Note: Descriptions are shown in the official language in which they were submitted.
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HOECHST AKTIENGESELLSCHAET HOE 88/F 313 K Dr.KL/PP
Description
A process for the preparation of an insulin precursor in
Streptomycetes
A process for the preparation of fusion proteins has
already been proposed ~hich comprises C-terminal coupling
of the structural gene for the desired protein to the
optionally modified tendamistat gene, brinying about the
expression of this gene structure in a Streptomycetes
host cell, and isolating the secreted fusion protein from
the culture supernatant (DE 37 14 866 A1 or EP 0,289,936
A2). The older application additionally relates to gene
structures containing the optionally modified tendamistat
gene to which a structural gene for another protein is C-
terminally coupled, vectors which contain a gene ~truc-
ture of this type, Streptomycetes cells which contain a
vector of this type, and fusion proteins which have an N-
terminal portion of optionally modified tendamistat. The
older application contains examples of fusion proteins in
which the amino acid sequence of tendamistat is coupled
via a bridging member to that of monkey proinsulin.
In a further development of the concept of this invention
it has now been found that this process can be u~ed
particularly well to prepare a fu~ion protein in which
the tendamistat portion is followed by a shortened
proinsulin whose C chain comprises only one or two ly~ine
residues. These precursors can be converted particularly
straightforwardly and economically into human insulin.
Particular embodiments of the invention relate to advan-
tageous gene tructures for the amplification and expres-
sion of the gene which codes for the fusion ~rotein.
Further preferred embodiments of the invention are
explained hereinafter and defined in the patent claims.
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The gene structure employed according to the invention is
advantageously based on a synthetic gene which codes for
the shortened proinsulin derivative. It iB expedient in
the construction of this gene to take account of the
S specific codon usage of Streptomycetes. It has emerged
that the yield of fusion protein is thereby increased.
It is also advantageous to incorporate a terminator
~equence in the synthetic gene structure, because an
increase in the synthesis rate is also thereby achieved.
A great advantage of the process according to the inven-
tion comprises the possibility of detecting the fusion
proteins using the plate test which is described in EP-
Al 0,161,629 in Example 3 and in German Offenlegungs-
schrift 3,536,182. This considerably facilitates not only
the selection of the interesting clones but also the
working up because the effect of the different parameters
on the yield can easily be established.
The fusion proteins obtained according to the invention
are apparently present in a conformation which corres-
ponds, or at least approximates, to that of matureinsulin. This not only considerably facilitates the
further processing to insulin but, moreover, at fermenta-
tion times long enough to provide a good yield, surpris-
ingly there is no noticeable attack by the proteases
excreted into the fermentation medium.
The modification according to the invention of the
proinsulin molecule with its ~hortened C chain permits
straightforward proces~ing to human insulin, namely by
chemical cleavage with hydroxylamine and/or by enzymatic
cleavage using trypsin or, advantageously, lysyl endo-
proteinases. Enzymatic cleavage is preferred. Lysyl
endoproteinases carry out specific carboxyl-terminal
cleavage after the amino acid lysine. The favorable
arrangement of the A and the B chain in the fusion
protein according to the invention means that the action
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of the said enzymes results in an insulin pr~cursor inwhich, surprisingly, the disulfide bridge~ are correctly
linked.
It is expedient in the construction of the gene to
provide between the tendamistat portion and the start of
the proinsulin molecule a bridging member which permits
the proinsulin derivative to be cleaved off from the
tendamistat portion with the same enzyme used to cleave
the proinsulin derivative into the two insulin chainsO
Cleavage of ~he fusion proteins according to the inven-
tion with a lysyl endopeptidase result~ - depending on
the construction of the modified C chain - in de-B30-
insulin which can be transformed into human insulin by
transpeptidation, or B31-Lys-insulin or B3l-Lys-B32-Lys-
insulin, each of which can be transformed into humaninsulin, for example, by the use of carboxypeptidase B.
Particularly high yields of the de~ired protein are
obtained when gene constructions with a shortened ten-
damistat gene are employed. This embodiment of the
proce~s according to the invention has the great advan-
tage that the portion o the modified insulin in the
fusion protein comprises about one half, and thus con-
tains considerably less "ballast". The correct folding of
the fusion pxotein i8 not impaired by the shortaning of
the tendamistat portion, BO that the advantageous working
Up i8 therefore also possible with the smaller fusion
protein according to the invention. Nor is this advantage
achieved at the expense of an increased rate of degra-
dation by the proteases intrinsic to the host - in fact,
unexpectedly, it has emerged that the stability to these
protesses i~ increased.
The invention thu~ allows a whole series of advantageous
gene constructions which result in insulin precursors
which can easily be separated from the "balla~t portion"
of the fusion protein. This straiqhtforward working up
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additionally improves the yield of human insulin.
The separation of the fusion protein from the culture
medium, it6 further processing to the insulin precursor
and the transformation thereof into human insulin can be
carried out by methods known per se. Thus, the fusion
protein can advantageously be isolated by adsorption or
ion exchange chromatography and/or gel filtration, and
the proinsulin portion can be cleaved off chemically or,
advantageously, enzymatically. The construction of
appropriate bridging members is generally known and
described, for example, in EP-A2 0,229,998.
EP-B 0,089,007 discloses analogs of prepro- and pro-
insulin which carry at the C end of the prechain (or at
the N-terminus of proinsulin) Lys or Arg (which is also
preferred in the constructions according to the inven-
tion), whose B chain terminates with B2~-Lys and where the
C peptide can be shortened to Lys or Arg 80 that there-
fore B2~-Lys in the proinsulin structure is, in the
simple6t case, followed by only Lys or Arg, to which the
A chain is attached. These compounds are used a6 precur-
~ors for preparing insulins with the aid of trypsin or
trypsin-like endopeptidases and of an ester of a natural
amino acid which, where appropriate, carries protective
groups.
Insulin precursors in which the B and A chain are con-
nected by the bridging member -X-Y-, in which X snd Y are
identical or different and represent Lys and Arg, are
disclosed in EP-A 0,195,691. These insulin precursors are
expressed from yeast and then converted into human
insulin by enzymatic transformation. Insulin precursors
with a shortened C chain are al~o disclosed in EP-A
0,163,529. EP-B 0,132,769 and 0,132,770 describe insulin
derivatives and pharmaceutical agents containing them.
.
The invention is illustrated in more detail in the
examples which follow. Unless stated otherwise,
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percentage data relate to weight.
The figure illustrateæ the gene construction according to
the invention in Example 1. It is not true to ~cale.
Example 1
The synthetic gene (1) depicted in ~able 1 is chemically
synthesized in a manner known per se by the phosphoami-
dite method. In the codon selection account was taken of
the preference of Streptomycetes for G and C. As with the
gene coding for monkey proinsulin ~n the earlier application
(Table 2 therein), the gene (1) ~hown in Table 1 also has
at the S' end a protruding sequence typical for the
restriction enzyme EcoRI. The structural gene is followed
by two stop codons and a linker sequence with the recog-
nition site for the enzyme SalI. The protruding sequence
corresponding to the restriction enzyme HindIII is
located at the 3' end.
The commercially available plasmid pUC19 is cut with the
enzymes ~coRI and HindIII, and the synthetic gene (1)
shown in Ta~le 1 i8 ligated in. The result is the plasmid
pll (2). After amplification, the synthetic gene is cut
out a~ fragment (3) with the enzyme~ EcoRI and SalI and
employed for t:he construction describ~d hereinafter.
The plasmid pUC19 i8 completely digested with SmaI and
ligated with the terminator sequence (4) depicted in
Tabla 2. Pla~mids which contain this sequence in the
correct orientation are called pTl (5). This plasmid (5)
is opened with EcoRI, and the cleavage site is filled in
with DNA polymerase (Klenow fragment). The plasmid pT2
(6) is obtained by religation. This plasmid i~ opened
with the enzymes SalI and SphI, and the large fragment
( 7 ) i8 isolated.
The plasmid pK~400 (8) Icf. earlier application, Figure 4,
(20)) i~ cut with SphI and EcoRIt and the small fragment
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(9~ with the tendamistat gene is isolated.
Ligation of fragments (3), (7~ and (93 re~ult6 in the
plasmid pRK500 (10) in which the tendamistat ~equence is
followed by the bridging member
Phe Asn Al a Met Al a Thr Gl y Asn Ser Asn Gl y Lys
TTC AAT GCG ATG GCC ACC GGG ATT TC~ AAC GGC AAG
AAG TTA CGC TAC CGG TGG CCC TAA AGC TTG 1: CG TTC
EcoR I
coding for 12 amino acids, and then by the gene for the
proinsulin modified according to the invention. The
correct arrangement is checked by cutting with SphI and
SstI, resulting in a fragment of 833 bp from the plasmid
about 3.5 kb in size. The sequence i8 confirmed as
correct by DNA sequencing using the dideoxy method~
Gene constructions, according to the invention, in which
the Lys acting a8 C peptide is supplemented by another
Lys are prepared analogously. For thi~ purpose, the
triplet AAG coding for Lys is doubled. The plasmid pI2,
and therefrom the vector pKR600, are obtained analo-
gously.
Example 2
In analogy to the vector pGF1 provosed m ~e earlier
application, the expression plasmids pGF2 and pGF3 are
prepared from the vectors pKX500 and pKR600. For this
purpose, double digestion with SphI and SstI of each of
the vec~ors pRK500 and pRK600 is carried out to isolate
the insert of 823 and 826 bp respectively, and these DNA
fra~ments are ligated into the exprQssion plasmid pIJ702
cleaved with the same enzymes. The ligation mi~ture is
transfonmed into S. lividan~ TK 24, and the plasmid DNA
is isolated from thiostrepton resistant transformants
which show tendamistat activity (plate test). All the
positive clones contain the insert from p~500 or pRK600
employed.
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The expres~ion of the coded fu~ion protein can be carried
out in a known manner. If the transformed strain S.
lividans TK 24 is incubated in a shaken flask at 28~C for
four days and the mycelium is separated from the culture
solution by centrifugation~ the fusion protein can be
detected in the clear solution as follows:
20 to 200 ~1 of 15~ ~trength trichloroacetic acid are
added to 10 to 100 ~1 of solution, and the precipitated
protein is concentrated by centrifugation, washed and
taken up in SDS-containing sample buffer (U. Laemmli,
Nature 227 (1970~ 680-685). Incubation at 90~C for 2
minutes is followed by fractionation by electrophoresis
on a 10-17~ SDS polyacrylamide gel. A protein of molecu-
lar weight 15 kD is obtained, that is to say in the
molecular weight range expected for the fusion protein
composed of tendamistat and proin~ulin. The fu ion
protein reacts both with antibodies against tend~mistat
and wi~h antibodies against insulin.
~xample 3
The expres~ion vector pTF2 (DE 37 14 866 A1, E~ample 4)
i8 digested with the restriction enzymes EcoRI and SstI,
snd the fra~ent which encodes monkey proinsulin is
removed. The fragment 5.65 kbp in size i8 used for the
ligation reac1:ion described below.
These ~ame restriction enzymes are used to cut a DNA
fragment which i6 285 bp in size and which contains the
shortened proinsulin gene, as well as the termination
sequence, out of the plasmid pRK500 (Example 1~.
Ligation of the fragment 5.65 kbp in size from pTF2 with
the fragment 285 bp in size from pKK500 yields the
expres~ion plasmid pTF3.
Transformation of protoplasts of Streptomyces lividans
TK24 with the ligation mixture results in clones which
are thiostrepton-resistant and secrete 8 fusion protein
which reacts with antibodies against proinsulin. This
fusion protein comprises the first 41 amino acids of
tendamistat, the bridging member
Pro-Ser-Leu-Asn-Ser-Asn-Gly-Lys
and the shortened proinsulin.
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Table 1
Bl 10
ASN SER ASN GLY LYS PHE VAL ASN GLN HIS LEU CYS GLY SER HIS
AAT TCG AAC GGC AAG TTC GTC AAC CAG CAC CTG TGC GGC TCG CAC
GC TTG CCG TTC AAG CAG TTG GTC GTG GAC ACG CCG AGC GTG
(EcoRI)
LEU VAL GLU ALA LEU TYR LEU VAL CYS GLY GLU ARG GLY PHE PHE
CTC GTG GAG GCC CTC TAC CTG GTG TGC GGG GAG CGC GGC TTC TTC
GAG CAC CTC CGG GAG ATG GAC CAC ACG CCC CTC GCG CCG AAG AAG
C Al 40
TYR THR PR0 LYS THR LYS GLY ILE VAL GLU GLN CYS CYS THR SER
TAC ACC CCC AAG ACC AAG GGC ATC GTG GAG CAG TGC TGT ACG TCC
ATG TGG GGG TTC T5G TTC CCG TAG CAC CTC GTC ACG ACA TGC AGG
ILE CYS SER LEU TYR GLN LEU GLU ASN TYR CYS ASN STP STP
ATC TGC TCC CTC TAC CAG CTC GAG AAC TAC TGC AAC TAG TAA
TAG ACG AGG GAG ATG GTC GAG CTC TTG ATG ACG TTG ATC ATT
CTC GAC CTG CAG CCA
CAG CTG GAC GTC GGT TCG A
SalI (HlndIII)
Table 2
: 5'-CGATAAACCGATACAATTAAAGGCTCCr~GAGCClllTrrl~GACATTTTCAACGTGGATC
GCTATTTGGCTATGTTAATTTCCGAGGAAAACCTCGGAAAAAAAAACCTCTAAAAGTTGCACCTAG-5'
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