Methods for the manufacture of proteolytically processed polypeptides

Abstract

The present invention relates to a novel proteolytically active polypeptide and various uses of the polypeptide (and others) in screening and manufacturing methods.

Claims

1. A method for manufacturing a di-chain botulinum neurotoxin serotype A (BoNT/A) or derivative thereof, the method comprising contacting a Lys-C polypeptide with a single-chain BoNT/A or derivative thereof; wherein the single-chain BoNT/A or derivative thereof is proteolytically processed by the Lys-C polypeptide to produce the di-chain BoNT/A or derivative thereof.

2. The method of claim 1, wherein the single-chain BoNT/A or derivative thereof is a naturally-occurring neurotoxin, a recombinant neurotoxin, a modified neurotoxin, a neurotoxin lacking the native H.sub.C domain or parts thereof, or a BoNT/A derivative with other amino acid residues replacing the neurotoxin H.sub.C domain.

3. The method of claim 2, wherein the single-chain BoNT/A or derivative thereof comprises an amino acid sequence having at least 50% sequence identity with a polypeptide sequence selected from any one of SEQ ID NOs: 3 to 10.

4. The method of claim 1, wherein the C-terminus of the L-chain and the N-terminus of the H-chain of the di-chain BoNT/A or derivative thereof are identical to the corresponding C-terminus of the L-chain and the N-terminus of the H-chain of di-chain BoNT/A isolated from wild-type clostridia.

5. The method of claim 1, wherein the di-chain BoNT/A or derivative thereof has an identical amino acid sequence as the corresponding di-chain BoNT/A generated from the same single-chain BoNT/A polypeptide in wild-type clostridia.

6. The method of claim 1, wherein the step of contacting the Lys-C polypeptide and the single-chain BoNT/A or derivative thereof occurs within a cell, in a cell lysate, in a purified cell lysate, or in a subject.

7. The method of claim 3, wherein the Lys-C polypeptide proteolytically cleaves the single-chain BoNT/A or derivative thereof at a position immediately C-terminal to a basic amino acid residue within the sequence of any one of SEQ ID NOs: 3 to 10.

Description

(1) The figures show:

(2) FIG. 1: Activity test of fractions collected from HiPrep 16/10 Q FF.

(3) 5 ?l of fractions collected from HiPrep 16/10 Q FF run were analysed for enzymatic activity by incubating 2 ?g scBoNTA (lane 2) for 1 h at 37? C. and subsequent 10% SDS-PAGE. Lane 1: low molecular weight marker (LMW): 116 kDa, 66 kDa, 45 kDa, 35 kDa.

(4) FIG. 2: Analysis of collected fractions from SEC (HiLoad 16/60 Superdex 75) regarding content of nBH with a molecular weight of ?37.3 kDa by 12.5% SDS-PAGE.

(5) Fractions 9 to 11 contain the majority of nBH. (Lane 1: LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4 kDa)

(6) FIG. 3: 12.5% SDS-PAGE analysis for determination of purity and protein concentration of three purification batches of nBH.

(7) lane 1, LMW (116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4 kDa); lane 2, nBH Lot TE311206 (192 ng/?l maturated NT02CB1446/CBO1444, amino acids 254-594 of Genbank acc. CAL82987.1, MW: 38.6 kDa); lane 3, nBH Lot TIK301009 (130 ng/?l maturated NT02CB1447/CBO1445, SEQ ID NO: 1, amino acids 249-581 of Genbank acc. CAL82988.1, MW: 37.3 kDa); lane 4, nBH Lot TIK280509 (114 ng/?l maturated NT02CB1447/CBO1445, SEQ ID NO: 1, amino acids 249-581 of Genbank acc. CAL82988.1, MW: 37.3 kDa).

(8) FIG. 4: ESI-MS/MS spectrum analysis report.

(9) 38.6 kDa protein band of nBH lot TE311206 was identified as NT02CB1446/CBO1444 with a Mascot score of 725 and a peptide MS/MS sequence coverage of 29.6% over the entire open reading frame (ORF). No peptide (grey box, identified MS peptide; red squares, identified amino acid y-/b-ion of peptide after MS/MS decay) was identified derived from the N-terminal 253 amino acids. The MS/MS analysis of lot TE311206 displayed a sequence coverage of 52% according to the C-terminal amino acids 254-594 forming the nBH.

(10) FIG. 5: ESI-MS/MS spectrum analysis report.

(11) 37.3 kDa protein band of nBH lot TIK301009 was identified as NT02CB1447/CBO1445 with a Mascot score of 555 and a peptide MS/MS sequence coverage of 28.4% over the entire open reading frame (ORF). Except one all peptides (grey box, identified MS peptide; red squares, identified amino acid y-/b-ion of peptide after MS/MS decay) identified derive from the C-terminal 333 amino acids. The MS/MS analysis of lot TIK301009 displayed a sequence coverage of 49.5% according to the C-terminal amino acids 249-581 forming the nBH.

(12) FIG. 6: Comparison of concentration dependent proteolytic activity of nBH derived from three purification batches.

(13) A 12.5% SDS-PAGE of activity test analysing nBH derived from the batches TIK301009, TIK280509 and TE311206 using the following dilutions of the concentrated nBH: 1:10, 1:30, 1:100, 1:300, 1:1000. The assay was performed by incubating 1 ?g scBoNT/A and 2 ?l dH.sub.2O and 1 ?l of correspondingly diluted nBH for 60 min at 37? C. For SDS-PAGE analysis, 3 ?l of a reducing 4?SDS Laemmli buffer was added to a final volume of 10 ?l. 150 kDa scBoNT/A was cleaved into 100 kDa heavy chain and 50 kDa light chain.

(14) B The optical density of the protein bands of heavy chain, light chain and scBoNT/A were quantified and the sum of light and heavy chain product bands was divided by sum of LC, HC and scBoNT/A protein bands. Higher dilution of first polypeptide decrease cleavage rate. The specific proteolytic activity of the three different batches is nearly identical

(15) FIG. 7: Time dependent cleavage of scBoNT/A wild-type and mutants containing a modified loop by nBH.

(16) A Modification of the loop sequence. In scBoNTAS Throm all lysine residues are removed and the thrombin recognition sequence LVPRGS is inserted whereas in scBoNT Res the loop lacks any basic amino acids. Shortening the loop to eight small residues or five amino acids with bulky side chains yields scBoNTAS (GGSG).sub.2 and scBoNTAS FQWYI, respectively. In scBoNTAS CGS-C the entire loop is deleted and the disulfide bridge forming cysteines are replaced by glycine and serine.

(17) B SDS-PAGE analysis of time-dependent cleavage of scBoNT/A wild-type and mutants.

(18) C scBoNTAS wild-type is activated by nBH in a time dependent manner into light chain and heavy chain within 120 min. Lack of lysines and insertion of a single arginine residue prolongs the cleavage of the loop (scBoNTAS Throm). A loop lacking any basic residue is still cleavable (scBoNTAS Res). Shortening the loop to 8mer peptide, introducing five amino acids with bulky side chains or deleting the entire loop yields an uncleavable scBoNT/A.

(19) FIG. 8: MS/MS analysis of the 50 kDa and 100 kDa cleavage products upon digestion of scBoNT/A with nBH.

(20) A Analysis of the 50 kDa cleavage product which was identified as light chain of BoNT/A with a Mascot score of 1460. The most C-terminal ascribed peptide covers amino acids G433 to K438 which corresponds to the physiologically observed C-terminus of BoNT/A LC.

(21) B Analysis of the 100 kDa cleavage product which was identified as heavy chain of BoNT/A with a Mascot score of 96. The most N-terminal ascribed peptide covers amino acids A449 to K456 which corresponds to the physiologically observed N-terminus of BoNT/A HC.

(22) FIG. 9: A The protein content (mg/ml) of anti-nBH-IgY of three subsequent pools was analysed by 12.5% SDS-PAGE. B ELISA: Nunc Maxisorp F96 microtiter plates were coated with nBH of various lots (500 ng/mL) in PBS overnight at 4? C. and then blocked for 1 h with blocking buffer of PBS containing 0.1% Tween-20 and 2% nonfat skimmed milk. After washing, an IgY dilution of each pool (10 ?g/ml in blocking buffer) was added for 1 h and detected using biotin-labelled donkey anti-chicken IgY, streptavidin-horseradish peroxidase (both Dianova, Hamburg, Germany) and 3,3,5,5-tetramethylbenzidine (Sigma).

(23) FIG. 10: A Recombinant expression and isolation of inactive BH 1-581 (63 kDa) by Talon IMAC. 10% SDS-PAGE analysis of Talon IMAC fractions (LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa; SS34, clear lysate; TD, flow through; W, wash fraction; E1-E7, imidazol eluted fractions 1 to 7). B No endoproteolyis of scBoNT/A into LC (50 kDa) and HC (100 kDa) with recombinant, iBH (SEQ ID NO: 2; E; 63 kDa) is observed at 37? C. after 1 h (lane 6)(LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa).

(24) FIG. 11: Use of purified active BoNTHydrolase (nBH) for obtaining proteolytically processed polypeptide

(25) A 200 ?g of recombinant purified scBoNT/A is incubated with 350 ng purified active BoNTHydrolase for 12 min at 37? C. To stop the reaction nBH is removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume=0.3 ml, flow rate=0.25 ml/min) and the amount of cleavage is analysed by 10% SDS-PAGE. B Fraction 1 (1800 ?l) containing ?40% processed BoNT/A is incubated with 350 ng purified active BoNTHydrolase for 15 min at 37? C. and concentrated to 300 ?l by ultrafiltration. To finally stop the reaction nBH is removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume=0.3 ml, flow rate=0.25 ml/min) and the amount of cleavage is analysed by 10% SDS-PAGE. C Fractions 1 and 2 (1800 ?l) containing ?80% processed BoNT/A are combined and incubated with 120 ng purified active BoNTHydrolase for 25 min at 37? C. and concentrated to 300 ?l by ultrafiltration. To finally stop the reaction nBH is removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume=0.3 ml, flow rate=0.25 ml/min) and the amount of cleavage is analysed by 10% SDS-PAGE. A >95% processed BoNT/A (SEQ ID NO. 3) is obtained.

THE SEQUENCE LISTING SHOWS

(26) SEQ ID NO: 1: proteolytically active polypeptide derived from a Clostridium botulinum strain ATCC 3502, GenBank accession No: CAL82988.1, lacking 248 N-terminal amino acid residues

(27) SEQ ID NO: 2: proteolytically inactive polypeptide derived from a Clostridium botulinum strain ATCC 3502, GenBank accession No: CAL82988.1

(28) SEQ ID NO: 3: BoNT/A of ATCC 3502, Genbank acc. AAA23262

(29) SEQ ID NO: 4: Loop of BoNT/A1

(30) SEQ ID NO: 5: Loop of BoNT/A2/A6

(31) SEQ ID NO: 6: Loop of BoNT/A3

(32) SEQ ID NO: 7: Loop of BoNT/A3

(33) SEQ ID NO: 8: Loop of BoNT/A4

(34) SEQ ID NO: 9: Loop of BoNT/A5

(35) SEQ ID NO: 10: Loop of BoNT/A7

(36) SEQ ID NO: 11: Loop of BoNT/B1/B4bv/B6

(37) SEQ ID NO: 12: Loop of BoNT/B2/B3

(38) SEQ ID NO: 13: Loop of BoNT/B5np

(39) SEQ ID NO: 14: Loop of BoNT/C/CD

(40) SEQ ID NO: 15: Loop of BoNT/D

(41) SEQ ID NO: 16: Loop of BoNT/DC

(42) SEQ ID NO: 17: Loop of BoNT/E1-E5

(43) SEQ ID NO: 18: Loop of BoNT/E6

(44) SEQ ID NO: 19: Loop of BoNT/F1/F6

(45) SEQ ID NO: 20: Loop of BoNT/F2/F3

(46) SEQ ID NO: 21: Loop of BoNT/F4

(47) SEQ ID NO: 22: Loop of BoNT/F5

(48) SEQ ID NO: 23: Loop of BoNT/F7

(49) SEQ ID NO: 24: Loop of BoNT/G

(50) SEQ ID NO: 25: Loop of TeNT

(51) SEQ ID NO: 26: nucleic acid sequence encoding SEQ ID NO: 1

(52) SEQ ID NO: 27: nucleic acid sequence encoding SEQ ID NO: 2

(53) The following Examples illustrate the invention and shall, whatsoever, not be construed to limit its scope.

EXAMPLES

Example 1: Purification and Characterisation of the Native BoNTHydrolase (nBH), which Specifically Cleaves Single Chain BoNT/a into its Active Di-Chain Form

(54) (1) Read-Out System/Activity Test:

(55) To specifically detect and purify an enzymatic activity hydrolysing botulinum neurotoxin A (BoNT/A) into the 50 kDa light chain (LC) and 100 kDa heavy chain (HC) in culture supernatants of C. botulinum and in between chromatographic steps we expressed the 150 kDa BoNT/A as single chain (sc) polypeptide in E. coli. Incubation of the recombinant scBoNT/A with the appropriate enzymatic activity (nBH) should yield a 50 kDa LC and a 100 kDa HC visualised by reducing 10-13% SDS-PAGE.

(56) (2) Clostridial Protease Expression:

(57) A single colony of C. botulinum strain ATCC 3502 was inoculated in 100 ml brain heart infusion (BHI) media and the culture was incubated over night at 37? C. under anaerobic conditions. 10 ml of 0/N culture was inoculated into 1 l BHI media and anaerobically incubated for 48-72 h.

(58) (3) Ammonium Sulphate Precipitation:

(59) The 11 culture supernatant was harvested by centrifugation (4? C., 6500?g, 25 min). Ammonium sulphate was added to a final concentration of 85% (here 575 g), the suspension was stirred for 6 hours at 4? C. and subsequently centrifuged (4? C., 6500?g, 30 min). The pelleted ammonium sulphate precipitate was dissolved in a small volume (here 5 ml) of 50 mM NaP pH 7.5 and dialysed against 50 mM NaP, 150 mM NaCl pH 7.5. Finally, the dialysate was centrifuged (4? C., 40000?g, 60 min) and the supernatant used for the IEC.

(60) (4) Ion Exchange Chromatography (IEC, Column HiPrep 16/10 Q FF):

(61) Supernatant of (3) (FIG. 1, lane 3) was applied to a HiPrep 16/10 Q FF anion-exchange column equilibrated and run with a buffer containing 50 mM NaP pH 7.5, 150 mM NaCl. The run was performed at a flow rate of 1 ml/min. An activity test was performed by incubating 5 ?l of every other fraction with 2 ?g scBoNTA for 1 h at 37? C. and subsequent analysis on SDS-PAGE (FIG. 1). Fractions 6-24 were combined and its volume concentrated to 3.5 ml by using ultrafiltration (Amicon-Ultra MWCO 10,000).

(62) (5) Size Exclusion Chromatography (SEC, HiLoad 16/60 Superdex 200):

(63) Subsequently, the concentrated protein solution of (4) was loaded on a HiLoad 16/60 Superdex 200 column, equilibrated with 50 mM NaP pH 7.5, 150 mM NaCl. Separation was performed at a flow rate of 1 ml/min. Fractions with a retention volume between 80 ml and 100 ml were analysed using the activity test (1) and the appropriate fractions containing the enzymatic activity (nBH) were combined (?10 ml) and concentrated to 3 ml by ultrafiltration. Subsequently, ammonium sulphate was added to a final concentration of 12.5%=500 mM (+0.2 g).

(64) (6) Hydrophobic Interaction Chromatography (HIC, HiTrap Phenyl Sepharose):

(65) nBH was bound to the Phenyl Sepharose in buffer A (50 mM NaP pH 7.5, 500 mM ammonium sulphate). Bound nBH was eluted by reducing the amount of ammonium sulphate due to a linear increasing gradient with buffer B (50 mM NaP pH 7.5) at a flow rate of 1 ml/min. All protein containing fractions were analysed using the activity test (1) and the appropriate fractions were combined and concentrated by ultrafiltration to 3.5 ml. The buffer of the solution was adjusted to 50 mM NaP pH 7.5; 150 mM NaCl.

(66) (7) SEC (HiLoad 16/60 Superdex 75):

(67) Finally, the nBH was purified by SEC using the HiLoad 16/60 Superdex 75 column at 50 mM NaP pH 7.5, 150 mM NaCl and a flow rate of 1 ml/min. Fractions with a retention volume between 70 ml and 80 ml were analysed by 12.5% SDS-PAGE (FIG. 2) and the fractions 8-12 containing the nBH which migrates at ?37.3 kDa were combined (?10 ml) and concentrated to 1 ml by ultrafiltration.

(68) (8) The prominent protein migrating at approximately 37.3 kDa (nBH) was analysed by N-terminal peptide sequencing according to Edman degradation protocol. The sequence of the identified peptide is V Q G Q S V K G V G and corresponds to the first ten residues of SEQ ID NO: 1.
(9) Two lots of nBH (NT02CB1447, 37.3 kDa, FIG. 3, lane 3: TIK301009, lane 4: TIK280509) were reproducibly isolated according to the procedure as described above. Following modifications of the isolation procedure yields the nBH isoform NT02CB1446 (38.6 kDa, FIG. 3, lane 2, lot TE311206): (i) growth of C. botulinum culture: 18 h instead of 48 to 72 h; (ii) variation of chromatographic steps: IEC.fwdarw.SEC Superdex 75.fwdarw.HIC Phenyl Sepharose instead of IEC.fwdarw.SEC Superdex 200.fwdarw.HIC Phenyl Sepharose.fwdarw.SEC Superdex 75.

Example 2: Sequence Identification of nBH from C. botulinum by Mass Spectrometry (MS)

(69) (1) Tryptic Digestion:

(70) The protein bands migrating at approximately 38 kDa (nBH) in SDS-PAGE were excised for tryptic digestion and destained by gently shaking in 50 mM NH.sub.4HCO.sub.3, 50% acetonitrile for 30 min at 37? C. Destaining was repeated until gel spots were clear. Acetonitrile (100%) was added and removed after 3 min. Subsequently, spots were dried in a speed vac system (Eppendorf, Germany). Trypsin (10 ng/?l) in 50 mM NH.sub.4HCO.sub.3 was added and incubated on ice for 1 h. Then, the remaining trypsin solution was removed, a small volume 50 mM NH.sub.4HCO.sub.3 was added and digestion was carried out at 37? C. over night. The supernatant was collected and gel pieces were extracted using 5% TFA, 10% acetonitrile for two times. All fluids were combined, dried in a speed vac and extracted peptides were stored at 4? C.

(71) (2) Matrix Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF/TOF) MS:

(72) Samples were analyzed in an MALDI-TOF/TOF mass spectrometer (Ultraflex1 Bruker Daltonik GmbH) in linear mode with an acceleration voltage of 25 kV. Masses were detected from 700 m/z to 4,500 m/z. Samples (2 ?l) were cocrystallised with 2 ?l of sinnapinic acid solution containing 50% acetonitril and 0.2% trifluoric acetic acid (TFA) directly on a stainless steel MALDI target plate. 500 laser shots were collected for each sample.

(73) (3) Peptide Separation by Reversed Phase Chromatography:

(74) Peptide separation was done by reversed phase chromatography using a nano-HPLC system (Agilent Technologies, Waldbronn, Germany) that consist of an autosampler and a gradient pump. The sample was dissolved in buffer A (5% acetonitril, 0.1% formic acid) and an aliquot of up to 10 ?l was injected onto a C18 column (Zorbax SB-C18, 5 ?m, 300 A, 0.5 mm inner diameter, length 15 cm) at a flow rate of 5 ?l/min. After loading, the column was washed for 15 min with buffer A and the peptides were eluted using a gradient of eluent A and eluent B (70% (v/v) acetonitrile in 0.1% (v/v) formic acid) from 0% to 100% eluent B in 75 min.

(75) (4) Electrospray Ionisation (ESI)-Interface and Ion Trap Mass Spectrometry:

(76) The HPLC outlet was directly connected to the nanoESI source of an ion trap mass spectrometer and the Agilent coaxial sheath-liquid sprayer was used (Agilent Technologies). The outlet capillary was hold by a surrounding steel needle and looked 0.1 to 0.2 mm out of it. The spray was stabilized by N.sub.2 as nebulizer gas (5 l/min). Ionization voltage was set to 4,500 V and dry gas was applied at 5 psi and 250? C. Spectra were collected with an Esquire3000+ ion trap mass spectrometer (Bruker Daltonik) at a scan speed of 13,000 m/z per second. Using ESI in positive mode, mass spectra were acquired from 50 to 1600 m/z in scanning mode and data dependent switching between MS and MS/MS analysis. To increase the quality of MS/MS spectra only two precursor ions from one spectrum were selected for MS/MS analysis and active exclusion was set to 2 min to exclude precursor ions that had already been measured.

(77) (4) Data Processing:

(78) Data processing was performed with the Data Analysis (version 3.0) and BioTools (version 3.0) software packages (Bruker Daltonik). Protein identification was done using MASCOT software (version 2.1) and MSDB data base (Matrix Science, London, UK).

(79) (5) Results:

(80) TABLE-US-00002 TABLE 2 nBH idntified by MS Protein Genbank aa of MW Mascot lane Lot nBH concentr. Name of ORF acc. ORF [kDa] score 2 TE311206 192 ng/?l NT02CB1446 CAL82987.1 254-594 38.6 725 CBO1444 3 TIK301009 130 ng/?l NT02CB1447 CAL82988.1 249-581 37.3 555 CBO1445 4 TIK280509 114 ng/?l NT02CB1447 CAL82988.1 249-581 37.3 609 CBO1445

(81) The 38.6 kDa protein band of lane 2 (nBH lot TE311206) was identified as NT02CB1446/CBO1444 with a Mascot score of 725 and a peptide MS/MS sequence coverage of 29.6% over the entire open reading frame (ORF). No peptide was identified derived from the N-terminal 253 amino acids (FIG. 4). The MS/MS analysis of lot TE311206 displayed a sequence coverage of 52% according to the C-terminal amino acids 254-594 forming the nBH.

(82) The 37.3 kDa protein bands of lane 3 (nBH lot TIK301009) and lane 4 (nBH lot TIK280509) were identified as NT02CB1447/CBO1445 with a Mascot score of 555 and 609, respectively. Except one all peptides identified derive from the C-terminal 333 amino acids (FIG. 5). The MS/MS analysis of lot TIK301009 displayed a sequence coverage of 49.5% according to the C-terminal amino acids 249-581 forming the nBH.

Example 3: Characterisation of Enzymatic Specificity of nBH

(83) (1) The concentration dependent proteolytic activity of nBH derived from three purification batches was compared (FIG. 6). An activity test analysing nBH derived from the batches TIK301009, TIK280509 and TE311206 using various dilutions of nBH demonstrates that higher dilutions decrease the cleavage rate. The proteolytic activity of the three different batches is nearly identical indicating that the maturated isoform NT02CB1446 (TE311206) displays a similar specific activity as the maturated NT02CB1447 (SEQ ID NO: 1).
(2) The time-dependent cleavage of scBoNT/A wild-type and mutants by nBH was analysed employing the activity test (FIG. 7). scBoNTAS wild-type is activated by nBH in a time dependent manner into light chain and heavy chain within 120 min by more than 95%. The loop sequence was modified to characterise the cleavage site. In scBoNTAS Throm all lysine residues are removed and the thrombin recognition sequence LVPRGS is inserted which prolonged the cleavage rate. In scBoNT Res the loop lacks any basic amino acids which drastically delays the complete hydrolysis indicating a strong recognition preference of nBH for basic residues like lysine and arginine at the cleavage site. Furthermore, accessibility of nBH to the loop by is impaired by shortening the loop to eight small residues or five amino acids with bulky side chains (scBoNTAS (GGSG).sub.2 and scBoNTAS FQWYI).
(3) The MS/MS analysis of the 50 kDa cleavage product upon digestion of scBoNT/A with nBH exhibited that the most C-terminal ascribed peptide covers amino acids G433 to K438 which corresponds to the physiologically observed C-terminus of BoNT/A LC (FIG. 8A). Analysis of the 100 kDa cleavage product which was identified as heavy chain of BoNT/A demonstrated that the most N-terminal ascribed peptide covers amino acids A449 to K456 which corresponds to the physiologically observed N-terminus of BoNT/A HC (FIG. 8B). Thus, the isolated nBH yields physiologically processed BoNT/A and preferentially hydrolyses peptide bonds C-terminal to lysine and arginine residues.

Example 4: Evolutionary Conservation of BoNTHydrolase and its Isoforms

(84) Protein sequence analysis of SEQ ID NO: 2 (Genbank acc. CAL82988.1/YP_001253958.1) revealed three conserved domains. Residues 18-573 correspond to a Zinc metalloprotease (elastase) or LasB involved in amino acid transport and metabolism with a Blast score of 738. Residues 148-212 correspond to a peptidase propeptide and YPEB domain or PepSY (Blast score 97. Residues 336-573 are part of the peptidase M4 family including thermolysin, protealysin, aureolysin and neutral proteases (Blast score 803).

(85) The genome sequencing of C. botulinum ATCC 3502 has revealed the existence of six ORFs encoding iBH isoforms (Sebaihia et al., 2007, Genome Res. 17(7):1082-1092). Further genome data is available for 10 group I C. botulinum strains as well as the non-BoNT secreting C. sporogenes which all contain between five to seven ORFs encoding iBH. The nBH (SEQ ID NO: 1) shares minimum 64% amino acid sequence identity with the other 63 isoforms.

Example 5: Generation of Antibodies Specific for the BoNTHydrolase

(86) (1) Generation of IgY:

(87) Sixteen-week-old chickens [ISA Brown and Lohmann Selected Leghorn (LSL), Spreenhagener Vermehrungsbetrieb f?r Legehennen GmbH, Bestensee, Germany] were kept in individual cages, exclusively constructed for the maintenance of chickens (Ebeco, Castrop-Rauxel, Germany). Food (ssniff Legeh?hner-Zucht 1 and 2; ssniff Spezialit?ten GmbH, Soest, Germany) and water were available ad libitum, and the chickens started laying eggs between 23 and 25 weeks of age. Eggs were collected daily, labelled, and stored at 4? C. until they were further processed. All animal maintenance and experiments were performed in accordance with the guidelines of local authorities, Berlin (No. H0069/03). Chickens were immunized and boosted via the i.m. route (pectoralis muscle, left and right side) a total of 10 times over a 1-year period, with intervals between 4 and 8 weeks. The interval used was based on previous work that showed no demonstrable memory cells until at least 3 weeks postimmunisation (Pei and Collisson, 2005). The antigen concentration used was approximately 20 ?g per injection (nBH). No more than 500 ?l of antigen solution was injected per immunization. Freund's complete adjuvant was used for the first immunisation, and FIA was used for the subsequent booster injections. The method for IgY purification was adapted from Polson et al. (1980). Briefly, the egg yolk was diluted 1:2 with sterile PBS (pH 7.4, Roche, Mannheim, Germany). For elimination of lipids and lipoprotein, 3.5% (w/v) polyethylene glycol (PEG) 6000 (Roth, Karlsruhe, Germany) was added. After gentle shaking followed by centrifugation (10,000?g for 20 min at 4? C.), the supernatant was decanted and solid PEG 6000 was added to a final concentration of 12% (w/v). This mixture was then centrifuged as above. The precipitate was dissolved in 10 ml of PBS, PEG was added to 12% (wt/vol), and the solution was centrifuged. Finally, the precipitate was dissolved in 1.2 ml of PBS, transferred into a microdialysis device (QuixSep, Roth, Germany) and dialysed against PBS at 4? C. The protein content (mg/ml) was analysed by 12.5% SDS-PAGE (FIG. 9A) and measured photometrically at 280 nm and was calculated according to the Lambert-Beer law with an extinction coefficient of 1.33 for IgY.

(88) (2) ELISA:

(89) Nunc Maxisorp F96 microtiter plates (VWR International GmbH, Darmstadt, Germany) were coated with nBH of various lots (500 ng/mL) in PBS overnight at 4? C. and then blocked for 1 h with blocking buffer of PBS containing 0.1% Tween-20 and 2% nonfat skimmed milk (Merck, Darmstadt, Germany). After washing, a IgY dilution (10 ?g/ml in blocking buffer) was added for 1 h and detected using biotin-labeled donkey antichicken IgY, streptavidin-horseradish peroxidase (both Dianova, Hamburg, Germany) and 3,3,5,5-tetramethylbenzidine (Sigma). Detected nBH is illustrated in FIG. 9B.

(90) (3) Western Blot:

(91) nBH was separated per 12.5% SDS-PAGE, and were transferred onto a polyvinylidene fluoride membrane (Invitrogen GmbH, Karlsruhe, Germany) using standard immunoblotting techniques. The membrane was blocked overnight at 4? C., and incubated with IgY (1:5,000 in blocking buffer) for 1 h. After washing, the membrane was probed with biotin-labelled donkey anti-chicken IgY for 30 min and was developed using alkaline phosphatase and CDP-Star (Perkin Elmer, Waltham, Mass.).

Example 6: Recombinant Expression of BoNTHydrolase

(92) (1) Plasmid Constructions:

(93) The gene portions encoding native BH (SEQ ID NO: 1) and its propeptide (SEQ ID NO: 2) were amplified by PCR using suitable oligonucleotides and genomic DNA of C. botulinum ATCC 3502, fused to an oligonucleotide coding for the His6Tag and inserted into pQE3 (Qiagen) yielding the expression plasmid pQ-BH1445H6-249-581 and pQ-BH1445H6-1-581, respectively. Nucleotide sequences were verified by DNA sequencing.

(94) (2) Purification of Recombinant Proteins:

(95) nBH and iBH, fused to a carboxyl-terminal His6Tag, were produced utilizing the E. coli strain M15pREP4 (Qiagen) during ten hours of incubation at room temperature, and were purified on Talon-sepharose beads (Clontech Inc.) following to the manufacturer's instructions. Fractions containing the desired proteins were pooled, frozen in liquid nitrogen, and kept at ?70? C. iBH was isolated as recombinant protein with a MW of 63 kDa (FIG. 10A). The inactivity of iBH was demonstrated using the activity test: after 1 h at 37? C. no scBoNT/A wt was hydrolysed in LC and HC (FIG. 10B).

Example 7: Inhibition of BoNTHydrolase

(96) (1) Screening for peptide inhibitors of BH: Peptides based on SEQ ID NOs: 4 to 25 will be synthesised lacking one or more basic residues. Each peptide will be added to the mixture according to the activity test. A peptide being able to decrease the amount of processed scBoNT/A, prolong the duration required for fully processing scBoNT/A or block processing scBoNT/A is considered to be an inhibitor of nBH.
(2) Screening for antibody-based inhibitors: Antibodies generated against epitopes derived from nBH like IgY of Example 5 are incubated with nBH and subsequently subjected to the activity test. An antibody being able to decrease the amount of processed scBoNT/A, prolong the duration required for fully processing scBoNT/A or block processing scBoNT/A is considered to be an inhibitor of nBH.

Example 8: Use of Purified Active BoNTHydrolase (nBH) for Obtaining Proteolytically Processed Polypeptide

(97) (1) 200 ?g of recombinant purified scBoNT/A is incubated with 350 ng purified active BoNTHydrolase for 12 min at 37? C. To stop the reaction nBH is removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume=0.3 ml, flow rate=0.25 ml/min) and the amount of cleavage is analysed by 10% SDS-PAGE (FIG. 11A).
(2) Fraction 1 (1800 ?l) containing ?40% processed BoNT/A is incubated with 350 ng purified active BoNTHydrolase for 15 min at 37? C. and concentrated to 300 ?l by ultrafiltration. To finally stop the reaction nBH is removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume=0.3 ml, flow rate=0.25 ml/min) and the amount of cleavage is analysed by 10% SDS-PAGE (FIG. 11B).
(2) Fractions 1 and 2 (1800 ?l) containing ?80% processed BoNT/A are combined and incubated with 120 ng purified active BoNTHydrolase for 25 min at 37? C. and concentrated to 300 ?l by ultrafiltration. To finally stop the reaction nBH is removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume=0.3 ml, flow rate=0.25 ml/min) and the amount of cleavage is analysed by 10% SDS-PAGE (FIG. 11C). A >95% processed BoNT/A (Seq ID NO. 3) is obtained. The identical fully processed second polypeptide (>95% processed BoNT/A) is obtained if the second polypeptide is processed in one step for 50 min at 37? C. (200 ?g scBoNT/A incubated with 350 ng nBH). After an incubation time of 1 h at 37? C., more than 97% of BoNT/A is processed.