BIOLOGICAL SYNTHESIS OF AMINO ACID CHAINS FOR PREPARATION OF PEPTIDES AND PROTEINS

Abstract

The invention relates to fusion polypeptides, nucleic acid molecules encoding said fusion polypeptides and genetically modified cells including said nucleic acid molecules. Moreover, the invention relates to a method for preparing target polypeptides using the fusion polypeptides.

Claims

1-18. (canceled)

19. A fusion polypeptide comprising in direction from the N-terminus to the C-terminus: (i) a purification domain, (ii) an autoprotease domain, and (iii) a target peptide domain, wherein the purification domain (i) binds to a carbohydrate.

20. The fusion polypeptide according to claim 19, wherein the purification domain (i) binds to an oligo- or polysaccharide, in particular to cellulose, chitin and/or starch.

21. The fusion polypeptide according to claim 19, wherein the purification domain (i) binds to starch and comprises a glucoamylase and/or an amylase, and/or a starch-binding domain, a surface-binding domain and/or a carbohydrate-binding domain thereof.

22. The fusion polypeptide according to claim 19, wherein the autoprotease domain (ii) comprises a viral autoprotease, preferably an autoprotease derived from a virus of the family Flaviviridae, more preferably an autoprotease derived from a pestivirus, and even more preferably an N.sup.pro autoprotease or an active fragment or an active mutant of such autoprotease.

23. The fusion polypeptide according to claim 19, wherein the autoprotease domain (ii) comprises an N.sup.pro autoprotease derived from CSFV or a mutant of an N.sup.pro autoprotease, in particular the CSFV N.sup.pro mutant EDDIE.

24. The fusion polypeptide according to claim 19, wherein the autoprotease domain (ii) cleaves the fusion polypeptide after the C-terminus of the autoprotease and before the N-terminus of the target peptide (iii).

25. The fusion polypeptide according to claim 19, wherein the target peptide (iii) has a chain length of (a) 2-1000 amino acids, (b) 100-500 amino acids, or (c) more than 500 amino acids.

26. The fusion polypeptide according to claim 19, wherein the target peptide (iii) has an amount of a. hydrophobic amino acids of 0%, preferably 20%, more preferably 30%, even more preferably 40%, b. hydrophilic amino acids of 0%, preferably 20%, more preferably 30%, even more preferably 40%, and/or c. a combination of (a) and (b).

27. A recombinant nucleic acid molecule encoding a fusion polypeptide according to claim 19, optionally operatively linked to an expression control sequence.

28. The nucleic acid molecule according to claim 27, arranged on a vector, in particular a plasmid.

29. A genetically modified cell, including a nucleic acid molecule according to claim 27, the cell being a prokaryotic or eukaryotic cell, preferably a prokaryotic cell, more preferably an E. coli cell.

30. The genetically modified cell according to claim 29 expressing a fusion polypeptide comprising in direction from the N-terminus to the C-terminus: (i) a purification domain, (ii) an autoprotease domain, and (iii) a target peptide domain, wherein the purification domain (i) binds to a carbohydrate.

31. A method for preparing a target peptide comprising the steps of: a. providing a genetically modified cell expressing a fusion polypeptide according to claim 19, b. culturing the cell in a suitable culture medium and under conditions suitable for expression of the fusion polypeptide and for formation of inclusion bodies comprising the fusion polypeptide, c. solubilizing the inclusion bodies comprising the fusion polypeptide, d. contacting the solubilized fusion polypeptide with a carbohydrate-based matrix having affinity to the purification domain (i) under conditions wherein the fusion polypeptide binds to the matrix, e. cleaving the fusion polypeptide by the autoprotease domain (ii) and releasing the target peptide (iii), and f. collecting the target peptide (iii).

32. The method according to claim 31, wherein the contacting (d) comprises chromatography over said matrix.

33. The method according to claim 31, wherein the cleaving (e) occurs due to an addition of autoproteolysis buffer, and/or wherein the collecting (f) comprises separating and/or isolating the target peptide from the matrix.

34. The method according to claim 31, wherein the target peptide (iii) collected in step (f) has an authentic N-terminus or an additional cysteine residue at the N-terminus.

35. A recombinant nucleic acid molecule, encoding a fusion polypeptide comprising the domains (i) and (ii) according to claim 19 and a cloning site for incorporation of a nucleic acid molecule comprising the domain (iii) according to claim 1, optionally operatively linked to an expression control sequence.

36. The nucleic acid molecule according to claim 35 arranged on a vector, in particular a plasmid.

Description

EXAMPLE 1: CONSTRUCTION OF FUSION POLYPEPTIDE ENCODING GENE SEQUENCES

[0075] Gene sequences encoding a fusion polypeptide having three sections were prepared. The N-terminal section consists of a purification domain (i), the middle section consists of an N.sup.pro autoprotease domain (ii), and the C-terminal section consists of the target peptide domain (iii). The domains (i) and (ii) are optionally interconnected with a linker (SEQ ID NO: 3/SEQ ID NO: 4).

[0076] Human -amylase (AMY1c; SEQ ID NO: 5/SEQ ID N.: 6), glucoamylase 1 (GA1) derived from Aspergillus niger (SEQ ID NO: 7/SEQ ID NO: 8), as well as the carbohydrate-binding units CBM20 (SEQ ID NO: 9/SEQ ID NO: 10) and CBM26 were used as purification domain.

[0077] N.sup.pro (CSFV Alfort 187, SEQ ID NO: 11/SEQ ID NO: 12), as well as the N.sup.pro mutant EDDIE (SEQ ID NO: 13/SEQ ID NO: 14) according to WO 2006/113957 were used as autoprotease domain.

[0078] A methionine-35 oxidized form of amyloid- (1-42), a heptameric valine peptide (Val.sub.7), a hydrophobic Ile.sub.13Thr.sub.8 peptide and the known Green Fluorescence Protein (GFP) were used as target peptides.

EXAMPLE 2: PREPARATION OF TARGET PEPTIDES

[0079] The gene sequences described in Example 1 were expressed in genetically modified host cells.

[0080] In a first step, a vector (e.g. the vector pet28a(+)) containing the respective gene sequence was introduced in a host cell, e.g. E. coli, BL21 DE 3. The gene sequence is arranged on this vector under control of the isopropyl--D-1-thiogalactopyranoside (IPTG) inducible lac promoter. The cells containing the vector were selected, e.g. by plating on kanamycin containing agarose plates. Colonies on this plate were used for the expression.

[0081] The bacterial cells were cultured under standard conditions in a suitable culture medium, e.g. LB medium, until an optical density OD.sub.600 of 0.6 was reached. For this purpose, inducing the gene expression took place at 37 C. for a period of 12 h by addition of IPTG (1 mM final concentration).

[0082] Following expression the cells were harvested by centrifugation, mixed with a lysis buffer (e.g. 2 mM MgCl.sub.2, 5 mM EDTA, 75 mM NaOAc, 20 mM HEPES pH 7.3) and disrupted by sonification. The fusion polypeptide was produced during the expression phase in the form of inclusion bodies (IBs) inside the cells and thus, was present in an insoluble and crystalline form within the cells. Then, the IBs were solubilized in a solubilizing buffer (e.g. 8 M urea, 6 M guanidinium HCl, 20 mM HEPES, 50 mM dithiothreitol pH 7.3), preferably under reducing conditions to be further processed. 10-30 ml of buffer were used for the cell mass derived from 1 l of culture.

[0083] In the denaturation buffer the autoprotease domain of the fusion polypeptide is inactive. For conversion into the native conformation and thus, for purification and activation of the autoprotease the solution of the solubilized IBs was added to a suspension of an autoproteolysis buffer (e.g. 0.5 M arginine, 100 mM HEPES, 10 mM sucrose, 5 mM EDTA pH 7.3) and starch, e.g. corn starch. Other sources of starch are likewise suitable. In doing so, the fusion protein was bound to the starch by its purification domain (amylase, glucoamylase or starch-binding domain).

[0084] Then, the fusion protein was incubated in the suspension of autoproteolysis buffer and starch for 10 min at 37 C. under constant agitation. Subsequently, the suspension was centrifuged and the supernatant was decanted. The centrifugate was resuspended in water for two or more times and re-centrifuged. The respective supernatants were discarded. By this means, possible impurities were removed. As a next step, the centrifugate was resuspended in autoproteolysis buffer and was stored at 8 C. for 60 min. After resuspension and subsequent centrifugation the supernatant was precipitated in alcohol and again centrifuged. By this means, a target peptide was obtained which may be lyophilized and thus be made storable.

[0085] In case the target peptide is a water-insoluble peptide or protein (e.g. amyloid--peptide), the starch was re-extracted in a suitable solvent (such as hexafluoroisopropanol, HFIP) prior to precipitation in alcohol, centrifuged and subsequently precipitated.

EXAMPLE 3: CHARACTERIZATION OF THE TARGET PEPTIDES

[0086] The identity of the target peptides obtained in Example 2 was verified by spectroscopic and spectrometric methods.

[0087] FIG. 1 depicts a MALDI-TOF spectrum of amyloid- (1-42) oxidized at the methionine residue 35 (+16 Da). The sample was dissolved in acetonitrile/water (1:1, 0.1% trifluoroacetic acid (TFA)) and co-crystallized with 2,5-dihydroxybenzoic acid (DHB) as a matrix (10 mg/ml) in a ratio of 1:50. The measurement was performed at 100 Hz by 1000 laser pulses.

[0088] FIG. 2 depicts a MALDI-TOF spectrum of Ile.sub.13Thr.sub.8. The sample was dissolved in acetonitrile/water 1:1, 0.1% TFA and co-crystallized with DHB as a matrix (10 mg/ml) in a ratio of 1:50. The measurement was performed at 100 Hz by 1000 laser pulses. Two signals were detected corresponding to Ile.sub.13Thr.sub.8 (M/Z=2) and Ile.sub.13Thr.sub.8+Na (M/Z=2).

[0089] FIG. 3 depicts a fluorescence emission spectrum of GFP at an excitation wavelength of 485 nm and a detected emission wavelength of 510 nm.

EXAMPLE 4: PREPARATION OF TARGET PEPTIDES

[0090] Gene sequences according to Example 1 with the target peptide being Ile.sub.13Thr.sub.8, Val.sub.7, melittin or GFP were introduced in host cells and fusion polypeptides were expressed according to Example 2.

[0091] Following expression the cells were harvested by centrifugation, resuspended in lysis buffer (e.g. 2 mM MgCl.sub.2, 5 mM EDTA, 75 mM NaOAc, 20 mM HEPES pH 7.5) in a ratio of e.g. 1:10 (w/v) and disrupted by sonification. During the expression phase the fusion polypeptides were produced in the form of inclusion bodies (IBs) within the cells. The IBs were solubilized in a solubilizing buffer (e.g. 8 M urea, 6 M guanidinium HCl, 20 mM HEPES, 50 mM dithiothreitol pH 7.5), preferably under reducing conditions to be further processed, for e.g. 40 min at room temperature. 10-30 ml of buffer were used for the cell mass derived from 1 l of culture.

[0092] In the solubilizing buffer the autoprotease domain of the fusion polypeptide is inactive. For conversion into the native conformation and thus, for purification and activation of the autoprotease the solution of the solubilized IBs was added to a suspension of an autoproteolysis buffer (e.g. 5 M arginine, 1.7 M HEPES, 1.6 mM sucrose pH 7.5) and starch, e.g. corn starch. Other sources of starch are likewise suitable. In doing so, the fusion protein was bound to the starch by its purification domain (amylase, glucoamylase or starch-binding domain).

[0093] Then, the fusion protein was incubated in the suspension of autoproteolysis buffer and starch for 10 min at 37 C. under constant agitation. Subsequently, the suspension was centrifuged and the supernatant was decanted. The centrifugate was resuspended in water for two or more times and re-centrifuged. The respective supernatants were discarded. By this means, possible impurities were removed. As a next step, the centrifugate was resuspended in autoproteolysis buffer (1.2 ml buffer per 100 mg of centrifugate) and stored at 37 C. for 30 min under constant agitation. After subsequent centrifugation the supernatant was precipitated in alcohol, preferably ethanol, and again centrifuged. By this means, a target peptide was obtained which may be lyophilized and thus be made storable.

[0094] In case the target peptide is a water-insoluble peptide or protein (e.g. amyloid--peptide), the starch was re-extracted in a suitable solvent (such as HFIP) prior to precipitation in alcohol, centrifuged and subsequently precipitated.

EXAMPLE 5: CHARACTERIZATION OF THE TARGET PEPTIDES

[0095] The identity of the target peptides obtained in Example 4 was verified by spectroscopic and spectrometric methods.

[0096] FIG. 4 depicts a MALDI-TOF spectrum of Ile.sub.13Thr.sub.8. The sample was dissolved in acetonitrile/water 1:1, 0.1% TFA (100 g/ml) and co-crystallized with DHB as a matrix. The measurement was performed at positive reflector mode. Two signals were detected: m/z=1148 (avg) corresponding to Ile.sub.13Thr.sub.8 (M+2, H); and m/z=1160 corresponding to Ile.sub.13Thr.sub.8+(M+2, Na).

[0097] FIG. 5 depicts a MALDI-TOF spectrum of Val.sub.e. The sample was dissolved in acetonitrile/water 1:1, 0.1% TFA (100 g/ml) and co-crystallized with DHB as a matrix. The measurement was performed at positive reflector mode. One signal was detected: m/z=712 (avg) corresponding to Val.sub.7 (M+1, H).

[0098] FIG. 6 depicts a MALDI-TOF spectrum of melittin. The sample was dissolved in acetonitrile/water 1:1, 0.1% TFA (100 g/ml) and co-crystallized with DHB as a matrix. The measurement was performed at positive reflector mode. A single signal was detected: m/z=2843 (avg) corresponding to melittin (M+1, H).

[0099] FIG. 7 depicts an UV spectra of melittin at 286 nm (tryptophan) after HPLC purification (flow rate 2 ml/min, linear gradient 5-80% buffer B over 20 min; buffer A: water, 0.1% TFA; buffer B: acetonitrile/water 80:20+0.1% TFA; sample concentration 1 mg/ml)

[0100] FIG. 8 depicts a fluorescence emission spectrum of GFP (10 mg/ml) at an excitation wavelength of 395 nm. A single emission band was detectable at a wavelength of 509 nm.

[0101] FIG. 9 depicts a MALDI-TOF spectrum of amyloid- (1-42). The sample was dissolved in acetonitrile/water 1:1, 0.1% TFA (100 g/ml) and co-crystallized with DHB as a matrix. The measurement was performed at positive reflector mode. A single signal was detected: m/z=4512 (avg) corresponding to amyloid- (1-42) (M+1, H).