Note: Descriptions are shown in the official language in which they were submitted.
CA 02232841 1998-03-20
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
CA 02232841 1998-03-20
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
CA 02232841 1998-03-20
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
CA 02232841 1998-03-20
-- 4
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
CA 02232841 1998-03-20
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
CA 02232841 1998-03-20
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
CA 02232841 1998-03-20
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
CA 02232841 1998-03-20
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.
CA 02232841 1998-03-20
_ 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
CA 02232841 1998-03-20
-- 10 --
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
CA 02232841 1998-03-20
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
CA 02232841 1998-03-20
- 12 -
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
CA 02232841 1998-03-20
(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.
CA 02232841 1998-03-20
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.
CA 02232841 1998-03-20
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
- 16 -
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
- 17 -
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
- 18 -
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
CA 02232841 1998-03-20
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.
CA 02232841 1998-03-20
- 20 -
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
laboratory manual. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York
7) EP-B O 198 015
CA 02232841 1998-03-20
8) EP-A 0 612 325
9) Sano, T. et al., Biochem. Biophys. Res. Commun. 176
(1991) 571-577
10) Sano, T. et al., Proc. Natl. Acad. Sci., USA 89 (1992)
1534-1538
11) WO 93/09144
12) Carter, P. In: Ladisch, M.R.; Willson, R.C.; Painton,
C.C.; Builder, S.E. eds. Protein Purification: From
Molecular Mechanisms to Large-Scale Processes. ACS
Symposium Series No. 427, American Chemical Society,
pp. 181-193 (1990)
13) Dougherty, W.G. et al., EMB0 J. 7, (1988) 1281-1287
14) Murray, N.E. et al., Mol. Gen. Genet. 150 (1977) 53-61
15) Laemmli, U.K., Nature 227 (1970) 680-685
16) Rokkones, E. et al., J. Biotechnol. 33 (1994) 293-306
17) Forsberg, G. et al., J. Prot. Chem. 10 (1991) 517-526
18) Gardella, T.J. et al., J. Biol. Chem. 265 (1990)
15854-15859
19) Gram, H. et al., Bio/Technology 12 (1994) 1017-1023
20) Forssmann, K. et al., Clin. Wochensch. 66 (1988)
752-752
21) Drummer, C. et al., Pflugers Archiv., European J. of
Physiol. 423 (1993) 372-377
22) Heim, J.M., Biochem. Biophys. Res. Commun. 163 (1989)
37-41
23) Argarana, C.E. et al., Nucl. Acids Res. 14 (1986)
1871-1882
24) Bub, A. et al., Histochem. J. (Suppl.) 24 (1992) 517
25) Drummer, C. et al., J. Am. Soc. Nephrol. 1 (1991)
1109-1113
26) Am. J. Physiol. 262 (1992) F 744-F 754
27) Emmeluth, C. et al., Am. J. Physiol. 262 (1992)
F 513-F 516
28) Goetz, K.L. et al., J. Am. Soc. Nephrol. 1 (1990)
867-874
CA 02232841 1998-03-20
29) Sharma, A. et al., Proc. Natl. Acad. Sci. USA 91
(1994) 9337-9341
30) Gram, H., Bio/Technology 12 (1994) 1017-1023
31) Ladisch,-M.R. (ed.) Protein Purification ACS-
Symposium, Series 427, American Chemical Society,
Washington D.C. (1990) 189
32) Allen, G. et al., J. Cell. Sci. Suppl. 3 (1985) 29
33) Beattie, K.L.;Fowler, R.F., Nature 352 (1991) 548-549
34) EP-B 0 424 990
35) Itakura, K. et al., Science 198 (1977) 1056-1063
36) US-Patent No. 5,077,392
37) EP-B 0 114 506
38) Marston, F.AØ, Biochem. J. 214 (1986) 1-12
39) Light, A., Biotechniques 3 (1985) 297-306
40) Allen, G. et al., J. Cell. Sci. Suppl. 3 (1985) 29-38
41) Drummer, C. et al., Pflugers Archiv, European J. of
Physiol. 423 (1993) 372-377
42) Bark, J.E. et al., J. Forensic Sci. Soc. 16 (1976)
115-120
43) Laemmli, U.K., Nature 227 (1970) 680-685
CA 02232841 1998-03-20
- 23 -
SEQUENCE LISTING
(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
CA 02232841 1998-03-20
- 24 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met
1 5 10 15
Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr
20 25 30
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 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: 3:
AATTCGCTAG CGTTGACGAC GATGACAAAA CGGCGCCGCG TTCCCTGCGT AGATCTTCCT 60
GCTTCGGC 68
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 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: 4:
GGCCGCCGAA GCAGGAAGAT CTACGCAGGG AACGCGGCGC CGTTTTGTCA TCGTCGTCAA 60
CGCTAGCG 68
CA 02232841 1998-03-20
- 25 -
(2) INFORMATION FOR SEQ ID NO: 5:
(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: 5:
GGCCGCATGG ACCGTATCGG TGCTCAGTCC GGACTGGGTT GCAACTCCTT CCGTTACTAA 60
TGA 63
(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:
AGCTTCATTA GTAACGGAAG GAGTTGCAAC CCAGTCCGGA CTGAGCACCG ATACGGTCCA 60
TGC 63
