Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1~3~340
The invention relates to a process for the pro-
puritan of proteins by co-cloning DNA sequences for the
desired protein Thea those for two particular other pep-
tides by means of genetic engineering methods, a combined
protein of the three constituents being expressed. Thus
combined protein is then absorbed biospec;fically onto a
carrier-bound, biospecifically complementary substance
and, after removal of the remaining constituents, is split
such that only a fez fragments are present, one of which
us the desired protein.
on the preparation of proteins by genetic engineer-
in, it is always a problem to isolate the expressed pro-
tern on the pure form from a multi-component mixture.
The invention thus provides a conceptually new
process for the preparation and isolation of a protein
by genetic engineering.
This new process is characterized on that
a) a DNA sequence uh;ch codes for this protein, a
DNA sequence which codes for a short-cha;n peptize and a DNA
sequence which codes for a biospecific palpated are linked
with one another such that the DNA sequence for the short-
chain peptize is between the other two DNA sequences;
b) the new DNA fragment thus obtained us intro-
duped into the DNA of a host organism by means of suit-
able genetic engineering methods such that transcription translation and expression of a combined protein kinesis-
tin of the biospecific palpated, the short-chain pep-
tide and the desired protein can take place;
c) the host organism us cultured such that it pro-
dupes the combined protein;
d) the part of the culture system containing the
combined protein is brought into contact, if appropriate
after removal of other constituents, with an imnob;l;sed
system consisting of a suitable carrier material, to
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which a substance is bound which it complementary to the
biospecific palpated and strongly binds to this
palpated under the given reaction conditions;
e) the immobilized system to which the combined
protein has become bound is separated from the other con-
stituents in a suitable manner and treated with an agent
which specifically splits off the short-chain putted
from the bound combined protein and
f) the desired protein liberated in this manner
is separated from the other constituents and isolated,
in a suitable manner.
The process of the invention thus comprises the
measure of alloying the desired protein to be expressed
not in isolated form but in association with two other
elements, the biospecific palpated and the short-chain
peptize.
In this concept, the short-chain peptize located
between the desired protein and the biospecific poLypep-
tide represents the intentional cutting site. The
short-chain peptize must therefore be such that it can be
excised in a highly specific manner in a Later step of
the process. This excision is preferably effected
Vito an enzyme. The choice of the short-chain peptize
thus depends on ~hethsr an agent, for example an enzyme,
is available which is capable of recognizing and splitting
the amino acid sequence of the short-cha;n peptize. Ho-
ever, it is also sufficient merely to split the bond bet-
teen the short-chain peptize and the desired protein on
one side.
An example of such a highly specific enzyme is
the enzyme kallikrein EKE. 3.4.21.8.~ obtained from the
pancreas. This enzyme splits off the sequence of the decay
peptize Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg from the
peptize chain of kininogen. This decapeptide is prefer-
ably co-cloned according to the invention.
The biospecific palpated also co-cloned
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according to the invention has the task of binding to a
carri~r-bound, biospecif;cally complementary substance,
so that the carrier can be separated from the other con-
stituents of the medium, together with the combined pro-
loin bound thereto. It follows that the system of boo-
specific polypeptide/complementary substance is to be
subject to the requirement that strong binding between
them takes place under the given reaction conditions.
Biologically active polypeptides, for example
enzymes, can, with biologically active complementary sub-
stances, for example with inhibitors, form biospecific come
plexus, for example enzyme-;nhibitor complexes.
Such biospecific enzyme-inhibitor complexes
have a high binding affinity. Their dissociation con-
slant is accordingly lo.
An example of an enzyme-inhibitor complex is the
tryps;n-aprot;nin complex preferred according to the
invention, which is formed from the enzyme trypsin and
the enzyme inhibitor appertain The dissociation constant
Kit of this complex is only 6 x 10 14 molt pi - 8.0,
t = 25), and the rate of complexion has t 1/2 = 6.3
seconds. The trypsin-aprotinin complex is accordingly
formed very rapidly and has a very high stability under
the given conditions. However, the stability of the
trypsin-aprotinin complex depends on the pi value of the
solution and decreases as the pi value falls. The
trypsin-aprotin;n complex is almost completely dissociated
at 2 pi value of about 2Ø
such Bavaria is particularly desirable in the
context of the invention, since regeneration of the
carr;er-bound complementary substance us easily possible
as a result. This substance can then be reused in
the process. It is furthermore desirable for the rate of
formation of the bond between the biospecific palpated
and the carrier-bound complementary substance to be high.
Possible competing reactions of the carrier-bound
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substance with other sites of the combined palpated
can in this way be reduced
The enzyme/inhibitor system described is merely
an example. Antigen/antibody or peptize hormone/
receptor systems, for example, can likewise be used.
The biospecific absorption is preferably effected
using uater-insoluble carriers, to which the biospecifi-
calmly complementary substance is bound. Thus binding can
be effected by the known methods of immobilization, with
or without spacer molecules.
The DNA sequence of the three components mentioned
are co-cLoned by methods of genetic engineering and the
DNA thus obtained us transferred unto the DNA of a host
organism, for example unto a plasm by methods of gene-
tic engineering. Suitable microorganisms, for examplebacter;a, in particular E. golf, or sac. subtilis, or
yeasts, can be used as the host organism. The transcript
lion, translation and, finally, expression of the co-
cloned bra take place during culture of the microorganism.
If the fermentation solution contains the desired protein
with the two co-cloned peptizes, the cells are separated
off, for example by centrifugation or by cross-flou lit-
traction, and the culture filtrate us used for the Swahili-
lion. If the cell itself contains the desired protein,
with the two co-cLoned peptizes, the cells are separated
off and ruptured and the cell fragments are removed
by separation. The culture filtrate or the cell extract
contains not only the desired co-cloned combined protein,
but moreover a large number of other proteins, a large
3û number of other products and also adhering constituents
of the nutrient medium. The desired combined protein is
no isolated from thus solution of complex composition
and is purified. This is effected by adding the carrier-
bound biospecifically complementary substance, for example
the immobilized trypsin, to the culture filtrate or
extract. A uater-insoluble complex of the fulling
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five components forms immediately with a high rate
of completing: carrier + biospecifically complement
try substance i biospecific palpated short-chain
peptize desired protein, for example the complex of the
five components carrier trypsin aprotinin, decapep-
tide I desired protein. The carrier-bound biospec;fically
complementary substance can be added at room temperature,
but also, Thea the high rate of complex;ng, at lo them-
portrays, for example at 12C. The uater-insoluble
complex can be separated off from other proteins in the
fermentation solution and also from all the other impure-
ties by simple filtration.
f a proteolytic enzyme, for example trypsin, is
used as the biospecifically complementary substance, this
can be saturated with a lo molecular weight inhibitor
before the addition to the fermentation solution. How-
ever, the dissociation constant of the lo molecular
weight inhibitor must be greater than that of the boo-
specific palpated. because of the different dissocia-
lion constants, the lo molecular weight inhibitor is displaced from the biospecific palpated on addition of
the fermentation solution. Examples of Lou molecular
weight inhibitors which can be used for the carrier-bound
tryps;n are benzam;dine, ugh a dissociation constant of
18 x 10 6 molehill, or phenylguanidine with a dissociation
constant of 72 x 10-6.
Alternatively, a column can be filled with the
carr;er-bound, b;ospec;fically complementary substance.
The culture filtrate or cell extract is then passed
through the column. In this case also, exclusively the
desired combined protein is bound to the solid carrier
via the biospec;fic palpated, whilst all the other pro-
terns and all the other ;mpur;t;es run through the column.
The biospec;f;cally complementary palpated bound to
the carrier should as far as possible be completely sat-
rated with the complex to be bound. The column can be
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freed from residual adhering impurities by washing ugh
buffer solutions or by washing with other suitable soul-
lions. with a high rate of completing, the column can
also be charged at lo temperatures.
Finally, the combined protein bound to the carrier
us cleaved by the action of a specific enzyme, the specie
lie enzyme cleaving exclusively the peptize bonds of the
short-cha;n peptize.
If the total complex is built up from the come
1û pennants carrier + trypsin aprotinin + decapeptide +
desired protein, the enzyme kaLlikrein can be used, as
mentioned, as the specific enzyme. Kallikrein hydrolyses
the decapep~ide on both sides and detaches the terminally
bound desired protein from the combined protein.
A cleavage at one side of the linkage between the short
chain peptize and the desired protein is sufficient. Such
a short chain peptize possibly contains the last three
C-terminal amino acids from the above mentioned decapeptide
with the sequence Pro-Phe-Arg. Thus kallikrein splits high
specifically the desired linkage between Argo in the
hexapeptide
Pro-Phe-Arg-Se~-Tyr-Gln KM = 0,25 my
or Pro-Phe-Arg-Ala-Asn-Leu KM = û,15 my.
The desired protein is eluded together with the decapeptide
and can be separated from the decapeptide by known methods,
for example by molecular sieve chromatography. The
desired protein is thus obtained in a pure form. The
advantage in choosing a short-chain peptize as the intent
tonal cutting site in the combined protein is that,
after cleavage, the desired protein can easily be removed,
as a rule in the form of a large molecule, from the small
short-chain peptize.
The biospecific palpated remains bound to the
carrier via the complementary substance under the elusion
conditions. Only by changing, for example, the pi value
of a buffer solution, for example to a value of 2-3, is
the biospecific palpated detached and washed out of
the column. After further washing operations, the column
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can then be brought to a suitable pi value, to pi 8.0 of
tryps;n/aprot;n;n is used, with buffer solutions and can
be reused for another experiment.
As proteins Shea can be prepared by the process
according to the invention there may be mentioned, inter
alga, palpated sequences of hormones, far example of
human somatrop;n, of erythropoitin, of corticotropn, of
pre-proinsul;n, of pre-minipro;nsulin and of the A and B
chain of insulin, palpated sequences of neutrotrans-
millers, for example of substance P or of endomorphic
palpated sequences of interferon, of IN INF-~gor
IN of hybrid ;nterferons and of in~erleukins, polyp
peptize sequences of plasma proteins, for example of
human ~1-antitryps;r,, of albumin and of the isolation
factors, fur example of Sue plasminogen activator and
of factor VOW polypentide sequences of enzymes, for
example of D-xylose isomers, of penicillin azaleas of
urok;nase and of reunion, and palpated sequences of
antibodies for example of monoclonal antibodies and of
vaccines.
Figure 1 illustrates the process according Jo the
invention in diagrammatic form.
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