu 805 810 815 Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys 820 825 830 Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser 835 840 845 Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 <210> 7 <211> 5479 <212> DNA <213> Escherichia coli <400> 7 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180 gatgacgata aggatcgatg gggatccgag ctcgagccga ttaccatcaa taattttcgt 240 tattcagatc cggtgaataa tgataccatc atcatgatgg aaccaccgta ctgtaaaggg 300 ctggatattt attataaagc gttcaaaatc accgaccgca tctggatcgt gccggaacgc 360 tacgaattcg gcaccaaacc ggaagatttt aatccgccga gtagtctgat cgaaggtgca 420 tcggaatact acgatccgaa ttatctgcgt actgactctg ataaagatcg ctttctgcaa 480 acgatggtaa aactgttcaa tcgtatcaaa aacaatgtag caggcgaagc cctgctggat 540 aaaatcatca acgccattcc gtatctggga aacagttatt ctctgctgga taaattcgat 600 acaaactcga actctgtgtc attcaacctg ctggaacagg acccgagcgg cgcgaccact 660 aagagtgcga tgctgactaa cctgattatt ttcggtccgg gaccggtact gaataaaaat 720 gaagttcgcg gcattgtact gcgtgtcgat aataaaaact atttcccatg tcgtgatggc 780 ttcggcagca tcatgcagat ggccttttgt ccggaatatg tgccaacttt cgataatgtg 840 attgagaaca tcacctctct gacgattggt aaaagtaaat atttccagga tccggctctg 900 ctgctgatgc atgaactgat ccatgttctg catggcctgt atggcatgca ggtttcatcc 960 cacgaaatta tcccatccaa acaggaaatt tacatgcagc atacatatcc gattagtgcc 1020 gaagaactgt tcacttttgg cggccaggat gcgaacctga tttcgattga cattaaaaac 1080 gatctgtatg aaaaaactct gaacgattat aaagcgattg ccaacaaact gtctcaggta 1140 acctcctgta acgatccgaa tattgatatt gacagttata aacaaattta tcagcagaag 1200 tatcagttcg ataaagactc taatggccag tatattgtta acgaagataa attccagatt 1260 ctgtacaata gcattatgta tggctttact gagatcgaac tgggtaaaaa atttaacatc 1320 aagactcgtc tgagctattt tagcatgaat catgatccag tgaaaatccc gaatctgctg 1380 gatgatacga tttataatga taccgaagga tttaacatcg aaagcaagga tctgaaatcc 1440 gaatataaag ggcagaacat gcgcgttaat accaatgcat ttcgcaatgt tgatggttca 1500 ggcctggtgt cgaaactgat tgggctgtgt aagaaaatca ttccaccgac aaatattcgc 1560 gaaaatctgt acaaccgtac ggcgagcctg accgatctgg ggggagaact gtgtattaaa 1620 atcaaaaatg aagatctgac cttcattgct gagaagaata gcttttccga agagccattc 1680 caggacgaaa tcgtgtctta taacaccaag aataaaccgc tgaatttcaa ctactccctg 1740 gacaaaatca ttgtggatta caacctgcag agtaaaatta ccctgccgaa tgatcgtacc 1800 accccggtga cgaaaggcat cccttacgca ccagaatata aatcaaatgc agcctcgact 1860 atcgagatcc ataatattga tgacaacact atttaccagt acctgtatgc tcagaaatct 1920 ccgacgacgc tgcagcgcat caccatgact aacagcgtgg acgatgccct gattaatagc 1980 accaaaatct actcttactt tccgtcggtg atctctaagg ttaatcaggg cgcgcaaggt 2040 atcctgtttc tgcaatgggt gcgtgatatt attgatgatt tcactaatga atctagccag 2100 aaaacgacaa ttgataaaat ttcggatgtt tccaccatcg tgccttacat cggcccagcg 2160 ctgaacatcg tgaagcaggg ttatgagggt aactttatcg gagcactgga aacgaccggc 2220 gtggttctgc tgctggaata tattccggag attactctgc cagttattgc ggctctgtcg 2280 attgcagaga gctcaacgca gaaagaaaaa attattaaga cgatcgacaa tttcctggaa 2340 aagcgctacg aaaaatggat cgaagtgtat aagctggtga aagcgaaatg gctggggacc 2400 gtgaacaccc agttccaaaa acgttcctat caaatgtatc gtagcctgga atatcaggtg 2460 gacgccatta aaaagatcat cgattacgaa tataagatct actccggtcc ggacaaagaa 2520 cagattgcgg acgaaattaa caatctgaaa aataaactgg aggaaaaagc caacaaagcg 2580 atgattaata tcaatatttt catgcgtgaa agcagccgta gcttcctggt caatcagatg 2640 attaatgaag cgaagaaaca actgctggaa tttgatacgc aatctaaaaa tattctgatg 2700 caatacatca aagccaattc taaatttatt gggatcacgg aactgaaaaa gctggaatcg 2760 aaaatcaata aagtctttag caccccgatt ccgttctcct actcgtaagg taccatggaa 2820 ttcgaagctt gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac 2880 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 2940 gctgaaagga ggaactatat ccggatctgg cgtaatagcg aagaggcccg caccgatcgc 3000 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta 3060 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 3120 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 3180 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 3240 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 3300 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 3360 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 3420 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 3480 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 3540 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 3600 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 3660 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 3720 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 3780 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 3840 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 3900 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 3960 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 4020 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 4080 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 4140 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 4200 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 4260 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 4320 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 4380 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 4440 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 4500 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 4560 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 4620 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 4680 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 4740 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 4800 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 4860 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 4920 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 4980 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 5040 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 5100 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 5160 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 5220 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 5280 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 5340 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 5400 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 5460 ggccgattca ttaatgcag 5479 <210> 8 <211> 451 <212> PRT <213> Escherichia coli <400> 8 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 1 5 10 15 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 20 25 30 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 35 40 45 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 50 55 60 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 65 70 75 80 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 85 90 95 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 100 105 110 Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 115 120 125 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 130 135 140 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 145 150 155 160 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg 165 170 175 Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala 180 185 190 Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu 195 200 205 Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys 210 215 220 Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu 225 230 235 240 Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn 245 250 255 Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser 260 265 270 Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr 275 280 285 Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg 290 295 300 Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn 305 310 315 320 Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val 325 330 335 Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn 340 345 350 Ala Phe Asn Asn Leu Asp Arg Ile Leu Glu Val Gly Tyr Asn Ala Pro 355 360 365 Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 370 375 380 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 385 390 395 400 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn 405 410 415 Arg Asp Ile Leu Ile Ala Ser Asn Ala Tyr Phe Asn His Leu Lys Asp 420 425 430 Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp 435 440 445 Thr Asn Asp 450 <210> 9 <211> 4234 <212> DNA <213> Escherichia coli <400> 9 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180 gatgacgata aggatcgatg gggatccgag ctcgagaaaa atttggattg ctgggtcgac 240 aatgaggagg acattgatgt gatattaaag aaatccacta tcttaaatct tgatataaac 300 aatgacatca tctcggatat atctgggttc aattcttccg tcataactta ccctgatgct 360 caactggtac cgggaattaa tgggaaggcc atacacttag tcaacaacga atcttccgag 420 gtgattgtac ataaggcaat ggacatagag tacaatgaca tgtttaataa ctttactgtc 480 tcgttctggt tacgcgtgcc caaagtatct gcctcacacc tggaacagta tggcacaaat 540 gaatattcta ttatcagtag tatgaagaaa cactcgcttt ctataggatc cggctggagt 600 gtttcgctga agggcaataa cttgatctgg acccttaaag attcagcggg agaagtaaga 660 caaataactt tccgggattt gcctgataag ttcaacgcat acctggccaa taagtgggtt 720 ttcataacta taacaaatga ccgcttgtct agcgccaact tatacatcaa tggagtattg 780 atgggttccg ctgagattac aggcttgggt gccataagag aggacaataa tatcaccctg 840 aaactggacc gctgcaataa taacaatcag tacgtgagca tagataaatt ccgtattttt 900 tgcaaagccc tgaacccgaa agaaatcgaa aaactgtata cttcatatct gagcataaca 960 tttctgcgtg atttttgggg taacccgctg cgttatgata ccgaatacta cctgattccg 1020 gttgccagca gcagcaaaga tgttcagctg aaaaatatta ccgactatat gtatctgaca 1080 aatgcgccgt cttataccaa tggcaaactg aatatttatt atcgccggtt gtacaacggg 1140 ctgaagttca ttattaaacg gtacaccccg aacaacgaaa tcgattcatt tgttaaatcc 1200 ggggatttca taaagttata cgtgagctat aataacaacg agcatattgt aggttatccg 1260 aaagacggta atgctttcaa caacttggat cggatactgg aagttggtta caacgcccca 1320 ggtattccac tgtataagaa aatggaagcc gtcaagttgc gtgatttaaa gacgtactca 1380 gtacagctta aattatacga cgataagaat gcaagccttg gattggttgg gacccacaat 1440 ggtcagattg gaaatgaccc caatcgggac attctgatag catccaacgc gtacttcaac 1500 catttgaaag ataaaatcct gggctgtgat tggtactttg taccgactga tgaaggatgg 1560 acgaatgact aagaattcga agcttgatcc ggctgctaac aaagcccgaa aggaagctga 1620 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 1680 cttgaggggt tttttgctga aaggaggaac tatatccgga tctggcgtaa tagcgaagag 1740 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 1800 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 1860 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 1920 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 1980 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 2040 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 2100 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 2160 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 2220 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa 2280 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 2340 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 2400 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 2460 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 2520 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 2580 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 2640 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 2700 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 2760 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 2820 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 2880 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 2940 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 3000 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 3060 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 3120 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 3180 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 3240 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 3300 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 3360 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 3420 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 3480 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 3540 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 3600 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 3660 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 3720 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 3780 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 3840 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 3900 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 3960 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 4020 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 4080 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 4140 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 4200 ctctccccgc gcgttggccg attcattaat gcag 4234 <210> 10 <211> 1314 <212> PRT <213> Escherichia coli <400> 10 Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp 1 5 10 15 Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr 20 25 30 Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg 35 40 45 Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu 50 55 60 Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp 65 70 75 80 Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg 85 90 95 Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn 100 105 110 Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe Asp 115 120 125 Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Ser 130 135 140 Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly 145 150 155 160 Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg 165 170 175 Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile 180 185 190 Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val 195 200 205 Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln 210 215 220 Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly 225 230 235 240 Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys Gln 245 250 255 Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe 260 265 270 Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn 275 280 285 Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys 290 295 300 Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser 305 310 315 320 Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser Asn 325 330 335 Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser 340 345 350 Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile 355 360 365 Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile 370 375 380 Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn 385 390 395 400 Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg 405 410 415 Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val Ser 420 425 430 Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg 435 440 445 Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu 450 455 460 Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys 465 470 475 480 Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn 485 490 495 Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile 500 505 510 Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr 515 520 525 Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn 530 535 540 Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr 545 550 555 560 Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr 565 570 575 Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr 580 585 590 Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly 595 600 605 Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn 610 615 620 Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr 625 630 635 640 Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr 645 650 655 Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu 660 665 670 Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser 675 680 685 Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp 690 695 700 Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu 705 710 715 720 Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg 725 730 735 Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys 740 745 750 Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu 755 760 765 Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys 770 775 780 Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser 785 790 795 800 Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu 805 810 815 Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys 820 825 830 Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser 835 840 845 Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys 850 855 860 Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu 865 870 875 880 Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser 885 890 895 Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln 900 905 910 Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu 915 920 925 Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp 930 935 940 Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val 945 950 955 960 Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile 965 970 975 Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val 980 985 990 Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly 995 1000 1005 Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 1010 1015 1020 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp 1025 1030 1035 Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly 1040 1045 1050 Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn 1055 1060 1065 Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val 1070 1075 1080 Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys 1085 1090 1095 Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu 1100 1105 1110 Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr 1115 1120 1125 Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn 1130 1135 1140 Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn 1145 1150 1155 Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys 1160 1165 1170 Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe 1175 1180 1185 Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn 1190 1195 1200 Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn 1205 1210 1215 Asn Leu Asp Arg Ile Leu Glu Val Gly Tyr Asn Ala Pro Gly Ile 1220 1225 1230 Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys 1235 1240 1245 Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 1250 1255 1260 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro 1265 1270 1275 Asn Arg Asp Ile Leu Ile Ala Ser Asn Ala Tyr Phe Asn His Leu 1280 1285 1290 Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp 1295 1300 1305 Glu Gly Trp Thr Asn Asp 1310 <210> 11 <211> 6816 <212> DNA <213> Escherichia coli <400> 11 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180 gatgacgata aggatcgatg gggatccgag ctcgagccga tcaccatcaa caacttccgt 240 tactctgacc cggttaacaa cgacaccatc atcatgatgg aaccgccgta ctgcaaaggt 300 ctggacatct actacaaagc gttcaaaatc accgaccgta tctggatcgt tccggaacgt 360 tacgaattcg gtaccaaacc ggaagacttc aacccgccgt cttctctgat cgaaggtgcg 420 tctgaatact acgacccgaa ctacctgcgt accgactctg acaaagaccg tttcctgcag 480 accatggtta aactgttcaa ccgtatcaaa aacaacgttg cgggtgaagc gctgctggac 540 aaaatcatca acgcgatccc gtacctgggt aactcttact ctctgctgga caaattcgac 600 accaactcta actctgtttc tttcaacctg ctggaacagg acccgtctgg tgcgaccacc 660 aaatctgcga tgctgaccaa cctgatcatc ttcggtccgg gtccggttct gaacaaaaac 720 gaagttcgtg gtatcgttct gcgtgttgac aacaaaaact acttcccgtg ccgtgacggt 780 ttcggttcta tcatgcagat ggcgttctgc ccggaatacg ttccgacctt cgacaacgtt 840 atcgaaaaca tcacctctct gaccatcggt aaatctaaat acttccagga cccggcgctg 900 ctgctgatgc acgaactgat ccacgttctg cacggtctgt acggtatgca ggtttcttct 960 cacgaaatca tcccgtctaa acaggaaatc tacatgcagc acacctaccc gatctctgcg 1020 gaagaactgt tcaccttcgg tggtcaggac gcgaacctga tctctatcga catcaaaaac 1080 gacctgtacg aaaaaaccct gaacgactac aaagcgatcg cgaacaaact gtctcaggtt 1140 acctcttgca acgacccgaa catcgacatc gactcttaca aacagatcta ccagcagaaa 1200 taccagttcg acaaagactc taacggtcag tacatcgtta acgaagacaa attccagatc 1260 ctgtacaact ctatcatgta cggtttcacc gaaatcgaac tgggtaaaaa attcaacatc 1320 aaaacccgtc tgtcttactt ctctatgaac cacgacccgg ttaaaatccc gaacctgctg 1380 gacgacacca tctacaacga caccgaaggt ttcaacatcg aatctaaaga cctgaaatct 1440 gaatacaaag gtcagaacat gcgtgttaac accaacgcgt tccgtaacgt tgacggttct 1500 ggtctggttt ctaaactgat cggtctgtgc aaaaaaatca tcccgccgac caacatccgt 1560 gaaaacctgt acaaccgtac cgcgtctctg accgacctgg gtggtgaact gtgcatcaaa 1620 atcaaaaacg aagacctgac cttcatcgcg gaaaaaaact ctttctctga agaaccgttc 1680 caggacgaaa tcgtttctta caacaccaaa aacaaaccgc tgaacttcaa ctactctctg 1740 gacaaaatca tcgttgacta caacctgcag tctaaaatca ccctgccgaa cgaccgtacc 1800 accccggtta ccaaaggtat cccgtacgcg ccggaataca aatctaacgc ggcgtctacc 1860 atcgaaatcc acaacatcga cgacaacacc atctaccagt acctgtacgc gcagaaatct 1920 ccgaccaccc tgcagcgtat caccatgacc aactctgttg acgacgcgct gatcaactct 1980 accaaaatct actcttactt cccgtctgtt atctctaaag ttaaccaggg tgcgcagggt 2040 atcctgttcc tgcagtgggt tcgtgacatc atcgacgact tcaccaacga atcttctcag 2100 aaaaccacca tcgacaaaat ctctgacgtt tctaccatcg ttccgtacat cggtccggcg 2160 ctgaacatcg ttaaacaggg ttacgaaggt aacttcatcg gtgcgctgga aaccaccggt 2220 gttgttctgc tgctggaata catcccggaa atcaccctgc cggttatcgc ggcgctgtct 2280 atcgcggaat cttctaccca gaaagaaaaa atcatcaaaa ccatcgacaa cttcctggaa 2340 aaacgttacg aaaaatggat cgaagtttac aaactggtta aagcgaaatg gctgggtacc 2400 gttaacaccc agttccagaa acgttcttac cagatgtacc gttctctgga ataccaggtt 2460 gacgcgatca aaaaaatcat cgactacgaa tacaaaatct actctggtcc ggacaaagaa 2520 cagatcgcgg acgaaatcaa caacctgaaa aacaaactgg aagaaaaagc gaacaaagcg 2580 atgatcaaca tcaacatctt catgcgtgaa tcttctcgtt ctttcctggt taaccagatg 2640 atcaacgaag cgaaaaaaca gctgctggaa ttcgacaccc agtctaaaaa catcctgatg 2700 cagtacatca aagcgaactc taaattcatc ggtatcaccg aactgaaaaa actggaatct 2760 aaaatcaaca aagttttctc taccccgatc ccgttctctt actctaaaaa cctggactgc 2820 tgggttgaca acgaagaaga catcgacgtt atcctgaaaa aatctaccat cctgaacctg 2880 gacatcaaca acgacatcat ctctgacatc tctggtttca actcttctgt tatcacctac 2940 ccggacgcgc agctggttcc gggtatcaac ggtaaagcga tccacctggt taacaacgaa 3000 tcttctgaag ttatcgttca caaagcgatg gacatcgaat acaacgacat gttcaacaac 3060 ttcaccgttt ctttctggct gcgtgttccg aaagtttctg cgtctcacct ggaacagtac 3120 ggtaccaacg aatactctat catctcttct atgaaaaaac actctctgtc tatcggttct 3180 ggttggtctg tttctctgaa aggtaacaac ctgatctgga ccctgaaaga ctctgcgggt 3240 gaagttcgtc agatcacctt ccgtgacctg ccggacaaat tcaacgcgta cctggcgaac 3300 aaatgggttt tcatcaccat caccaacgac cgtctgtctt ctgcgaacct gtacatcaac 3360 ggtgttctga tgggttctgc ggaaatcacc ggtctgggtg cgatccgtga agacaacaac 3420 atcaccctga aactggaccg ttgcaacaac aacaaccagt acgtttctat cgacaaattc 3480 cgtatcttct gcaaagcgct gaacccgaaa gaaatcgaaa aactgtacac ctcttacctg 3540 tctatcacct tcctgcgtga cttctggggt aacccgctgc gttacgacac cgaatactac 3600 ctgatcccgg ttgcgtcttc ttctaaagac gttcagctga aaaacatcac cgactacatg 3660 tacctgacca acgcgccgtc ttacaccaac ggtaaactga acatctacta ccgtcgtctg 3720 tacaacggtc tgaaattcat catcaaacgt tacaccccga acaacgaaat cgactctttc 3780 gttaaatctg gtgacttcat caaactgtac gtttcttaca acaacaacga acacatcgtt 3840 ggttacccga aagacggtaa cgcgttcaac aacctggacc gtatcctgga agttggttac 3900 aacgcgccgg gtatcccgct gtacaaaaaa atggaagcgg ttaaactgcg tgacctgaaa 3960 acctactctg ttcagctgaa actgtacgac gacaaaaacg cgtctctggg tctggttggt 4020 acccacaacg gtcagatcgg taacgacccg aaccgtgaca tcctgatcgc gtctaacgcg 4080 tacttcaacc acctgaaaga caaaatcctg ggttgcgact ggtacttcgt tccgaccgac 4140 gaaggttgga ccaacgacta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260 gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780 gcctctcccc gcgcgttggc cgattcatta atgcag 6816 <210> 12 <211> 1314 <212> PRT <213> Escherichia coli <400> 12 Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp 1 5 10 15 Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr 20 25 30 Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg 35 40 45 Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu 50 55 60 Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp 65 70 75 80 Cys Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg 85 90 95 Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn 100 105 110 Ala Ile Pro Tyr Leu Gly Asn Cys Tyr Ser Leu Leu Asp Lys Phe Asp 115 120 125 Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Cys 130 135 140 Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly 145 150 155 160 Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg 165 170 175 Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile 180 185 190 Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val 195 200 205 Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln 210 215 220 Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly 225 230 235 240 Leu Tyr Gly Met Gln Val Ser Cys His Glu Ile Ile Pro Ser Lys Gln 245 250 255 Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe 260 265 270 Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn 275 280 285 Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys 290 295 300 Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser 305 310 315 320 Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Cys Asn 325 330 335 Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser 340 345 350 Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile 355 360 365 Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile 370 375 380 Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn 385 390 395 400 Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg 405 410 415 Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Cys Gly Leu Val Ser 420 425 430 Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg 435 440 445 Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu 450 455 460 Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys 465 470 475 480 Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn 485 490 495 Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile 500 505 510 Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr 515 520 525 Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn 530 535 540 Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr 545 550 555 560 Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr 565 570 575 Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr 580 585 590 Ser Tyr Phe Pro Ser Val Ile Cys Lys Val Asn Gln Gly Ala Gln Gly 595 600 605 Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn 610 615 620 Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr 625 630 635 640 Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr 645 650 655 Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu 660 665 670 Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser 675 680 685 Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp 690 695 700 Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu 705 710 715 720 Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg 725 730 735 Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys 740 745 750 Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu 755 760 765 Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys 770 775 780 Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser 785 790 795 800 Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu 805 810 815 Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys 820 825 830 Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser 835 840 845 Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys 850 855 860 Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu 865 870 875 880 Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser 885 890 895 Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln 900 905 910 Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu 915 920 925 Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp 930 935 940 Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val 945 950 955 960 Ser Ala Cys His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile 965 970 975 Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val 980 985 990 Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly 995 1000 1005 Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 1010 1015 1020 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp 1025 1030 1035 Arg Leu Cys Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly 1040 1045 1050 Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn 1055 1060 1065 Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val 1070 1075 1080 Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys 1085 1090 1095 Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu 1100 1105 1110 Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr 1115 1120 1125 Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn 1130 1135 1140 Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Cys Tyr Thr Asn 1145 1150 1155 Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys 1160 1165 1170 Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Cys Phe 1175 1180 1185 Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn 1190 1195 1200 Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn 1205 1210 1215 Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile 1220 1225 1230 Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys 1235 1240 1245 Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 1250 1255 1260 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro 1265 1270 1275 Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu 1280 1285 1290 Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp 1295 1300 1305 Glu Gly Trp Thr Asn Asp 1310 <210> 13 <211> 6816 <212> DNA <213> Escherichia coli <400> 13 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180 gatgacgata aggatcgatg gggatccgag ctcgagccga tcaccatcaa caacttccgt 240 tactctgacc cggttaacaa cgacaccatc atcatgatgg aaccgccgta ctgcaaaggt 300 ctggacatct actacaaagc gttcaaaatc accgaccgta tctggatcgt tccggaacgt 360 tacgaattcg gtaccaaacc ggaagacttc aacccgccgt cttctctgat cgaaggtgcg 420 tctgaatact acgacccgaa ctacctgcgt accgactgcg acaaagaccg tttcctgcag 480 accatggtta aactgttcaa ccgtatcaaa aacaacgttg cgggtgaagc gctgctggac 540 aaaatcatca acgcgatccc gtacctgggt aactgctact ctctgctgga caaattcgac 600 accaactcta actctgtttc tttcaacctg ctggaacagg acccgtgcgg tgcgaccacc 660 aaatctgcga tgctgaccaa cctgatcatc ttcggtccgg gtccggttct gaacaaaaac 720 gaagttcgtg gtatcgttct gcgtgttgac aacaaaaact acttcccgtg ccgtgacggt 780 ttcggttcta tcatgcagat ggcgttctgc ccggaatacg ttccgacctt cgacaacgtt 840 atcgaaaaca tcacctctct gaccatcggt aaatctaaat acttccagga cccggcgctg 900 ctgctgatgc acgaactgat ccacgttctg cacggtctgt acggtatgca ggtttcttgc 960 cacgaaatca tcccgtctaa acaggaaatc tacatgcagc acacctaccc gatctctgcg 1020 gaagaactgt tcaccttcgg tggtcaggac gcgaacctga tctctatcga catcaaaaac 1080 gacctgtacg aaaaaaccct gaacgactac aaagcgatcg cgaacaaact gtctcaggtt 1140 acctcttgca acgacccgaa catcgacatc gactcttaca aacagatcta ccagcagaaa 1200 taccagttcg acaaagactg caacggtcag tacatcgtta acgaagacaa attccagatc 1260 ctgtacaact ctatcatgta cggtttcacc gaaatcgaac tgggtaaaaa attcaacatc 1320 aaaacccgtc tgtcttactt ctctatgaac cacgacccgg ttaaaatccc gaacctgctg 1380 gacgacacca tctacaacga caccgaaggt ttcaacatcg aatctaaaga cctgaaatct 1440 gaatacaaag gtcagaacat gcgtgttaac accaacgcgt tccgtaacgt tgacggttgc 1500 ggtctggttt ctaaactgat cggtctgtgc aaaaaaatca tcccgccgac caacatccgt 1560 gaaaacctgt acaaccgtac cgcgtctctg accgacctgg gtggtgaact gtgcatcaaa 1620 atcaaaaacg aagacctgac cttcatcgcg gaaaaaaact ctttctctga agaaccgttc 1680 caggacgaaa tcgtttctta caacaccaaa aacaaaccgc tgaacttcaa ctactctctg 1740 gacaaaatca tcgttgacta caacctgcag tctaaaatca ccctgccgaa cgaccgtacc 1800 accccggtta ccaaaggtat cccgtacgcg ccggaataca aatctaacgc ggcgtctacc 1860 atcgaaatcc acaacatcga cgacaacacc atctaccagt acctgtacgc gcagaaatct 1920 ccgaccaccc tgcagcgtat caccatgacc aactctgttg acgacgcgct gatcaactct 1980 accaaaatct actcttactt cccgtctgtt atctgcaaag ttaaccaggg tgcgcagggt 2040 atcctgttcc tgcagtgggt tcgtgacatc atcgacgact tcaccaacga atcttctcag 2100 aaaaccacca tcgacaaaat ctctgacgtt tctaccatcg ttccgtacat cggtccggcg 2160 ctgaacatcg ttaaacaggg ttacgaaggt aacttcatcg gtgcgctgga aaccaccggt 2220 gttgttctgc tgctggaata catcccggaa atcaccctgc cggttatcgc ggcgctgtct 2280 atcgcggaat cttctaccca gaaagaaaaa atcatcaaaa ccatcgacaa cttcctggaa 2340 aaacgttacg aaaaatggat cgaagtttac aaactggtta aagcgaaatg gctgggtacc 2400 gttaacaccc agttccagaa acgttcttac cagatgtacc gttctctgga ataccaggtt 2460 gacgcgatca aaaaaatcat cgactacgaa tacaaaatct actctggtcc ggacaaagaa 2520 cagatcgcgg acgaaatcaa caacctgaaa aacaaactgg aagaaaaagc gaacaaagcg 2580 atgatcaaca tcaacatctt catgcgtgaa tcttctcgtt ctttcctggt taaccagatg 2640 atcaacgaag cgaaaaaaca gctgctggaa ttcgacaccc agtctaaaaa catcctgatg 2700 cagtacatca aagcgaactc taaattcatc ggtatcaccg aactgaaaaa actggaatct 2760 aaaatcaaca aagttttctc taccccgatc ccgttctctt actctaaaaa cctggactgc 2820 tgggttgaca acgaagaaga catcgacgtt atcctgaaaa aatctaccat cctgaacctg 2880 gacatcaaca acgacatcat ctctgacatc tctggtttca actcttctgt tatcacctac 2940 ccggacgcgc agctggttcc gggtatcaac ggtaaagcga tccacctggt taacaacgaa 3000 tcttctgaag ttatcgttca caaagcgatg gacatcgaat acaacgacat gttcaacaac 3060 ttcaccgttt ctttctggct gcgtgttccg aaagtttctg cgtgccacct ggaacagtac 3120 ggtaccaacg aatactctat catctcttct atgaaaaaac actctctgtc tatcggttct 3180 ggttggtctg tttctctgaa aggtaacaac ctgatctgga ccctgaaaga ctctgcgggt 3240 gaagttcgtc agatcacctt ccgtgacctg ccggacaaat tcaacgcgta cctggcgaac 3300 aaatgggttt tcatcaccat caccaacgac cgtctgtgct ctgcgaacct gtacatcaac 3360 ggtgttctga tgggttctgc ggaaatcacc ggtctgggtg cgatccgtga agacaacaac 3420 atcaccctga aactggaccg ttgcaacaac aacaaccagt acgtttctat cgacaaattc 3480 cgtatcttct gcaaagcgct gaacccgaaa gaaatcgaaa aactgtacac ctcttacctg 3540 tctatcacct tcctgcgtga cttctggggt aacccgctgc gttacgacac cgaatactac 3600 ctgatcccgg ttgcgtcttc ttctaaagac gttcagctga aaaacatcac cgactacatg 3660 tacctgacca acgcgccgtg ctacaccaac ggtaaactga acatctacta ccgtcgtctg 3720 tacaacggtc tgaaattcat catcaaacgt tacaccccga acaacgaaat cgactgcttc 3780 gttaaatctg gtgacttcat caaactgtac gtttcttaca acaacaacga acacatcgtt 3840 ggttacccga aagacggtaa cgcgttcaac aacctggacc gtatcctgcg tgttggttac 3900 aacgcgccgg gtatcccgct gtacaaaaaa atggaagcgg ttaaactgcg tgacctgaaa 3960 acctactctg ttcagctgaa actgtacgac gacaaaaacg cgtctctggg tctggttggt 4020 acccacaacg gtcagatcgg taacgacccg aaccgtgaca tcctgatcgc gtctaactgg 4080 tacttcaacc acctgaaaga caaaatcctg ggttgcgact ggtacttcgt tccgaccgac 4140 gaaggttgga ccaacgacta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260 gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780 gcctctcccc gcgcgttggc cgattcatta atgcag 6816 <210> 14 <211> 1314 <212> PRT <213> Escherichia coli <400> 14 Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp 1 5 10 15 Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr 20 25 30 Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg 35 40 45 Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu 50 55 60 Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp 65 70 75 80 Cys Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg 85 90 95 Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn 100 105 110 Ala Ile Pro Tyr Leu Gly Asn Cys Tyr Ser Leu Leu Asp Lys Phe Asp 115 120 125 Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Cys 130 135 140 Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly 145 150 155 160 Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg 165 170 175 Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile 180 185 190 Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val 195 200 205 Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln 210 215 220 Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly 225 230 235 240 Leu Tyr Gly Met Gln Val Ser Cys His Glu Ile Ile Pro Ser Lys Gln 245 250 255 Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe 260 265 270 Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn 275 280 285 Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys 290 295 300 Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser 305 310 315 320 Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Cys Asn 325 330 335 Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser 340 345 350 Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile 355 360 365 Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile 370 375 380 Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn 385 390 395 400 Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg 405 410 415 Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Cys Gly Leu Val Ser 420 425 430 Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg 435 440 445 Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu 450 455 460 Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys 465 470 475 480 Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn 485 490 495 Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile 500 505 510 Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr 515 520 525 Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn 530 535 540 Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr 545 550 555 560 Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr 565 570 575 Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr 580 585 590 Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln Gly 595 600 605 Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn 610 615 620 Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr 625 630 635 640 Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr 645 650 655 Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu 660 665 670 Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser 675 680 685 Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp 690 695 700 Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu 705 710 715 720 Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg 725 730 735 Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys 740 745 750 Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu 755 760 765 Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys 770 775 780 Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser 785 790 795 800 Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu 805 810 815 Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys 820 825 830 Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser 835 840 845 Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys 850 855 860 Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu 865 870 875 880 Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser 885 890 895 Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln 900 905 910 Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu 915 920 925 Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp 930 935 940 Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val 945 950 955 960 Ser Ala Cys His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile 965 970 975 Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val 980 985 990 Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly 995 1000 1005 Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 1010 1015 1020 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp 1025 1030 1035 Arg Leu Cys Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly 1040 1045 1050 Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn 1055 1060 1065 Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val 1070 1075 1080 Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys 1085 1090 1095 Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu 1100 1105 1110 Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr 1115 1120 1125 Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn 1130 1135 1140 Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Cys Tyr Thr Asn 1145 1150 1155 Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys 1160 1165 1170 Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Cys Phe 1175 1180 1185 Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn 1190 1195 1200 Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn 1205 1210 1215 Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile 1220 1225 1230 Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys 1235 1240 1245 Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 1250 1255 1260 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro 1265 1270 1275 Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu 1280 1285 1290 Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp 1295 1300 1305 Glu Gly Trp Thr Asn Asp 1310 <210> 15 <211> 6816 <212> DNA <213> Escherichia coli <400> 15 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180 gatgacgata aggatcgatg gggatccgag ctcgagccga tcaccatcaa caacttccgt 240 tactctgacc cggttaacaa cgacaccatc atcatgatgg aaccgccgta ctgcaaaggt 300 ctggacatct actacaaagc gttcaaaatc accgaccgta tctggatcgt tccggaacgt 360 tacgaattcg gtaccaaacc ggaagacttc aacccgccgt cttctctgat cgaaggtgcg 420 tctgaatact acgacccgaa ctacctgcgt accgactgcg acaaagaccg tttcctgcag 480 accatggtta aactgttcaa ccgtatcaaa aacaacgttg cgggtgaagc gctgctggac 540 aaaatcatca acgcgatccc gtacctgggt aactgctact ctctgctgga caaattcgac 600 accaactcta actctgtttc tttcaacctg ctggaacagg acccgtgcgg tgcgaccacc 660 aaatctgcga tgctgaccaa cctgatcatc ttcggtccgg gtccggttct gaacaaaaac 720 gaagttcgtg gtatcgttct gcgtgttgac aacaaaaact acttcccgtg ccgtgacggt 780 ttcggttcta tcatgcagat ggcgttctgc ccggaatacg ttccgacctt cgacaacgtt 840 atcgaaaaca tcacctctct gaccatcggt aaatctaaat acttccagga cccggcgctg 900 ctgctgatgc acgaactgat ccacgttctg cacggtctgt acggtatgca ggtttcttgc 960 cacgaaatca tcccgtctaa acaggaaatc tacatgcagc acacctaccc gatctctgcg 1020 gaagaactgt tcaccttcgg tggtcaggac gcgaacctga tctctatcga catcaaaaac 1080 gacctgtacg aaaaaaccct gaacgactac aaagcgatcg cgaacaaact gtctcaggtt 1140 acctcttgca acgacccgaa catcgacatc gactcttaca aacagatcta ccagcagaaa 1200 taccagttcg acaaagactg caacggtcag tacatcgtta acgaagacaa attccagatc 1260 ctgtacaact ctatcatgta cggtttcacc gaaatcgaac tgggtaaaaa attcaacatc 1320 aaaacccgtc tgtcttactt ctctatgaac cacgacccgg ttaaaatccc gaacctgctg 1380 gacgacacca tctacaacga caccgaaggt ttcaacatcg aatctaaaga cctgaaatct 1440 gaatacaaag gtcagaacat gcgtgttaac accaacgcgt tccgtaacgt tgacggttgc 1500 ggtctggttt ctaaactgat cggtctgtgc aaaaaaatca tcccgccgac caacatccgt 1560 gaaaacctgt acaaccgtac cgcgtctctg accgacctgg gtggtgaact gtgcatcaaa 1620 atcaaaaacg aagacctgac cttcatcgcg gaaaaaaact ctttctctga agaaccgttc 1680 caggacgaaa tcgtttctta caacaccaaa aacaaaccgc tgaacttcaa ctactctctg 1740 gacaaaatca tcgttgacta caacctgcag tctaaaatca ccctgccgaa cgaccgtacc 1800 accccggtta ccaaaggtat cccgtacgcg ccggaataca aatctaacgc ggcgtctacc 1860 atcgaaatcc acaacatcga cgacaacacc atctaccagt acctgtacgc gcagaaatct 1920 ccgaccaccc tgcagcgtat caccatgacc aactctgttg acgacgcgct gatcaactct 1980 accaaaatct actcttactt cccgtctgtt atctctaaag ttaaccaggg tgcgcagggt 2040 atcctgttcc tgcagtgggt tcgtgacatc atcgacgact tcaccaacga atcttctcag 2100 aaaaccacca tcgacaaaat ctctgacgtt tctaccatcg ttccgtacat cggtccggcg 2160 ctgaacatcg ttaaacaggg ttacgaaggt aacttcatcg gtgcgctgga aaccaccggt 2220 gttgttctgc tgctggaata catcccggaa atcaccctgc cggttatcgc ggcgctgtct 2280 atcgcggaat cttctaccca gaaagaaaaa atcatcaaaa ccatcgacaa cttcctggaa 2340 aaacgttacg aaaaatggat cgaagtttac aaactggtta aagcgaaatg gctgggtacc 2400 gttaacaccc agttccagaa acgttcttac cagatgtacc gttctctgga ataccaggtt 2460 gacgcgatca aaaaaatcat cgactacgaa tacaaaatct actctggtcc ggacaaagaa 2520 cagatcgcgg acgaaatcaa caacctgaaa aacaaactgg aagaaaaagc gaacaaagcg 2580 atgatcaaca tcaacatctt catgcgtgaa tcttctcgtt ctttcctggt taaccagatg 2640 atcaacgaag cgaaaaaaca gctgctggaa ttcgacaccc agtctaaaaa catcctgatg 2700 cagtacatca aagcgaactc taaattcatc ggtatcaccg aactgaaaaa actggaatct 2760 aaaatcaaca aagttttctc taccccgatc ccgttctctt actctaaaaa cctggactgc 2820 tgggttgaca acgaagaaga catcgacgtt atcctgaaaa aatctaccat cctgaacctg 2880 gacatcaaca acgacatcat ctctgacatc tctggtttca actcttctgt tatcacctac 2940 ccggacgcgc agctggttcc gggtatcaac ggtaaagcga tccacctggt taacaacgaa 3000 tcttctgaag ttatcgttca caaagcgatg gacatcgaat acaacgacat gttcaacaac 3060 ttcaccgttt ctttctggct gcgtgttccg aaagtttctg cgtgccacct ggaacagtac 3120 ggtaccaacg aatactctat catctcttct atgaaaaaac actctctgtc tatcggttct 3180 ggttggtctg tttctctgaa aggtaacaac ctgatctgga ccctgaaaga ctctgcgggt 3240 gaagttcgtc agatcacctt ccgtgacctg ccggacaaat tcaacgcgta cctggcgaac 3300 aaatgggttt tcatcaccat caccaacgac cgtctgtgct ctgcgaacct gtacatcaac 3360 ggtgttctga tgggttctgc ggaaatcacc ggtctgggtg cgatccgtga agacaacaac 3420 atcaccctga aactggaccg ttgcaacaac aacaaccagt acgtttctat cgacaaattc 3480 cgtatcttct gcaaagcgct gaacccgaaa gaaatcgaaa aactgtacac ctcttacctg 3540 tctatcacct tcctgcgtga cttctggggt aacccgctgc gttacgacac cgaatactac 3600 ctgatcccgg ttgcgtcttc ttctaaagac gttcagctga aaaacatcac cgactacatg 3660 tacctgacca acgcgccgtg ctacaccaac ggtaaactga acatctacta ccgtcgtctg 3720 tacaacggtc tgaaattcat catcaaacgt tacaccccga acaacgaaat cgactgcttc 3780 gttaaatctg gtgacttcat caaactgtac gtttcttaca acaacaacga acacatcgtt 3840 ggttacccga aagacggtaa cgcgttcaac aacctggacc gtatcctgcg tgttggttac 3900 aacgcgccgg gtatcccgct gtacaaaaaa atggaagcgg ttaaactgcg tgacctgaaa 3960 acctactctg ttcagctgaa actgtacgac gacaaaaacg cgtctctggg tctggttggt 4020 acccacaacg gtcagatcgg taacgacccg aaccgtgaca tcctgatcgc gtctaactgg 4080 tacttcaacc acctgaaaga caaaatcctg ggttgcgact ggtacttcgt tccgaccgac 4140 gaaggttgga ccaacgacta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260 gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780 gcctctcccc gcgcgttggc cgattcatta atgcag 6816 <210> 16 <211> 1314 <212> PRT <213> Escherichia coli <400> 16 Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn Asp 1 5 10 15 Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile Tyr 20 25 30 Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu Arg 35 40 45 Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser Leu 50 55 60 Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr Asp 65 70 75 80 Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn Arg 85 90 95 Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile Asn 100 105 110 Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe Asp 115 120 125 Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro Ser 130 135 140 Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe Gly 145 150 155 160 Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu Arg 165 170 175 Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser Ile 180 185 190 Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn Val 195 200 205 Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe Gln 210 215 220 Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His Gly 225 230 235 240 Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys Gln 245 250 255 Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu Phe 260 265 270 Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys Asn 275 280 285 Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn Lys 290 295 300 Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp Ser 305 310 315 320 Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser Asn 325 330 335 Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn Ser 340 345 350 Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn Ile 355 360 365 Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys Ile 370 375 380 Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe Asn 385 390 395 400 Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met Arg 405 410 415 Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val Ser 420 425 430 Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg 435 440 445 Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu 450 455 460 Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu Lys 465 470 475 480 Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn 485 490 495 Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile 500 505 510 Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr 515 520 525 Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser Asn 530 535 540 Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile Tyr 545 550 555 560 Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile Thr 565 570 575 Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr 580 585 590 Ser Tyr Phe Pro Ser Val Ile Cys Lys Val Asn Gln Gly Ala Gln Gly 595 600 605 Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn 610 615 620 Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr 625 630 635 640 Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly Tyr 645 650 655 Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu Leu 660 665 670 Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu Ser 675 680 685 Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile Asp 690 695 700 Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys Leu 705 710 715 720 Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg 725 730 735 Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys 740 745 750 Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu 755 760 765 Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu Lys 770 775 780 Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser Ser 785 790 795 800 Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln Leu 805 810 815 Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile Lys 820 825 830 Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser 835 840 845 Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys 850 855 860 Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu 865 870 875 880 Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser 885 890 895 Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln 900 905 910 Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu 915 920 925 Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp 930 935 940 Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val 945 950 955 960 Ser Ala Cys His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile 965 970 975 Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val 980 985 990 Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly 995 1000 1005 Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn 1010 1015 1020 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp 1025 1030 1035 Arg Leu Cys Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly 1040 1045 1050 Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn 1055 1060 1065 Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val 1070 1075 1080 Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys 1085 1090 1095 Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu 1100 1105 1110 Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr 1115 1120 1125 Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn 1130 1135 1140 Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Cys Tyr Thr Asn 1145 1150 1155 Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys 1160 1165 1170 Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Cys Phe 1175 1180 1185 Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn 1190 1195 1200 Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn 1205 1210 1215 Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile 1220 1225 1230 Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys 1235 1240 1245 Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser 1250 1255 1260 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro 1265 1270 1275 Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu 1280 1285 1290 Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp 1295 1300 1305 Glu Gly Trp Thr Asn Asp 1310 <210> 17 <211> 6816 <212> DNA <213> Escherichia coli <400> 17 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 60 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat 120 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac 180 gatgacgata aggatcgatg gggatccgag ctcgagccga tcaccatcaa caacttccgt 240 tactctgacc cggttaacaa cgacaccatc atcatgatgg aaccgccgta ctgcaaaggt 300 ctggacatct actacaaagc gttcaaaatc accgaccgta tctggatcgt tccggaacgt 360 tacgaattcg gtaccaaacc ggaagacttc aacccgccgt cttctctgat cgaaggtgcg 420 tctgaatact acgacccgaa ctacctgcgt accgactctg acaaagaccg tttcctgcag 480 accatggtta aactgttcaa ccgtatcaaa aacaacgttg cgggtgaagc gctgctggac 540 aaaatcatca acgcgatccc gtacctgggt aactcttact ctctgctgga caaattcgac 600 accaactcta actctgtttc tttcaacctg ctggaacagg acccgtctgg tgcgaccacc 660 aaatctgcga tgctgaccaa cctgatcatc ttcggtccgg gtccggttct gaacaaaaac 720 gaagttcgtg gtatcgttct gcgtgttgac aacaaaaact acttcccgtg ccgtgacggt 780 ttcggttcta tcatgcagat ggcgttctgc ccggaatacg ttccgacctt cgacaacgtt 840 atcgaaaaca tcacctctct gaccatcggt aaatctaaat acttccagga cccggcgctg 900 ctgctgatgc acgaactgat ccacgttctg cacggtctgt acggtatgca ggtttcttct 960 cacgaaatca tcccgtctaa acaggaaatc tacatgcagc acacctaccc gatctctgcg 1020 gaagaactgt tcaccttcgg tggtcaggac gcgaacctga tctctatcga catcaaaaac 1080 gacctgtacg aaaaaaccct gaacgactac aaagcgatcg cgaacaaact gtctcaggtt 1140 acctcttgca acgacccgaa catcgacatc gactcttaca aacagatcta ccagcagaaa 1200 taccagttcg acaaagactc taacggtcag tacatcgtta acgaagacaa attccagatc 1260 ctgtacaact ctatcatgta cggtttcacc gaaatcgaac tgggtaaaaa attcaacatc 1320 aaaacccgtc tgtcttactt ctctatgaac cacgacccgg ttaaaatccc gaacctgctg 1380 gacgacacca tctacaacga caccgaaggt ttcaacatcg aatctaaaga cctgaaatct 1440 gaatacaaag gtcagaacat gcgtgttaac accaacgcgt tccgtaacgt tgacggttct 1500 ggtctggttt ctaaactgat cggtctgtgc aaaaaaatca tcccgccgac caacatccgt 1560 gaaaacctgt acaaccgtac cgcgtctctg accgacctgg gtggtgaact gtgcatcaaa 1620 atcaaaaacg aagacctgac cttcatcgcg gaaaaaaact ctttctctga agaaccgttc 1680 caggacgaaa tcgtttctta caacaccaaa aacaaaccgc tgaacttcaa ctactctctg 1740 gacaaaatca tcgttgacta caacctgcag tctaaaatca ccctgccgaa cgaccgtacc 1800 accccggtta ccaaaggtat cccgtacgcg ccggaataca aatctaacgc ggcgtctacc 1860 atcgaaatcc acaacatcga cgacaacacc atctaccagt acctgtacgc gcagaaatct 1920 ccgaccaccc tgcagcgtat caccatgacc aactctgttg acgacgcgct gatcaactct 1980 accaaaatct actcttactt cccgtctgtt atctgcaaag ttaaccaggg tgcgcagggt 2040 atcctgttcc tgcagtgggt tcgtgacatc atcgacgact tcaccaacga atcttctcag 2100 aaaaccacca tcgacaaaat ctctgacgtt tctaccatcg ttccgtacat cggtccggcg 2160 ctgaacatcg ttaaacaggg ttacgaaggt aacttcatcg gtgcgctgga aaccaccggt 2220 gttgttctgc tgctggaata catcccggaa atcaccctgc cggttatcgc ggcgctgtct 2280 atcgcggaat cttctaccca gaaagaaaaa atcatcaaaa ccatcgacaa cttcctggaa 2340 aaacgttacg aaaaatggat cgaagtttac aaactggtta aagcgaaatg gctgggtacc 2400 gttaacaccc agttccagaa acgttcttac cagatgtacc gttctctgga ataccaggtt 2460 gacgcgatca aaaaaatcat cgactacgaa tacaaaatct actctggtcc ggacaaagaa 2520 cagatcgcgg acgaaatcaa caacctgaaa aacaaactgg aagaaaaagc gaacaaagcg 2580 atgatcaaca tcaacatctt catgcgtgaa tcttctcgtt ctttcctggt taaccagatg 2640 atcaacgaag cgaaaaaaca gctgctggaa ttcgacaccc agtctaaaaa catcctgatg 2700 cagtacatca aagcgaactc taaattcatc ggtatcaccg aactgaaaaa actggaatct 2760 aaaatcaaca aagttttctc taccccgatc ccgttctctt actctaaaaa cctggactgc 2820 tgggttgaca acgaagaaga catcgacgtt atcctgaaaa aatctaccat cctgaacctg 2880 gacatcaaca acgacatcat ctctgacatc tctggtttca actcttctgt tatcacctac 2940 ccggacgcgc agctggttcc gggtatcaac ggtaaagcga tccacctggt taacaacgaa 3000 tcttctgaag ttatcgttca caaagcgatg gacatcgaat acaacgacat gttcaacaac 3060 ttcaccgttt ctttctggct gcgtgttccg aaagtttctg cgtgccacct ggaacagtac 3120 ggtaccaacg aatactctat catctcttct atgaaaaaac actctctgtc tatcggttct 3180 ggttggtctg tttctctgaa aggtaacaac ctgatctgga ccctgaaaga ctctgcgggt 3240 gaagttcgtc agatcacctt ccgtgacctg ccggacaaat tcaacgcgta cctggcgaac 3300 aaatgggttt tcatcaccat caccaacgac cgtctgtgct ctgcgaacct gtacatcaac 3360 ggtgttctga tgggttctgc ggaaatcacc ggtctgggtg cgatccgtga agacaacaac 3420 atcaccctga aactggaccg ttgcaacaac aacaaccagt acgtttctat cgacaaattc 3480 cgtatcttct gcaaagcgct gaacccgaaa gaaatcgaaa aactgtacac ctcttacctg 3540 tctatcacct tcctgcgtga cttctggggt aacccgctgc gttacgacac cgaatactac 3600 ctgatcccgg ttgcgtcttc ttctaaagac gttcagctga aaaacatcac cgactacatg 3660 tacctgacca acgcgccgtg ctacaccaac ggtaaactga acatctacta ccgtcgtctg 3720 tacaacggtc tgaaattcat catcaaacgt tacaccccga acaacgaaat cgactgcttc 3780 gttaaatctg gtgacttcat caaactgtac gtttcttaca acaacaacga acacatcgtt 3840 ggttacccga aagacggtaa cgcgttcaac aacctggacc gtatcctgcg tgttggttac 3900 aacgcgccgg gtatcccgct gtacaaaaaa atggaagcgg ttaaactgcg tgacctgaaa 3960 acctactctg ttcagctgaa actgtacgac gacaaaaacg cgtctctggg tctggttggt 4020 acccacaacg gtcagatcgg taacgacccg aaccgtgaca tcctgatcgc gtctaactgg 4080 tacttcaacc acctgaaaga caaaatcctg ggttgcgact ggtacttcgt tccgaccgac 4140 gaaggttgga ccaacgacta aaagcttgat ccggctgcta acaaagcccg aaaggaagct 4200 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 4260 gtcttgaggg gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag 4320 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4380 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4440 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4500 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4560 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4620 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4680 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4740 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4800 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4860 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4920 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4980 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5040 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5100 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5160 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 5220 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 5280 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5340 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5400 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5460 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5520 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5580 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5640 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5700 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5760 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5820 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5880 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5940 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6000 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6060 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6120 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6180 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6240 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6300 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6360 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6420 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6480 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6540 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6600 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6660 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6720 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6780 gcctctcccc gcgcgttggc cgattcatta atgcag 6816

Claims

1. A tetanus neurotoxin (TeNT), wherein the tetanus neurotoxin contains amino acid substitutions R1225E and W1288A relative to the tetanus neurotoxin of SEQ ID NO: 1, and wherein the TeNT is inactive.

2. A composition comprising: (i) a polyethylene glycolylated tetanus neurotoxin (PEG-TeNT), wherein the PEG-TeNT contains the following surface serine-to-cysteine amino acid substitutions relative to the tetanus neurotoxin (TeNT) of SEQ ID NO:1: S963C, S1041C, S1155C, and S1187C; S600C, S963C, S1041C, S1155C, and S1187C; S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C; or S81C, S120C, S144C, S248C, S335C, S428C, S600C, S963C, S1041C, S1155C, and S1187C; and wherein the substituted cysteines are conjugated to the PEG; and (ii) the TeNT according to claim 1.

3. The composition according to claim 2, wherein the molecular weight of the PEG is 2 kDa, 5 kDa, 10 kDa, or 20 kDa.

4. The composition according to claim 2, wherein the PEG is linear or branched.

5. The composition according to claim 2, wherein the PEG-TeNT comprises a polyethylene glycolylated TeNT light chain (LC), a polyethylene glycolylated TeNT heavy chain (HC), or a polyethylene glycolylated TeNT fragment c (c).

6. The composition according to claim 2, wherein the PEG-TeNT is PEG-TeNT-HC comprising a polyethylene glycolylated HC, or PEG-TeNT-LC-c comprising a polyethylene glycolylated LC and a polyethylene glycolylated c.

7. The composition according to claim 2, wherein the PEG-TeNT is PEG-TeNT-HC, and the composition further comprises PEG-TeNT-LC-c.

8. The composition according to claim 2, wherein the composition is a therapeutic composition.

9. Use of TeNT or a composition comprising TeNT in the preparation of a medicament for treating hypotonia selected from obstructive sleep apnea, apnea, and snoring, wherein the TeNT or composition comprises: (i) PEGylated tetanus neurotoxin (PEG-TeNT), wherein the PEG-TeNT comprises the following surface serine → cysteine amino acid substitutions relative to the tetanus neurotoxin (TeNT) of SEQ ID NO:1: S963C, S1041C, S1155C, and S1187C; S600C, S963C, S1041C, S1155C, and S1187C; S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C, and S1187C; or S81C, S120C, S144C, S248C, S335C, S428C, S600C, S963C, S1041C, S1155C, and S1187C; and wherein the substituted cysteines are conjugated to the PEG; and (ii) TeNT according to claim 1.

10. Use according to claim 9, wherein the medicament comprises: PEG-TeNT (PEG-TeNT-c) comprising PEGylated c; and / or PEG-TeNT (PEG-TeNT-HC) comprising PEGylated HC and PEG-TeNT (PEG-TeNT-LC-c) comprising PEGylated LC-c; and / or PEG-TeNT (PEG-TeNT-LC-HC) comprising PEGylated HC and PEGylated LC.

11. Use according to claim 9, wherein the hypotonia is obstructive sleep apnea.

12. A kit comprising TeNT according to claim 1 or a composition according to any one of claims 2 to 8.

Images