Note: Descriptions are shown in the official language in which they were submitted.
13 412 1
Description
Mini-proinsulin, its preparation and use
The invention relates to a novel "mini-proinsulin" in
which the unshortened B chain is only bonded to the A
chain via an arginine residue. Human insulin is access-
ible from this mini-proinsulin without difficult chemical
reaction.
Mini-proinsulins having a shortened C chain are known.
Thus R. Wetzel et al., Gene 16 (1981), 63-71 have de-
scribed a proinsulin having a C chain shortened to six
amino acids. In the European patent application having
the publication number (EP-A) 0,055,945 (Published 14 July
1982), corresponding proinsulins are disclosed, the C chain
of which is shortened by two amino acids.
In EP-A 0,163,529 (Published 04 December 1985), insulin
precursors having a shortened B chain are disclosed in which
the C chain is either missing completely or else shortened to
one amino acid. These precursors are converted into mature
human insulin by trypsin-catalysed transpeptidation using an
a-threonine ester.
On the other hand, the invention relates to human Des-
(32-65) proinsulin or mini-proinsulin of the formula I
B(1-30)-Arg-A(1-21) (I),
in which B(1-30) and A(1-21) denote the B and A chain of
human insulin. This compound is used not only as an
intermediate for the preparation of human insulin Arg831-
OH, called "mono-Arg-insulin" in the following, which has
been disclosed in European Patent Nos. (EP-B) 0,132,769
and 0,132,770 (Published 07 January 1988), and of human
insulin, but it also shows a certain insulin activity itself.
13412 1 1
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The invention therefore also relates to the compound of
the formula I for use as a pharmaceutical, in particular
for the treatment of diabetes mellitus, and furthermore
pharmaceuticals containing the compound of the formula
I, and pharmaceuticals composed of a pharmacologically
acceptable excipient and the compound of the formula I.
The invention furthermore relates to the use of the
compound of the formula I for the preparation of the
mono-Arg insulin of the formula II
S - S
I I
A(1 - 21)
I I
(II),
S S
t I
B(1 - 30) - Arg
in which A(1-21) and B(1-30) have the meanings mentioned
previously and the -S-S-bridges are arranged as in
insulin, and of human insulin by enzymatic cleavage. The
immediate conversion of the compound of the formula I
into insulin in a "one-pot reaction" is particularly
advantageous.
The invention furthermore relates to a process for the
preparation of the compound of the formula I which
comprises expressing a gene structure encoding for this
compound in a host cell, preferably in a bacterium such
as E. coli or in a yeast, in particular Saccharomyces
cerevisiae, and, if the gene structure encodes for a
fusion protein, liberating the compound of the formula I
from this fusion protein. The invention relates in
addition to DNA sequences which encode for the compound
of the formula I, gene structures or plasmids which
contain this DNA, and host cells, in particular bacteria
such as E. coli or yeast cells, particularly yeasts of
the strain Saccharomyces cerevisiae which contain such
gene structures or plasmids. The invention furthermore
13412 1 1
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relates to fusion proteins which contain a compound of
the formula I, preferably fusion proteins in which the
compound of the formula I is bonded via the bridging
member
- Met - Ile - Glu - Gly - Arg -
to the "ballast component" of the fusion protein.
Further preferred embodiments of the invention are
explained in more detail in the following.
The figures are used to explain the Examples, Fig. 1 (and
its continuation in Fig. la and lb) showing the construc-
tion of the E, coli expression vectors PIR10 and pSW3 and
Fig. 2 (and its continuation in Fig. 2a and 2b) showing
that of the yeast expression vector paf8102 or paf8104.
These vectors encode for mini-proinsulin.
It has been found that the mini-proinsulin has the
correct folding so that mono-Arg insulin is formed almost
quantitatively after cleavage with trypsin. A surpris-
ingly simple process for the preparation of mono-Arg
insulin thus results. Human insulin can be prepared from
this in a manner known per se. Mono-Arg insulin is
furthermore used as an active compound in pharmaceuticals
(EP-B 0,132,769, Published 07 January 1988).
The expression vector pK50 was described in EP-A
0,229,998 (Published 29 July 1987). Mini-proinsulin can
be prepared in a bacterium such as E. coli in the form of
a fusion protein corresponding to this construction.
The poorly soluble fusion protein can be concentrated by
washing with neutral buffer solutions. The mini-pro-
insulin is liberated by cyanogen halide cleavage
(E. Gross and B. Wittkop, J. Am. Chem. Soc. 82 (1961)
1510-1517). This still does not exist in the
,,
i34 92 1 1
- 4 -
biologically active form, but consists of a non-uniform
mixture having various inter- and intramolecular disul-
fide bridges, possibly also with other protein fragments .
The S-sulfonate form of the molecule is prepared as a
chemically uniform, relatively stable derivative (P. G.
Ratsoyannis et al., Biochemistry 6_ (1967) 2635-2641).
This derivative can be purified very easily by ion
exchange chromatography and is an approved starting
material for the folding into the native spatial struc-
ture with the formation of the correct disulfide bridges
(Y. C. Du et al., Sci. Sin. 15 (1965) 229-236; H.P.
Gattner et al., Hoppe-Seylers Z. physiol. Chem. 362
(1981) 1943-1049; B.H. Frank et al. in: "Peptides:
Synthesis-Structure-Function", D.H. Rich and E. Gross,
Publishers, (1981) 1043-1049). The success of this
folding is safeguarded by HPLC analysis of the fragments
resulting after cleavage with S. aureus protease V8
(U. Grau, Diabetes ~ (1985) 1174-1180).
The liberation of mono-Arg insulin or insulin by the
action of trypsin or carboxypeptidase B or by enzymes
having the same action (W. Remmler et al., J. Biol. Chem.
246 (1971) 2780-2795) proceeds in a decidedly uncompli-
cated manner, since it proves particularly advantageous
for this purpose that the number of possible cleavage
sites is reduced compared to normal proinsulin. Because
of this, the cleavage is considerably more simple to
control (with respect to the formation of side products
as in the preparation of Des-B30 insulin or Desocta-B23-
B30 insulin). Both mono-Arg insulin and also insulin can
be isolated in a known manner in highly pure form by ion
exchange chromatography. The formation of the insulin
and the mono-Arg derivative, the course of the purifica-
tion and the quality of the final product are checked
using customary RP-HPLC methods (G. Seipke et al., Angew.
Chem. ~8 (1986) 530-548).
Surprisingly, hardly any insulin Des-B30 is formed in the
cleavage of mini-proinsulin in contrast to natural
_5- 1341211
proinsulin. Since the latter can only be separated from
insulin in a very difficult manner, the ~two-pot reac-
tion" is preferred in the production of insulin from
natural proinsulin, i.e. the principal products of the
tryptic cleavage, insulin-Arge31-ArgH32 and mono-Arg insulin
are first separated from insulin Des-830 via ion exchan-
gers in a known manner and then cleaved to give human
insulin by means of carboxypeptidase B (EP-A 0,195,691, Published 24
September 1986). In comparison, mini-proinsulin can be converted to
human insulin in an ideal manner in a "one-pot reaction" simultaneously
using trypsin and carboxypeptidase B or by means of enzymes having the
same action.
The expression of the compound of the formula I in yeast
with subsequent secretion is particularly advantageous,
since the correctly folded proinsulin derivative can be
isolated directly. Yeasts are used as host systems, for
example B. Pichia pastoris, Hansenula polymorphis,
Schizosaccharomyces pombe or, preferably Saccharomyces
cerevisiae.
Vectors for expression in yeasts are known in large
numbers. The preparation of the insulin derivative
according to the invention is described in the following
with the aid of the yeast a-factor system which, however,
is only to be taken as an example, since other expression
systems can also be employed in a manner known per se.
The structure of the yeast pheromone gene 1~'a is known
from the publication Kurjan and Herskovitz, Cell 30
(1982) 933-943, where the possibility of the expression
of other genes and the secretion of the gene products are
also discussed. Regarding this, reference can also be
made to Brake et al., Proc. Natl. Acad. Sci. USA 81
(1984), 4642-4646.
x
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Alternatively, a yeast "Rillertoxin" system can be used,
or secretion via the acidic phosphatase or invertase
system can be utilized.
As yeast vectors, so-called "shuttle" vectors are advan-
tageously used which have a bacterial plasmid and a yeast
plasmid replication origin and also a gene or genes for
selection in both host systems. In addition, such
vectors contain the promoter sequences necessary for the
expression of foreign genes and, if appropriate, a
terminator sequence to improve the yield so that the
heterologous gene - expediently fused to secretory
signals - is arranged between promotor and terminator.
Such vectors are described, for example, in US-A
4,766,073.
The genetic code is, as is known, "degenerate", i.e. a
single nucleotide sequence encodes only for two amino
acids, while the residual 18 encodable amino acids are
allocated two to six triplets. For the synthesis of the
gene for mini-proinsulin, there is thus a large variety
of codon combinations to be chosen. It has now been
found that the DNA sequence I encoding for mini-pro-
insulin (which is reproduced in the Appendix in the form
of the two gene fragments IR I (Table 1) and IR II (Table
2)) is particularly advantageous, since it is optimized
to codon use of both yeast and E. coli.
A "protruding" DNA sequence corresponding to the restric-
tion endonuclease RpnI is situated at the 5' end of the
encoding strand of the DNA sequence I. In comparison,
the single-stranded sequence corresponding to the restri-
ction enzyme HindIII is protruding at the 3' end of the
encoding strand. These two different recognition sequen-
ces ensure the insertion of the DNA sequence I into
plasmids in the desired orientation. Two translation/-
termination codons (stop codons) follow triplet No. 65
for asparagine in the encoding sequence. An internal
unique cleavage site for the restriction enzyme Pst I
134121 1
_ 7 _
(codon 41/42) makes possible the sub-cloning of two gene
fragments which can be incorporated in well investigated
plasmids such as pUCl8 or derivatives of these plasmids.
Additionally, a number of further unique recognition
sequences for restriction enzymes can be incorporated
within the structure gene, which on the one hand provide
access to partial sequences of proinsulin and on the
other hand permit mutations to be carried out:
Restriction enzyme Cut after nucleotide no.
(encoding strand)
AccI 201
DraIII 46
FnuDII 107
HgaI 105
HindIII 213
Hinf I 17
HpaI 22
HphI 76
MaeI 155
MaeIII 191
MboII 89
MluI 106
NcoI 207
NIaIII 208
PvuII 175
SalI 201
SpeI 154
StyI 207
TaqI 202
The DNA sequence I was modified from the natural sequence
at essential points. In this way, the insertion of the
numerous unique cleavage sites for restriction enzymes
was possible.
The DNA sequence I can be constructed from a total of 6
oligonucleotides having a chain length of 47 to 96
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- g -
nucleotide units. For this purpose, the procedure is as
described in the following.
The gene fragment IR I (Table 1) can be constructed from
4 oligonucleotides having a chain length of 47 to 74
units by first synthesizing these chemically and then
linking them enzymatically via "sticky ends" of 3 nucleo-
tides. The sticky ends correspond to those of the
restriction enzyme DraIII, which is advantageous for
later modifications.
The gene fragment IR II (Table 2) can be obtained from
two chemically synthesized oligonucleotides having a
length of 88 and 96 nucleotide units.
Example 1
a) Chemical synthesis of a single-stranded oligo-
nucleotide
The synthesis of the DNA building blocks is explained
using oligonucleotide No. 4 (Table 1) as an example. For
the solid phase synthesis, the nucleoside at the 3' end,
i.e. adenine (nucleotide No. 125) in the present case, is
used bonded covalently to a support via the 3'-hydroxyl
function. The support material is CPG ("controlled pore
glass") functionalized with long-chain aminoalkyl radi-
cals.
In the following synthetic steps, the base component is
employed as ~B-cyanoethyl N,N'-dialkyl-5'-0-dimethoxy-
tritylnucleoside-3'-phosphoramidite, where the adenine is
present as the N6-benzoyl compound, the cytosine as the
N4-benzoyl compound, the guanine as the NZ-isobutyl
compound and the thymine without a protective group.
25 mg of the polymeric support, which contains 0.2 ~mol
of 5'-0-dimethoxytrityl-N'°-benzoyl-2'-desoxyadenosine
- 1341' 1 1
bonded, are successively treated with the following
agents:
A) acetonitrile
B) 3% trichloroacetic acid in dichloromethane
C) acetonitrile
D) 5 ~mol of the appropriate nucleoside-3'-0-phosphite
and 25 ~mol of tetrazole in 0.15 ml of anhydrous
acetonitrile
E) acetonitrile
F) 20% acetic anhydride in tetrahydrofuran containing 40%
lutidine and 10% dimethylaminopyridine
G) acetonitrile
H) 3% iodine in lutidine/water/tetrahydrofuran in a
volume ratio of 5:4:1
In this connection, "phosphite" is taken to mean ~-cyano-
ethyl 2'-desoxyribose-3'-monophosphite, the third valency
being satisfied by a diisopropylamino radical. The
yields of the individual synthesis steps can in each case
be determined spectophotometrically by the detritylation
reaction 8) by measuring the absorption of the dimeth-
oxytrityl cation at the wavelength of 496 nm.
After synthesis has been concluded, the cleavage of the
dimethoxytrityl group is carried out as described in A)
to C). The oligonucleotide is cleaved from the support
by treatment with ammonia and the p-cyanoethyl groups are
eliminated at the same time. The amino protective groups
of the bases are cleaved quantitatively by treatment of
the oligomers with concentrated ammonia at 50°C for 16
hours. The crude product thus obtained is purified by
polyacrylamide gel electrophoresis.
The oligonucleotides 1 -3 ( Table 1 ) , 5 and 6 ( Table 2 )
are prepared in an analagous manner.
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b) Enzymatic linkage of the single-stranded oligo-
nucleotides
For enzymatic phosphorylation of the oligonucleotides at
the 5' terminus, each 1 ~mol of the oligonucleotides 1
and 4 is treated for 30 minutes at 37°C with four units
of T4 polynucleotide kinase in 20 ~1 of 50 mM tris-HC1
buffer (pH 7.6), 10 mM of magnesium chloride and 10 mM of
dithiothreitol (DTT). The enzyme is inactivated by
heating to 95°C for 5 minutes . The oligonucleotides 2
and 3, which form the "protruding" single-stranded
sequences, are not phosphorylated. This prevents the
formation of larger gene fragments in the subsequent
ligation.
The oligonucleotides 1 to 4 are ligated as follows: each
1 ~mol of the oligonucleotides 1 and 2 or 3 and 4 are
hybridized in pairs by dissolving these in each case in
~1 of 50 mM tris-HC1 buffer (pH 7.6), 10 mM of mag-
nesium chloride and 10 mM of DTT, heating this solution
to 95°C for 5 minutes and cooling to room temperature
20 within 2 hours. For this purpose, the oligonucleotides
1 and 4 are employed in the form of their 5'-phosphates.
For further linking of the bihelical DNA fragments
formed, the solutions of these are combined, warmed to
60°C for 15 minutes and cooled to room temperature. 2 ~1
of 0.1 M DDT, 16 ~1 of 2.5 mM adenosine triphosphate (pH
7) and 1 ~1 of T4 DNA ligase (400 units) are then added
and the mixture is incubated at 22°C for 16 hours.
The purification of the gene fragments thus obtained
(Tables 1 and 2) is carried out by gel electrophoresis on
a 10% strength polyacrylamide gel (without addition of
urea, 40 x 20 x 0.1 cm), X174 DNA (BRL) cleaved using
HinfI, or pBR322, cut using HaeIII, being used as a
labeling substance.
13412 1 1
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Example 2
a) Cloning of the synthesized DNA fragments
The commercial plasmid pUCl9 is opened using the restric-
tion enzymes RpnI and PstI and the large fragment (1) is
separated through a 0.8% strength "Seaplaque" gel. This
fragment is reacted with T4 DNA ligase using the DNA (2)
synthesized according to Table 1 and the ligation mixture
is incubated with competent E. coli 79/02 cells. The
transformation mixture is plated out on IPTG/Xgal plates
which contain 20 mg/1 of ampicillin. The plasmid DNA is
isolated from the white colonies and characterized by
restriction and DNA sequence analysis. The desired
plasmids are called pIRl.
Accordingly, the DNA (5) according to Table 2 is ligated
into pUCl9 which has been opened using PstI and HindIII
(4). The plasmid pIR2 (6) is obtained.
b) Construction of the mini-proinsulin gene
The DNA sequences (2) and (5) according to Table 1 and 2
are reisolated from the plasmids pIRl ( 3 ) and pIR2 ( 6 )
and ligated with pUCl9, which has been opened using RpnI
and HindIII (7). The plasmid pIR3 (8) is thus obtained
which encodes for a modified human insulin sequence.
The plasmid pIR3 ( 8 ) is opened using MluI and SpeI and
the large fragment (9) is isolated. This is ligated with
the DNA sequence (10)
B30 A1 A2 A3 A4 A5 A6 A7 A8 A9
(Thr)(Arg) Gly Ile Val Glu Gln Cys Cys (Thr) (Ser) (10)
5' CG CGT GGT ATC GTT GAA CAA TGT TGT A 3'
3' A CCA TAG CAA CTT GTT ACA ACA TGA TC 5'
(MLUI ) (~I)
1341211
- 12 -
which supplements the last codon of the B chain (B30) by
one arginine codon and replaces the excised codon for the
first 7 amino acids of the A chain and supplements the
codon for the amino acids 8 and 9 of this chain.
The plasmid pIR4 (11) is thus obtained, which encodes for
the human mini-proinsulin according to the invention.
c) Expression vectors for mini-proinsulin
pIR I:
The plasmid pR50 (12) known from EP-A 0,229,998 (Example
3 therein; Figure 3 (33)) is cleaved using EcoRI and
HindIII. Both fragments (13) and (14) are isolated. The
small fragment (14) containing the IL-2 partial sequence
is subsequently cleaved with MluI and the IL-2 partial
sequence (15) is isolated.
The plasmid pIR4 (11) is cleaved using EcoRI and HpaI and
the large fragment (16) is isolated. This is now ligated
with the IL-2 partial sequence (15) and the synthetic DNA
(17)
Bi Ba
Met Ile Glu Gly Arg Phe Val
5' CG CGT ATG ATT GAG GGC CGT TTC GTT 3' (17)
3' A TAC TAA CTC CCG GCA AAG CAA 5'
(MluI) (HpaI)
the plasmid pIR8 (18) being obtained. This encodes for
a fusion protein in which a bridging member Met-Ile-Glu-
Gly-Arg and then the amino acid sequence of the mini-
proinsulin follow the first 38 amino acids of the IL-2.
The EcoRI-HindIII fragment which encodes for the fusion
protein mentioned is excised from the plasmid pIR8 (18).
This fragment is ligated with the large fragment (13)
which was obtained in the cleavage of pR50. The expres-
1341211
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sion vector pIRlO (20) which encodes for the previously
characterized fusion protein is thus obtained.
pSW3:
If the NdeI-BstEII segment which includes the "bom site"
is removed from the vector pIRlO (20), a vector is
obtained which is present in the cell in a relatively
high copy number and - on account of the missing "bom
site" - can no longer be mobilized by conjugative plas-
mids.
For this purpose, the vector pIRlO (20) is cleaved using
BstEII and NdeI, which is precipitated using ethanol,
tranferred in DNA polymerase buffer and subjected to a
Rlenow polymerise reaction. The truncated DNA fragments
thus formed are separated by gel electrophoresis and the
larger fragment (21) is isolated. The vector pSW3 (22)
is obtained by ligation. After transformation of com-
petent E. coli-Mc1061 cells and amplification, the
plasmid pSW3 (22) is isolated and characterized.
Example 3: Expression in the strain E. coli W3110
An overnight culture of E. coli cells which contain the
plasmid pIRlO (20) or pSW3 (22) is diluted in a ratio of
about 1:100 using LB medium (J.H. Miller, Experiments in
Molecular Genetics, Cold Spring Harbor Laboratory, 1972)
which contains 50 ~g/ml of ampicillin and the growth is
followed by means of OD measurement. At OD = 0.5, the
culture is adjusted to 1 mM IPTG and the bacteria are
centrifuged off after 150 to 180 minutes. The bacteria
are boiled for 5 minutes in a buffer mixture (7 M urea,
0.1% SDS, 0.1 M sodium phosphate, pH 7.0) and samples are
applied to an SDS gel electrophoresis plate. After
analysis by gel electrophoresis, an additional band is
observed in the region from about 10 Rd, which corres-
ponds to the fusion protein expected. This band reacts
with antibodies directed against insulin in the "Western
Blot" experiment. If the cells are disintegrated under
1341211
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pressure and the debris is centrifuged off, the fusion
protein is found in the sediment in addition to other
insoluble cell constituents.
The induction conditions indicated apply to shaken
cultures; with larger fermentations the choice of other
media and conditions, for example in order to obtain
changed O.D. values, is expedient.
Example 4:
a) Preparation of mono-Arg insulin
40 g of the fusion protein concentrated by centrifugation
and washing with phosphate buffer ( pH 7 ) or water ( dry
substance content about 25% ) are dissolved in 75 ml of
98-100% strength phosphoric acid and 5 g of BrCN are
added. After reacting for 6 hours at room temperature,
2 1 of water are added to the mixture and it is freeze-
dried.
The fragment mixture ( 10 g) is dissolved in 1 1 of buffer
solution (8 M urea, 0.2 M tris-HC1 (pH 8.5)), warmed to
30°C and 10 g of sodium sulfite and 2.5 g of sodium
tetrathionate are added. After 90 minutes at 30°C, 3 1
of cold water are added and the pH is adjusted to 7 . 0 .
The resulting precipitate is centrifuged off. The hexa-
S-sulfonate of the mini-proinsulin is precipiated from
the supernatant by adjusting the pH to 3.5. The mixture
is centrifuged after incubating for 15 hours at +4°C.
The precipitate is washed with 200 ml of water and
freeze-dried. 4.8 g of a substance mixture in which a
mini-proinsulin content of 900 mg is determined by RP-
HPLC are obtained. The concentration of the S sulfonate
is carried out in two steps:
1. Anion exchange chromatrography through a 5 x 60 cm
column containing 'Fractogel TSR DEAF 650 M in 3 M
urea; 0.05 M tris-HC1 (pH 8.3). The elution is
13412 1 1
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performed using a gradient of 0.05-0.5 M NaCl (each
6 1). After analysis of the eluate by isoelectric
focusing, the product is precipitated from the com
bined fractions by diluting to 1 M urea and adjusting
the pH to 3.5.
2. Removal of high and low molecular weight impurities by
gel filtration through 'Sephacryl S200 in 3 M urea;
0.05 M tris-HC1; 0.05 M NaCl (pH 8.3). Analysis of
the fractions and isolation of the product are carried
out as in the preceding step. The precipitate is
washed with 20 ml of water and freeze-dried. 1.10 g
of product cocentrated to 69% purity are obtained.
For folding and disulfide bridge formation, the S sul-
fonate is dissolved in 50 ml of 8 M urea; 0.02 M tris-HC1
at pH 8.6. After addition of a few drops of octanol,
purified nitrogen is passed into the mixture for 15
minutes. Complete reduction is carried out in the course
of 1 hour at room temperature by addition of 1.1 ml (16
mMol) of 2-mercaptoethanol. The solution is applied to
a 'Sephadex G25 column ( 5 x 60 cm) and eluted using 0. 05 M
glycine/NaOH (pH 10.6). The protein fraction in 300 ml
of the glycine buffer is kept for 2 days at 4°C after
checking and, if necessary, correction of the pH value
(10.6). The solution is then adjusted to a pH of 6.8 and
the solution is incubated at room temperature for 4 hours
with 1 mg (3.5 U) of trypsin (Merck, treated with L-1-p-
tosylamino-2-phenylethylchloromethyl ketone (TPCR). The
pH is then adjusted to 3.5, 1 mg of Soya bean trypsin
inhibitor ( Sigma ) and 3 ml 10% ZnCl2 are added and the
solution is readjusted again to pH 6.8. The resulting
precipitate is separated by centrifugation. It contains
predominantly mono-Arg insulin which is purified by ion
exchange chromatography on S-Sepharose' (2.5 x 40 cm) in
a buffer composed of 50 mM lactic acid and 30% isoprop-
anol (pH 3.5). Elution is carried out by means of a
gradient of 0.05-0.50 M of NaCl (each 1 1). The elutate
is, analyzed by HPLC; the mono-Arg insulin is precipitated
13412 1 1
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from the product-containing fractions after 1:1 dilution
with HZO by adding 10 ml of 10% ZnCl2 per 1 1 and adjust-
ing the pH to 6.8. The precipitate separated by centri-
fugation is crystallized at pH 6 from a buffer composed
of 1 g/1 of phenol, 10.5 g/1 of citric acid and 200 mg/1
of ZnCl2. 390 mg of mono-Arg insulin in over 90% purity
are obtained after freeze-drying the crystals washed with
some water.
Example 5:
Preparation of insulin
200 mg of mono-Arg insulin (see Example 4) are dissolved
in 100 ml of 0.05 M tris-HC1 (pH 8.5). 1 U (about 4 gig)
of carboxypeptidase B is then added and the solution is
stirred slowly at room temperature. After 3 hours, the
human insulin is crystallized by acidifying to pH 3.5 and
adding 1 ml of 10% ZnClZ at pH 5.5. 200 mg of crystalline
insulin having a purity of more than 85% are obtained.
This material is sub jected to purification by ion exchan-
ge chromatography on a column containing Fractogel TSK
DEAE 650 M (2.5 x 40 cm) in 0.1% 'Lutensol ON 100 (BASF
AG; oxethylate of a linear saturated fatty alcohol of
essentially 12 carbon atoms); 0.05 M tris-HCl (pH 8.3),
the elution being carried out using a gradient of 0-0.4
M NaCl (each 1 1). The insulin is crystallized at pH 5.5
from the product-containing fractions identified by means
of HPLC after addition of 10 ml of 10% ZnCl2 and 1 ml of
10% citric acid. After slowly stirring overnight, the
mixture is centrifuged and the sediment obtained is
recrystalized at pH 5.5 from 20 ml of a buffer composed
of 5 g/1 of citric acid, 125 ml/1 of acetone and 200 mg/1
of ZnCl2. 160 mg of insulin having a purity of more than
95% are obtained.
1341211
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Example 6:
Preparation of insulin from mono-Arg insulin by combined
use of trypsin and carboxypeptidase B
mg of mono-Arg insulin are dissolved in 20 ml of 0.1 M
5 of tris-HC1 (pH 8.0) and the solution is warmed to 30°C.
At the same time, 2.5 ~1 of trypsin solution (1 U/ml) and
150 ~1 of carboxypeptidase B solution (1 U/ml) are added.
After 3 hours, the solution is adjusted to pH 3.5 and 2.5
~1 of trypsin inhibitor solution (1 U/ml) and 200 ~1 of
10% strength ZnCl2 solution are added. The human insulin
is precipitated by adjusting to pH 6.8, centrifuged off
and crystallized as in Example 5. The crystallized
insulin has a purity > 95%.
Example 7z Construction of a yeast expression vector
The DNA sequence (23) (Table 3) is first synthesized by
the phosphite method. This DNA sequence (23) encodes for
the amino acids 49 to 80 of the MFa precursor protein and
corresponds essentially to the natural DNA sequence.
The DNA sequence (23) is first used as a probe for the
isolation of the gene for the a factor and is labeled
with g2P for this purpose. With the aid of this probe,
the gene is isolated from a genomic agtll yeast gene bank
(as are meanwhile commercially available and obtainable,
for example, at Clontech Laboratories Inc., 4055 Fabian
Way, Palo Alto, CA94303). To this end, agtll phages
which carry the a factor gene are identified in a plaque
hybridization experiment. Phages from plaques identified
as positive are isolated, replicated and the DNA ob-
tained. This is cleaved using EcoRI and analyzed on a
0.8% strength agarose gel. The membrane is hybridized
against the 32P-labeled DNA sequence (23) by a "Southern
transfer" experiment. Phage DNA which contains a frag-
ment of about 1.75 kb (24) which is hybridized against
the DNA sequence ( 23 ) is again cleaved with the enzyme
134121'1
- 18 -
and the corresponding fragment (24) is isolated. The
vector pUC 19 is opened using EcoRI (25) and reacted with
the 1.75 kb fragment (24) using T4 ligase. The cloning
vector (26) is obtained.
The strain E. coli 79/02 is transformed using the liga-
tion mixture. White colonies are isolated, the plasmid
DNA is obtained from these and plasmids (26) which
contain the 1.75 kb EcoRI fragment, are identified.
The natural DNA sequence of the precursor protein for MFa
contains a PstI cleavage site in the region of amino
acids 8 to 10 and a TaqI cleavage site in the region of
amino acids 48/49. From the isolated plasmid DNA (26),
the fragment (27) which encodes for the amino acids 9 to
48 of the I~'a precursor sequence is isolated by reaction
with PstI and TaqI. The vector pUClB is opened using
PstI and RpnI and reacted with the PstI-TaqI fragment
( 27 ) and with the synthetic DNA sequence ( 23 ) with the
aid of T4 ligase. E. coli 79/02 is transformed using the
ligation mixture. The transformation mixture is plated
out on to IPTG-Xgal-Ap-plates. White colonies are
isolated and the plasmid DNA of this clone is charac-
terized by restriction analysis. The cloning vector (29)
which encodes for the amino acids 8 to 80 of the I~'a
precursor sequence is thus obtained.
The encoding sequence (30) mentioned is excised from the
cloning vector ( 29 ) by reaction with PstI and RpnI and
incorporated in the ligation described in the following.
For this purpose, the cloning vector (26) is reacted with
EcoRI and partially with PstI and the fragment (31)
including the encoding sequence for the first 8 amino
acids of the 1~'a precursor sequence is isolated. Fur-
thermore, the vector pUCl9 is opened using EcoRI and Rpnl
(32) and ligated with the two fragments (30) and (31)
described, the cloning vector (33) being formed. This
encodes for the total precursor sequence of l~'a up to
amino acid 80.
-19- 1341211
The cloning vector (33) is opened using RpnI and HindIII
and the large fragment (34) is isolated. This is ligated
using the RpnI-HindIII fragment (35) from the plasmid
(11) which encodes for the mini-proinsulin. The plasmid
pIR20 (36), the structure of which is confirmed by
restriction analysis, is thus obtained.
The plasmid Yepl3 (Broach et al., Gene 8 (1979) 121) is
opened using BamHI and the protruding ends are filled in
with Rlenow polymerase (38). The DNA is precipitated
using ethanol and treated with alkaline bovine phospha-
tase.
The fragment encoding for the insulin derivative and the
precusor sequence of MFa is excised from the cloning
vector (36) using HindIII and EcoRI and the protruding
ends are filled in as described (37).
The two truncated DNA sequences (37) and (38) are ligated
with one another, the plasmids pafB102 (39) and pafB104
(40) being formed. These two plasmids differ only in the
orientation of the inserted fragment.
As described in EP-A 0,171,024 (Published 12 February
1986), a terminator can be inserted behind the inserted
sequence (Figures 4 to 6 of EP-A 0,171,024). For this
purpose, the NcoI and/or the BamHI cleavage sites are
suitable.
After amplification of the plasmid DNA in E. cola MM294,
the plasmid pafB102 (39) is transformed in the leucine-
requiring yeast strain Y79 (a,trpl,leu2-1) (Cantrell et
al., Proc. Acad. Natl. Sci. USA 82 (1985) 6250) and DM6-6
(a/a leu2-3,112: sura3+/leu2: slys2+, trill-/trill-, his3-11,
15/his3-11, 15, ura3-/ura3-, lys2-/lys2~, arg4-17/arg4+,
adel/adel+) (Maya Hanna, Dept. Mol. Biol. Massachusetts
General Hospital, Boston, USA) by the lithium method of
Ito, H. et al., J. Bacteriol., 153 (1983) 163. Colonies
which can grow on selective medium without addition of
..
k. '',,~1,.,~. ~,
-20- 1341211
leucine, are isolated and combined. Yeast minimum medium
is inoculated with the individual colonies and incubated
at 28°C for 24 hours. The cells are centrifuged off and
the supernatant is tested for insulin activity in an RIA
test. The plasmid DNA is reisolated from yeast clones
whose supernatant shows insulin activity, and charac-
terized by restriction analysis. The transformed yeast
strains are employed for the following expression.
Example 8: Expression in yeast
10 ml of yeast complete medium is inoculated with cells
which have been removed from a fresh overnight culture of
a strain of selective medium obtained according to
Example 7, in such a way that an optical density
ODsoo = 0.1 is achieved. The culture is shaken at 28°C for
8 hours, after which 90 ml of fresh medium are added.
The culture is then shaken for a further 20 hours. The
cells are centrifuged off and the insulin concentration
is determined in the supernatant. The conditions are
modified for a larger fermentation, for example fresh
medium can be added continuously.
Example 9: Purification of mono-Arg insulin from yeast
supernatant
The fermentation supernatant is added through an adsorp-
tion column containing a porous adsorber resin composed
of a copolymer of styrene and divinylbenzene ('Diaion
HP 20). The column was previously equilibrated with a
20-50 mM acetate buffer (pH 5). After washing with tris
buffer (pH 8), an isopropanol gradient (0-50%) is applied
using a 10-fold column volume. Insulin-containing
fractions are adjusted to pH 6, ~MATREX CELI~UFINE AM
(Amicon) is added, and the mixture is stirred and fil-
tered off with suction and washed with 50 mM acetate
buffer (pH 6). The wash fraction and the main fraction
are combined, adjusted to pH 3.5 using lactic acid and
134121 1
- 21 -
added through an S-SEPHAROSE column which has been ..
equilibrated using 50 mM lactic acid (pH 5)/30% isopropa-
nol.
The elution is carried out by means of a 0-0.6 M NaCl
gradient. Mini-proinsulin elutes in the range 0.25-0.3
M.
The proinsulin-containing fractions are concentrated to
1/4 of the volume and added through a column containing
Biogel* P10 (Bio-Rad), equilibrated in 6% acetic acid
(pH 2). The insulin-containing eluate is lyophilised and
purified through a preparative "reversed phase" HPLC step
(RP18 material, 0.1% TFA, acetonitrile gradient 20-40%).
After subsequent freeze-drying, the lyophilisate is
dissolved in tris buffer (pH 6.8) and incubated for 3 to
5 hours at room temperature with 4 units of trypsin per
gramme of mono-Arg proinsulin. The course of the reac-
tion is checked by "reversed-phase" analysis. It shows
that mono-Arg insulin is formed nearly quantitatively.
At the end of the reaction, the pH is adjusted to 3.5 and
the reaction is completed by addition of an equivalent
amount of trypsin inhibitor. The zinc chloride con-
centration is then adjusted to 0.21 g/1 and the pH to
6.8. A flocculent precipitate is obtained which is
dissolved in lactic acid buffer. The components are
separated from one another by means of S-SEPHAROSE
chromatography. Fractions which contain mono-Arg insulin
are combined and mixed with water in the ratio lsl.
10 ml of 10% ZnCl2 per 1 1 are then added to the solution.
Mono-Arg insulin then precipitates at pH 6.8 and is
recrystallized in a known (for insulin) manner.
Example lOs Preparation of human insulin
The mono-Arg insulin prepared according to Example 9 is
used as a starting substance for the carboxypeptidase
B cleavage. To this end, the insulin derivative is
dissolved in tris buffer (pH 8.5) and 5 units of
* Denotes Trade-mark
1
134121 1
- 22 -
carboxypeptidase per gramme of mono-Arg insulin are
added. The reaction is carried out over the course of 3
hours with slow stirring at room temperature. The
product is then precipitated with ZnCl2 as described in
Example 9. Human insulin is then purified in a known
manner (DE-B 2,629,568).
13412 1 1
- 23 -
Table 1: Gene fragment IR I (2)
B1
Phe
20 30 40
~______________________________2___________________________
CT TTG GAC AAG AGA TTC GTT AAC CAA CAC TTG TGT GGT TCT CAC
CAT GGA AAC CTG TTC TCT AAG CAA TTG GTT GTG AAC ACA CCA AGA GTG
~_____________________________1_______________________________>
(KpnI) HpaI
50 60 70 80 90
__> c______________________________________________4________
TTG GTG GAA GCG TTG TAC TTG GTT TGT GGT GAG CGT GGT TTC TTC
AAC CAC CTT CGC AAC ATG AAC CAA ACA CCA CTC GCA CCA AAG AAG
~______________________________________________3____________
$30
Thr Arg Lys Gly Ser Leu
100 110 120
________________________________________>
TAC ACT CCA AAG ACG CGT AAG GGT TCT CTG CA
ATG TGA GGT TTC TGC GCA TTC CCA AGA G
__________________________________>
MluI (PstI)
1341211
- 24 -
Table 2: Gene fragment IK II (5)
A1
Gln Lys Arg Gly
130 140 150 160
____________________________________________________
G AAG CGT GGT ATC GTT GAA CAA TGT TGT ACT AGT ATC TGT TCT
AC GTC TTC GCA CCA TAG CAA CTT GTT ACA ACA TGA TCA TAG ACA AGA
~_____________________________________________________________5
(PstI) SpeI
A21
Asn
170 180 190 200 210
__________________________________________________________>
TTG TAC CAG CTG GAA AAC TAC TGT AAC TGA TAG TCG ACC CAT GGA
AAC ATG GTC GAC CTT TTG ATG ACA TTG ACT ATC AGC TGG GTA CCT TCG A
________________________________________________________________>
(HindIII)
r
134121 1
- 25 -
Table 3: DNA sequence (23)
50 55
5' C GAT GTT GCT GTT TTG CCA TTC TCC
3' TA CAA CGA CAA AAC GGT AAG AGG
(TaqI)
60 65
AAC AGT ACT AAT AAC GGT TTA TTG TTC
TTG TCA TGA TTA TTG CCA AAT AAC AAG
ATT AAT ACT ACT ATT GCT AGC ATT GCT
TAA TTA TGA TGA TAA CGA TCG TAA CGA
80
GCT AAA GAA GAA GGG GTA C 3'
CGA TTT CTT CTT CCC 5'
{KpnI)
