CA2232841A1 - Process for producing natriuretic peptides via streptavidine fusion proteins - Google Patents

Abstract

The disclosure pertains to a process for the recombinant production of natriuretic peptides by expression in prokaryotic or eukaryotic cells of a DNA
that codes for a fusion protein from streptavidine and the peptide in question. The process is especially suitable for the production of urodilatin and its fragments.

Description

Process for the production of natriuretic peptides via streptavidin fu ion proteins The invention concerns a process for the recombinant production of natriuretic peptides (NP peptides) by expression of streptavidin fusion proteins and subsequent cleavage of the fusion proteins with a suitable restriction endoprotease.

Natriuretic peptides (NP peptides) are peptides with a natriuretic activity which are formed from a precursor polypeptide (prohormone) in the ventricle of the heart, the adrenal gland and the brain and contain a ring of 17 amino acids as a structural element which is formed by a disulfide bridge between two cysteine residues.
Precursor polypeptides are for example the "atrial"
natriuretic peptide (ANP 1 - 126) or cardiodilatin (CCD
1 - 126) and the "brain" natriuretic peptides of the B
and C type.

Urodilatin (CCD 95 - 126) is a natriuretic peptide which can be isolated from human urine (Forssmann, K. et al., Clin. Wochensch. 66 (1988) 752 - 759 (20). The peptide has a length of 32 amino acids, forms a ring of 17 amino acids by the formation of a disulfide bridge between two cysteine residues and is a member of the cardiodilatin/
"atrial" natriuretic peptide (CDD/ANP) family. Like a-ANP (99 - 126) it is formed from the ANP propeptide (ANP
1 - 126). Urodilatin (CCD 95 - 126) is presumably formed in vivo by cleavage of this propeptide between the amino acids 94 and 95. The ca. 3.5 kDa urodilatin peptide differs from the a-ANP (99 - 126) peptide by a 4 amino acid extension at the N-terminus. The amino acid sequence and the structure of urodilatin are described for example in Drummer, C. et al., Pflugers Archiv, European J. of Physiol. 423 (1993) 372 - 377 (21).
Urodilatin binds to the membranous ANP receptors A and B
and activates an intracellular guanylate cyclase coupled to the receptor. This causes the formation of the second messenger cGMP which mediates the diuretic and natriuretic effects in the kidney and the relaxing effect on the smooth vascular muscles (Heim, J.M., Biochem. Biophys. Res. Commun. 163 (1989) 37 - 41 (22J).
Consequently urodilatin is a preferred therapeutic for the prophylaxis and therapy of acute renal failure e.g.
in patients after heart or liver transplantations (Bub, A. et al., Histochem. J. (Suppl.) 24 (1992) 517 (24);
Drummer, C. et al., J. Am. Soc. Nephrol. 1 (1991) 1109 -1113 (25) and Am. J. Physiol. 262 (1992) F744 - 754 (26); Emmeluth, C. et al., Am. J. Physiol. 262 (1992) F513-F516 (27); Goetz, K.L. et al., J. Am. Soc. Nephrol.
1 (1990) 867 - 874 (28)).

The synthesis of C-terminal fragments of ANP (1 - 126) such as of the propeptide of ~-ANP (99 - 126) or urodilatin is usually carried out by chemical peptide synthesis (Kent, S.B.H. et al., Banburi Rep. 29 (1988) 3 - 20 (1); Hodson, J.H., Bio/Technology 11 (1993) 1309 -1310 (2J).

The disadvantages of chemical peptide synthesis are in particular the fact that undesired modifications (false sequences, non-cleaved protective groups) are frequently formed in the synthesis. Further problems are racemization during fragment coupling, difficulties in cleaving the protective groups and finally the complicated purification.

Various methods can be used for the recombinant production of peptides (Kopetzki, E. et al. (1994) (3J;
Winnacker, E.-L. (1987) (4); Harris, T.J.R. (1983) (5J).
For example a direct expression in the cytoplasm of microorganisms or cell lines can take place. However, a minimum polypeptide length of ca. 80 - 100 amino acids is required for this. Smaller peptides are not stable and are degraded by proteolysis. Moreover these proteins usually contain an additional N-terminal methionine and the yields are very low.

The production of such peptides can be improved by the expression of soluble fusion proteins with a selective cleavage sequence and subsequent release of the desired peptide by chemical or enzymatic cleavage (Sharma, A. et al., Proc. Natl. Acad. Sci. USA 91 (1994) 9337 - 9341 (29); Gram, H., Bio/Technology 12 (1994) 1017 - 1023 (30)). However, a particular disadvantage of soluble fusion proteins is that they can already be degraded by proteolysis in the cell or during the secretion and processing mainly in the non-structured peptide region.

The production of streptavidin fusion proteins is described by Sano, T. et al., Biochem. Biophys. Res.
Commun. 176 (1991) 571 - 577 (9J and Sano, T. et al., Proc. Natl. Acad. Sci., USA 89 (1992) 1534 - 1538 (10).
The chimeric protein comprises the amino acids 16 - 133 of streptavidin as the streptavidin moiety, a polylinker and the sequence of the target protein. The target proteins described by Sano are the mouse metallothionein I protein and the T7 gene 10 protein. However, these chimeric proteins contained no cleavage site by which the target protein can be cleaved again from the streptavidin moiety.

The object of the present invention is to provide a process by which NP peptides, preferably C-terminal ANP
(1 - 126) peptide fragments (amino acids (AA) 1 - 126) such as the fragments AA 95 - 126 (urodilatin), AA 99 -126 (a-ANP) or AA 102 - 126, can be produced in a high yield and purity.

The object is achieved according to the invention by a process for the recombinant production of an NP peptide by expression of a DNA in prokaryotes which codes for a fusion protein comprising streptavidin which is linked C-terminally to the N-terminus of the said NP peptide via a peptide sequence (also denoted linker in the following) which contains at least one lysine at the C-terminus and can be cleaved by endoproteinase LysC, isolation of the insoluble inactive protein, solubilization of the inactive protein, cleavage of the fusion protein with endoproteinase LysC and isolation of the desired NP peptide.

Endoproteinase LysC surprisingly completely cleaves the fusion proteins according to the invention although it is known that endoproteinase LysC usually only cleaves fusion proteins very ineffectively (Allen, G. et al., J.
Cell. Sci. Suppl. 3 (1985) 29 - 38 (40)). Furthermore endoproteinase LysC cleaves the fusion protein essentially only at the lysine of the linker. This is particularly surprising since it would have been expected that endoproteinase LysC also cleaves at the 4 lysine residues of the streptavidin moiety of the fusion protein. Since the cleavage is additionally rapid and proceeds almost completely, the combination of a streptavidin fusion protein and cleavage with endoproteinase LysC represents a particularly suitable system for the recombinant production of urodilatin.

Endoproteinase LysC is an endoproteinase which specifically cleaves proteins and peptides at the C-terminal end of lysine. Such an enzyme is for example known from fungi or bacteria (DE 30 34 045 C2).
Endoproteinase LysC from bacteria is a protein with a molecular weight of 35 - 38 kDa. The pH optimum is at 7.7 and the enzyme is inhibited by aprotinin. The specific activity measured with tosyl-glycyl-prolyl-lysyl-p-nitroaniline at 25~C is ca. 25 U/mg or ca. 50 Azocoll~ units/mg enzyme at 37~C. The enzyme can for example be isolated and purified from the culture broth of lysobacteraceae. Endoproteinase LysC (EC 3.4.21.50) from lysobacter enzymogenes is available from Boehringer ~nn~eim GmbH, Germany, order No. 476986. Endoproteinase LysC is used to cleave fusion proteins which contain no lysine residues (Ladisch, M.R. (editor) Protein Purification ACS-Symposium, series 427, American Chemical Society, Washington D.C. 1990, 189 (31); Allen, G. et al., J. Cell. Sci. Suppl 3 (1985) 29 (32)).

A linker in the sense of the present invention is understood as a short-chain peptide sequence which is preferably composed of 5 - 15 amino acids and contains at least one Lys as a cleavage site for endoproteinase LysC. This linker preferably contains a combination of several amino acids selected from amino acids Gly, Thr, Ser, Ala, Pro, Asp, Glu, Arg and Lys. A linker is particularly preferably used in which 2 - 8 of these amino acids are the negatively charged amino acids Asp and/or Glu. It is expedient that the linker ends C-terminally with Lys.

The specifications "5 - 15 amino acids" and "2 - 8 of these ami.no acids" mean that in a linker that is composed of 5 amino acids, at least one amino acid is Lys and in the preferred embodiment 2 - 3 of the amino acids are Asp andlor Glu. In a linker that is composed of 9 amino acids, 2 - 8 of the amino acids can be Asp and/or Glu in the preferred embodiment. The 9th amino acid is Lys.

Nucleic acids (preferably DNA) coding for the fusion protein can be produced by known processes as described in Sambrook, J. et al. (1989) (6).

Streptavidin as described in EP-B 0 198 015 (7) and EP-A 0 612 325 (8J can for example be used as streptavidin. Further streptavidin derivatives or streptavidin fragments as described for example by Sano, T. et al., (9J are also suitable. A streptavidin is preferably used as streptavidin which is truncated (shortened) at the N-terminus and/or C-terminus. This prevents aggregation and proteolysis (Sano, T. et al., (9)). A streptavidin is preferably used which begins with the amino acids 10 - 20 and ends with the amino acids 130 - 140 (numbering analogous to Argarana C.E. et al., Nucl. Acids. Res. 14 (1986) 1871 - 1882 (23)). A
streptavidin composed of the amino acids 16 - 133 or 13 - 13g is preferably used.

Natriuretic peptides (NP peptides) in the sense of the invention are peptides with a natriuretic activity which are formed from a precursor polypeptide (prohormone) in the ventricle of the heart, the adrenal gland and the brain and contain a ring of 17 amino acid as a structural element which is formed by a disulfide bridge between two cysteine residues. Precursor polypeptides are for example the "atrial" natriuretic peptide (ANP 1 - 126) or cardiodilatin (CCD 1 - 126) and the "brain"
natriuretic peptides of the B and C type. Preferred NP
peptides are derived from the human a atrial natriuretic peptide (hccANP). In this connection the C-terminal haANP
fragments of amino acids 95 - 126, 99 - 126 and 102 126 are particularly preferred.

The fusion proteins are produced by expression of a DNA
which codes for the fusion protein in prokaryotic or eukaryotic host cells preferably in prokaryotes. A DNA
that is suitable for the expression can preferably be produced by synthesis. Such processes are familiar to a person skilled in the art and are described for example in Beattie K.L. and Fowler, R.F., Nature 352 (1991) 548 - 549 r33); EP-B 0 424 990 (34); Itakura, K. et al., Science 198 (1977) 1056 - 1063 (35). The nucleic acid sequence of the proteins according to the invention can also be modified. Such modifications are for example:

- Modification of the nucleic acid sequence in order to introduce various recognition sequences of restriction enzymes to facilitate the steps of ligation, cloning and mutagenesis.

- Modification of the nucleic acid sequence to incorporate preferred codons for the host cell.

- Extension of the nucleic acid sequence with additional regulation and transcription elements in order to optimize the expression in the host cell.

All further steps in the process for the production of suitable expression vectors and for the expression are state of the art and familiar to a person skilled in the art. Such methods are described for example in Sambrook, J. et al. (1989) (6J.

E. coli, streptomyces or bacillus are for example suitable as prokaryotic host organisms. Suitable eukaryotic host cells are for example yeasts such as Saccharomyces, pichia, hansenula and kluyveromyces and fungi such as aspergillus and trichoderma. For the production of the fusion proteins according to the invention the prokaryotic cells are transfected in the usual manner with the vector which contains the DNA
coding for the fusion protein and subsequently fermented in the usual manner. After lysis of the cells the protein is isolated in the usual manner and optionally purified by means of immobilized biotin or derivatives thereof preferably by means of affinity chromatography.

If the protein is not expressed in a soluble form and accumulates in prokaryotes in an inactive form (IBs inclusion bodies), it is expedient to solubilize it by methods familiar to a person skilled in the art with a denaturing agent such as guanidine hydrochloride or urea and renature it by dilution on dialysis in a suitable buffer. In this process the dilution is carried out in such a manner that afterwards the denaturing agent is diluted at least to the extent that it no longer has a denaturing effect.

_ g _ The dilution is preferably carried out in a pulse-like manner for example by adding the solubilisate dropwise to buffer that contains no denaturing agent.

Such a pulse-like dilution enables an almost simultaneous removal of the effect of the denaturing agent and separation of the molecules to be renatured.
This largely avoids an undesired intermolecular interaction (aggregation) of the molecules to be renatured.

If the fusion protein solubilized in the denaturing agent cannot be renatured by dilution, the renaturation is carried out in the presence of renaturation aids.
Such renaturation processes and renaturation aids are known to a person skilled in the art and described for example in the US patent No. 5,077,392 (36), in EP-B 0 114 506 (37J as well as in Marston, F.AØ, Biochem. J.
214 (1986) 1 - 12 (38) and Light, A., Biotechniques 3 (1985) 297 - 306 (39)). For this it is expedient to solubilize the inclusion bodies with the denaturing agent optionally in the presence of a reducing agent in the case of peptides containing cysteine, to dilute the denaturing agent until it no longer has a denaturing action and allows the fusion protein to fold into a state in which its protein domains can adopt the natural state. Characteristics of this state are that the disulfide bridges are natively linked and that the fusion protein is soluble also without a high concentration of denaturing agent. It can subsequently be cleaved with endoproteinase LysC.

In the case of proteins/peptides containing disulfide bridges dithioerythritol, dithiothreitol or mercaptoethanol are preferably used as a reducing agent.
It is then expedient to carry out the renaturation in the presence of a redox system such as for example of oxidized and reduced glutathione or cysteine.

The following examples, publications, the sequence protocol and the figure elucidate the invention the protective scope of which results from the patent claims. The described methods are to be understood as examples which also after modifications still describe the subject matter of the invention.

Fig. 1 shows the DNA segments A and B obtained according to example 1.

Example Construction of the core-SA-UR0(95-126) fusion gene containing an endoproteinase linker (plasmid: p8A-Eg-~RO) core-SA: shortened streptavidin of amino acids Met-(13-139) URO (95-126): urodilatin or cardiodilatin fragment of amino acids 95 - 126 (sequence described by Drummer, C.
et al., Plugers Archiv, European J. of Physiol. 423 (1993) 372 - 377 (41)).

The expression vector for the core-SA-URO (95-126) fusion gene containing an endoproteinase LysC cleavage site is based on the expression vector pSAM-CORE for core streptavidin. The construction and description of the plasmid pSAM-CORE is described in WO 93/09144 (11).
In order to construct core-SA fusion proteins the singular NheI restriction cleavage site located at the 3' end before the stop codon of the core-SA gene is used.

A ca. 140 bp long DNA fragment coding for the linker ~VDDDDK] (SEQ ID NO:1) and the urodilatin (95 - 126) polypeptide [TAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY] (SEQ ID
N0:2) was composed of 2 ca. 70 bp long chemically synthesized DNA segments. The codons preferably used in E. coli (E. coli codon usage) were taken into account in the gene design and the ends of the individual DNA
segments were provided with suitable singular restriction endonuclease cleavage sites.

In two reaction mixtures the complementary oligonucleotides 1 (SEQ ID NO:3) and 2 (SEQ ID NO:4) AATTCGCTAGCGTTGACGACGATGACAAAACGGCGCCGCGTTCCCTGCGTAGATCT
TCCTGCTTCGGC (SEQ ID N0:3) GGCCGCCGAAGCAGGAAGATCTACGCAGGGAACGCGGCGCCGTTTTGTCATCGTCG
TCAACGCTAGCG (SEQ ID N0:4) were annealed to the DNA segment A (Fig. 1) and the oligonucleotides 3 (SEQ ID NO:5) and 4 (SEQ ID NO:6) GGCCGCATGGACCGTATCGGTGCTCAGTCCGGACTGGGTTGCAACTCCTTCCGTTA
CTAATGA (SEQ ID N0:5) AGCTTCATTAGTAACGGAAGGAGTTGCAACCCAGTCCGGACTGAGCACCGATACGG
TCCATGC (SEQ ID NO:6) were annealed to the DNA segment B (Fig. 1) (reaction buffer: 12.5 mmol/l Tris-HCl, pH 7.0 and 12.5 mmol/l MgCl2; oligonucleotide concentration: in each case 1 pmol/60 ~l) and the hybridization products A and B
were each subcloned into the polylinker region of the E.
coli pUCBM21 vector (Boehringer Mannheim GmbH, Mannheim, Germany) (DNA segment A, cleavage sites: EcoRI and NotI;
DNA segment B, cleavage sites: NotI and HindIII). The DNA sequence of the two subcloned DNA segments was confirmed by DNA sequencing. Afterwards the expression plasmid pSA-EK-URO for the core-SA-URO (95-126) fusion gene was assembled in a three fragment ligation from the Nhe/NotI-DNA segment A, the NotI/HindIII DNA segment B
and the ca. 2.9 kBp long NheI/HindIII-pSAM-CORE vector fragment. In this process the DNA segments A and B were isolated by double digestion with the appropriate endonucleases from the corresponding pUCBM21 plasmid derivatives. The desired plasmid pSA-EK-URO was identified by restriction mapping and the DNA sequence of the linker urodilatin region was again checked by DNA
sequencing.

ExamPle 2 Expres~ion of the core-SA fusion proteins in E. coli For the expression of the core-SA fusion protein the E. coli K12 strain RM82 (a methionine revertant of ED
8654, Murray, N.E. et al. (1977) (14)) was transformed with the expression plasmid pSA-EK-URO described in example 1 and with the lacIq repressor plasmid pUBS500 (kanamycin resistance, preparation and description see:
EP-A 0368342).

The RM82/pUBS500/pSA-EK-URO cells were cultured up to an optical density at 550 nm of 0.6 - 0.9 in DYT medium (1 % (w/v) yeast extract, 1 % (w/v) Bacto Tryptone (Difco, Detroit, USA) and 0.5 % NaCl containing 50 mg/l ampicillin and 50 mg/l kanamycin and subsequently induced with IPTG (isopropyl-~-D-thiogalactoside) (final concentration 1 - 5 mmol/l). After an induction phase of 4 - 8 hours, the cells were harvested by centrifugation and the cell pellets were washed with 25 mmol/l potassium phosphate buffer pH 7.5.

Expression analysis The cell pellets from in each case 1 ml centrifuged culture medium (RM82/pUBS500/pSA-EK-UR0 cells) were resuspended in 0.25 ml 10 mmol/l phosphate buffer, pH 6.8 and 1 mmol/l EDTA and the cells were lysed by ultrasonic treatment. After centrifugation 1/5 volume 5 x SDS sample buffer (1 x SDS sample buffer: 50 mmol/l Tris-HCl, pH 6.8, 1 % SDS, 1 ~ mercaptoethanol, 10 %
glycerol, 0.001 % bromophenol blue) was added to the supernatant. The insoluble cell debris fraction was resuspended in 0.3 ml 1 x SDS sample buffer containing 6 - 8 M urea, the samples were incubated'for 5 minutes at 95~C and centrifuged. Afterwards the proteins were separated by SDS polyacrylamide gel electrophoresis (PAGE) (Laemmli, U.K. (1970) (15J) and stained with Coomassie brilliant blue R dye.

The core-SA fusion protein synthesized in E. coli was homogeneous and was found exclusively in the insoluble cell debris fraction (IBs). The expression yield for the core-SA fusion protein was 30 - 50 % relative to the total E. coli protein.

Exam~le 3 Cell lysis and preparation of inclusion bodies (IBs) 200 g (wet weight) E. coli RM82/pUBS500/pSA-EK-URO cells was suspended in 1 l 0.1 mol/l Tris-HCl, pH 7.0 at 0~C, 300 mg lysozyme was added and incubated for 20 minutes at 0~C. Afterwards the cells were completely lysed mechanically by means of high pressure dispersion and the DNA was digested within 30 minutes at 25~C by adding 2 ml 1 mol/l MgCl2 and 10 mg DNAse (Boehringer Mannheim # 154709). Subsequently 500 ml 60 mmol/l EDTA, 6 %
Triton~ X100 and 1.5 mol/l NaCl, pH 7.0 was added to the lysis solution and incubated for a further 30 minutes at 0~C. Afterwards the insoluble components (cell debris and IBs) were sedimented by centrifugation.

The pellet was suspended in 1 l 0.1 mol/l Tris-HCl, 20 mmol/l EDTA pH 6.5, incubated for 30 minutes at 25~C
and the IB preparation was isolated by centrifugation.

8O1ubilization of the IBs 25 g IB pellet (wet weight) was suspended by stirring for 2 hours at 25~C in 200 ml 0.1 mol/l sodium phosphate buffer, 6 mol/l guanidine-HCl, 10 mmol/l EDTA pH 7Ø
The insoluble components were separated by centrifugation and the clear supernatant was processed further.

Example 4 Renaturation The renaturation was carried out in a BioFlo II
fermenter (New Brunswick Scientific Co., Inc. Edison, NJ, USA) at 16~C while stirring (300 rpm) by continuous addition of 200 ml core-SA fusion protein solubilisate to 5 l 20 mmol/l sodium phosphate pH 7.0, 5 mmol/l EDTA
using a pump (output: 15 - 20 ml/h).

After completion of the renaturation reaction, 50 g diatomaceous earth (standard Supercel from the T-~h~nn &
Foss Company (Hamburg, Germany)) was added and the insoluble components were separated by 2-fold filtration [prefiltration by means of a Buchner funnel fitted with a 520 B II round filter from the Schleicher & Schull Company (Dassel, Germany) and refiltration by means of a filtration apparatus from the Satorius Company (Gott~ngen, Germany) equipped with a K 250 deep filter from the Seitz Company (Bad Kreuznach, Germany)] and the clear supernatant containing the core-SA fusion protein was processed further.

Concentration and/or dialysis of the renaturation preparation The renaturation preparation was concentrated by cross-flow filtration in a Minisette (membrane type: Nova K10) from the Filtron Company (Karlstein, Germany) and dialysed against a desired buffer if necessary to remove guanidine HCl.

CA 0223284l l998-03-20 Example 5 Affinity chromatography of the core-8A fusion protein The core-SA fusion protein was purified directly from the filtered and concentrated renaturate by affinity chromatography on iminobiotin Sepharose.

Preparation of iminobiotin 8epharose 4B

500 ml epoxy-activated EAH Sepharose 4B (Pharmacia Biotech, Freiburg, Germany) was washed on a frit with 15 l 0.5 mol/l NaCl, 3 l deionized water and 1 l 50 mmol/l potassium phosphate buffer, pH 7.5 containing 150 mmol/l NaCl and resuspended in 5 l 10 mmol/l potassium phosphate buffer, pH 7.5 containing 150 mmol/l NaCl and 20 %
dimethylsulfoxide (DMS0). 0.5 g iminobiotin hydroxy-succinimide ester (Sigma, Deisenhofen, Germany) was dissolved in 60 ml DMS0, diluted with 600 ml 10 mmol/l potassium phosphate buffer, pH 7.5 and 150 mmol/l NaCl and added to the gel suspension. The gel suspension was incubated overnight at room temperature while gently shaking and subsequently washed on a frit with 5 l 10 mmol/l potassium phosphate buffer, pH 7.5 containing 150 mmol/l NaCl and 20 % DMSO, 3 l deionized water and 3 l 10 mmol/l potassium phosphate buffer, pH 7.5 containing 150 mmol/l NaCl.

In order to determine the core-SA loading capacity, a column was packed with exactly 1 ml iminobiotin Sepharose 4B and equilibrated with 50 mmol/l ethanolamine buffer, pH 9.5 containing 0.5 mmol/l NaCl.
Afterwards a core-SA solution having a concentration of 1 mg/ml was applied in the equilibration buffer. The loading capacity/ml affinity gel was determined by CA 0223284l l998-03-20 measuring the absorbance at 280 nm in the eluate and determining the application volume (30 - 40 mg core-SA/ml gel).

Affinity chromatography on iminobiotin 8epharo~e 4B

An iminobiotin Sepharose 4B column (4 x 24 cm; V =
300 ml) equilibrated with 25 mmol/l ethanolamine pH 9.5 was loaded with the concentrated renaturation preparation that had been titrated with ethanolamine to pH 9.5 (1 column volume/hour, 1 CV/h) and washed with equilibration buffer until the absorbance of the eluate at 280 nm reached the blank value of the buffer. The bound material was eluted with 0.1 mol/l potassium acetate buffer, pH 3.5. Afterwards the core-SA protein was dialysed against the buffer used for the enzymatic cleavage.

Example 6 Enzymatic cleavage of the core-8A fusion protein with endoproteinase LysC

The core-SA-EK-UR0 fusion protein was digested in 50 mmol/l Tris-HCl, pH 8.0 at 30 to 35~C at a concentration of 0.3 to 3 mg/ml and a substrate/protease ratio of l:lOOo to 1:25,000 (endoproteinase LysC from Lysobacter enzymogenes, sequencing grade; Boehringer Mannheim, Mannheim, Germany) and the time course of the enzymatic cleavage was analysed by analytical reversed phase HPLC (see example 8). For this purpose samples (10 to 100 ~1) were removed from the reaction mixture at intervals of 1 to 3 hours over a period of 6 to 24 hours.

CA 0223284l l998-03-20 Bxample 7 Purification of the peptide URO (95-126) The enzymatically released peptide can be further purified with chromatographic methods that are known to a person skilled in the art.

7.1 8eparation of the core-8A carrier protein by mean of affinity chromatography The core-SA carrier protein can be separated from the cleavage mixture by negative chromatography on iminobiotin Sepharose as described in example 5 for the core-SA fusion protein.

For this the reaction mixture is adjusted with ethanolamine to a pH value of 9 to 9.5 after the enzymatic cleavage and the core-SA carrier protein and non-cleaved core-SA fusion protein are separated by affinity binding to iminobiotin.

7.2 Purification of the peptide by cation exchange chromatography on Fractogel EMD-8O3- 65 (M) 1 mol/l sodium acetate, pH 5.0 was added to the cleavage mixture to a final concentration of 25 mmol/l, the pH
was adjusted to 5.0 and a Fractogel EMD-SO3- 650(M) column (3 x 40 cm, V = 283 ml) from the Merck Company (Darmstadt, Germany) equilibrated with 25 mmol/l, pH 5.0 was loaded with this (1 CV/h) and washed with equilibration buffer until the absorbance of the eluate at 280 nm reached the blank value of the buffer. The bound material was eluted by a gradient of 0 to 1 mol/l NaCl in equilibration buffer (10 to 20 CV, 1 CV/h).

7.3 Purification of the peptide by reversed phase HPLC

After pre-purification of the peptide by means of cation exchange chromatography (see: example 7.2), an aliquot of l to 2 ml (ca. 100 to 300 ~g) was further purified by semipreparative RP-HPLC with fractionation.

Chromatography conditions:

Column: Europher lO0-C8, 5 ~m (4 x 250 mm, V = 3.17) (Knauer, Berlin, Germany) Sample volume: 1 - 2 ml (100 - 300 ~g protein) Detector: W, 220 nm Flow rate: 0.5 ml/min Mobile solvent:
- A: 0.13 % TFA in H20 - B: 0.1 % TFA, 80 % acetonitrile, 20 % H2O
(v/v) Example 8 Analytical reversed phaoe HPLC

The analytical reversed phase HPLC was carried out with a Europher column (Europher 100-C8, 5 ~m (4 x 250 mm, V = 3.17 ml, Knauer, Berlin, Germany). The sample volume was 10 - 100 ~l corresponding to 1 - lO0 ~g protein. The detection was carried out with a W detector at 220 nm.
It was chromatographed at a flow rate of 0.5 ml/min.

Mobile solvent:
A: 0.13 % trifluoroacetic acid in H20 B: 0.1 % trifluoroacetic acid, 80 % acetonitrile, 20 % H20 (V/V) (gradient 100 - O % in 50 min).

Urodilatin (95 - 126) eluted at 31 min.

Exam~le 9 Characterization of the purified peptide The identity and purity of the purified peptide can for example be examined in comparison to a chemically synthesized standard by mass spectroscopy (PD-MS and laser desorption spectroscopy), analytical reversed phase HPLC, isoelectric focussing (Bark, J.E. et al., J.
Forensic Sci. Soc. 16 (1976) 115 - 120 (42), SDS PAGE
(Laemmli, U.K., Nature 227 (1970) 680 - 685 (43) and capillary electrophoresis.

List of reference~

1) Kent, S.B.H. et al., Banburi Rep. 29 (1988) 3-20 2) Hodson, J.H., Bio/Technology 11 (1993) 1309-1310 3) Kopetzki, E. et al., Clin. Chem. 40 (1994) 688-704 4) Winnacker, E.-L., (1987) VCH Publishers, Weinheim and New York 5) Harris, T.J.R. In: Genetic Engineering (Williamson, R. ed.), Academic Press, London, vol. 4 (1983) 127-185 6) Sambrook, J. et al. (1989) In: Molecular cloning: A
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(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: BOEHRINGER MANNHEIM GMBH
(B) STREET: Sandhofer Str. 116 (C) CITY: Mannheim (E) COUNTRY: Germany (F) POSTAL CODE (ZIP): D-68305 (G) TELEPHONE: 08856/60-3446 (H) TELEFAX: 08856/60-3451 (ii) TITLE OF INVENTION: Process for producing natriuretic peptides via streptavidin fusion proteins (iii) NUMBER OF SEQUENCES: 6 (iv) CO~ ~ READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) CONPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30B (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Val Asp Asp Asp Asp Lys (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
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(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = synthetic oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Claims (6)

Claims

1. Process for the recombinant production of a natriuretic peptide (NP peptide) by expression of a DNA in prokaryotes or eukaryotes which codes for a fusion protein of streptavidin and the said NP
peptide, wherein the C-terminus of streptavidin and the N-terminus of the NP peptide are linked via a peptide sequence that can be cleaved by endoproteinase LysC, cleavage of the fusion protein with endoproteinase LysC and isolation of the desired protein.

2. Process as claimed in claim 1, wherein the expression is carried out in prokaryotes where the fusion protein forms as an insoluble inactive fusion protein, solubilization of the inactive fusion protein, renaturation of the fusion protein, optionally purification of the fusion protein by means of immobilized biotin or derivatives thereof, cleavage of the fusion protein with endoproteinase LysC and isolation of the desired peptide.

3. Process as claimed in claim 2, wherein the renaturation of the solubilized, inactive fusion protein is achieved by diluting the solubilized, inactive fusion protein in aqueous buffer solution.

4. Process as claimed in claims 1 - 3, wherein the fusion protein is purified before cleavage by affinity chromatography on immobilized biotin or biotin derivatives.

5. Process as claimed in claims 1 - 4, wherein urodilatin or fragments derived therefrom are used as the NP peptide.

6. Process as claimed in claims 1 - 5, wherein fragments of urodilatin of amino acids 95 - 126, 99 - 126 or 102 - 126 are used as peptide.