Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1 329 1 57
The invention relates to a method for activating gene
technologiGally produced eucaryotic proteins, containing
disulphide bridges, aFter expression into procaryotes.
When heterologous proteins are expressed in pro-
caryotes, these proteins often form, in the host cells, inactive
poorly soluble aggregates (so called "refractile bodies") which
are also conta~ninated with proteins from the host cells. It is
assumed that the formation of such "refractile bodies" is a
result of the high protein concentration in the cell arising
during expression. It is known that the formation of large
quantities of enzymes in the cell is Followed by aggregation of
the enzymes into insoluble, high-molecular, mainly inactive
particles. Before such proteins can be used, for therapeutical
purposes for example, they must therefore be purified and con-
verted into their active form.
According to known methods, activation of such pro-
teins, present as aggregates, may be carried out in several
steps (cf., for example, B.R. JAENICKE, FEBS Federation of
European Biochemical Societies, Vol. 52 (1979) 187 to 198; R.
RUDOL.PH, Biochemistry 18 (1979) 5572 to 5575).
In the First step, solubilizing is achieved by the
addition of strong denaturing agents, for example, guanidine
hydrochloride or urea in high concentration, or by the addition
of highly acid agents, for example, glycine/phosphoric acid
mixtures. Reducing SH reagents, for example, dithioerythritol,
DTE and EDTA, have been found satisfactory as additional aids,
for example in the renaturing of LDH. If the protein is
.
'
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- 2 i 1 32q 1 57
contaminated by proteins from the host cell, the next step is
purification by conventional methods known per se, for example,
gel- or ion-exchange chromatography. This is followed by con-
siderable dilution in order to reduce the concentration of the
denaturing agent. If guanidine hydrochloride is used, dilution
is carried out to values of less than 0.5 mol/l. In the case of
enzymes with free SH groups, it was found advantageous to add
agents which protect the SH groups (cf. e.g. R. JAENICKE,
Journal Polymer Science, Part C 16 (1967) 2143 to 2160).
European Published Patent Specification 0114506
describes methods for isolating, purifying and activating certain
heterologous products expressed from cultures of bacteria; for
purposes of activation, the solutions of "refractile bodies" in
a strong denaturing agent a) is transferred direc-tly in a solution
in a weaker denaturing agent which is then subjected to oxidizing
conditions for the reformation of dilsulphide bridges, b) the
protein is sul~honated, is then transferred in a solution in a
weak denaturing agent, and the S-sulphonate groups are converted,
by treatment with a sulfhydryl reagent, into their reduced and
oxidized form; or c) the solution is treated, in a weak de-
naturing agent, directly with the sulfhydryl reagent, for example,with reauced glutathione/glutathione disulphide (GSH/GSSG). A typical
example of this, in which the above mentioned problems arise, is t-PA.
The main constituent o-F the protein matrix of con-
gealed blood is polymeric fibrin. This protein matrix is dis-
solved by plasmin which is formed from plasminogen by activation
with so called plasminogen activators, for example, by t-PA
,
, ~ . . . .
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1 32q 1 57
("tissue-type plasminogen activator"). The enzymatic activity
of natural t-PA, or of t-PA obtained gene technologically from
eucaryotes (the catalytic activation of plasminogen to plasmin) -
is very sl,ght in the absence of fibrin or fibrin-fission pro-
ducts (FFP), but can be substantially increased in the presence
of these stimulators (by more than a factor of 10). This so
called ability to stimulate activity is a decided advantage of
t-PA over other known plasminogen activators such as urokinase
or streptokinase (c.f. e.g. M. HOYLAERTS et al, J.Biol. Chem. 257
(1982) 2912 to 2919; NIEUWENHIUZEN et al, Biochenlica et Bio-
physica Acta 755 (1983) 531 to 533). The stimulatability
factor with Br~N fission products therefore varies in the rele-
vant literaturej up to the number 35.
A t-PA-like, non-glycosylated product is also formed
in genetically manipulated procar~otes (after the introduction
of c-DNA); however, a product of this kind does not have the
activity stimulating effect of a t-PA from eucaryotes. ~his
may conceivably be attributable to the fact that the redox condi-
tions in the procaryotic cell differ from the eucaryotic cell,
from which the gene originates, in such a manner that an inactive
product is formed right from the start. This, in turn, may be
attributable to the fact that numerous SS bridges containing the
naturally active molecule are linked erroneously or are not even
formed. However, for therapeutic use of t-PA, not only is
enzymatic activity necessary, but also stimulatability. Refer-
ence is made in The EMBO Journal 4, No. 3 (1985) 775 to 780, in
connection with other substances, to the fact that the pro-
."
1 329 1 57
caryotic cell presumably does not provide the correct conditionsto produce eucaryotic protein activity in the correct rnanner.
According to European Published Patent Specification
0093639, in order to activate t-PA, the cell pellets obtained
from E. coli are suspended in 6 mol/l of guanidine hydrochloride,
are treated with ultrasonics" incubated, and then dialyzed for
four hours against a solution of tris/HC1 (pH = 8.0), sodium
chloride, EDTA and Tween* 80. This is followed by centrifuging,
the plasminogen activator activity being found in the remainder.
Thus, although natured t-PA is proteolytically active, it shows
no measurable stimulatability by BrCN fission products (BrCN-FFP)
of fibrin, according to the method described in J.H. VERHEIJEN,
Thromb. Haemostas., 48. (3), 260-269 (1982).
No generally applicable method for activating denatured
proteins is known from the state of the art; this applies in
particular to t-PA because the nat-ive protein has a highly
complex structure, it contains a free thiol group and 17 S-S
bridges which, in theory, may be linked together in 2.2 x 102
different ways, although only one structure corresponds to the
native condition. Although methods known from the state of the
art for activating t-PA lead to a proteolytically active t-PA,
this shows no measurable stimulatability; an activating method
leading to stimulatable t-PA is not known.
It is therefore the purpose of the present invention
to provide a method for complete activation of gene technologi-
cally produced, heterologous, eucaryotic . proteins, containing
disulphide bridges, after expression in procaryotes.
*trade mark
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i 1 3~9 1 57
According to the invention there is provided a method
for activating gene technologically produced heterologous,
eucaryotic proteins, containing disulphide bridges, after
expression into procaryotes, by cell decomposition, solutilizing
under denaturing and reducing conditions, and activating or
renaturing under oxidizing conditions in the presence of
GSH/GSSG, characterized in that one operates, in the activating
stage, at a pH value of 9 to 12, preferably 9.5 to 11, a GSH
concentration of 0.1 to 20, preferably 0.2 to 10 mmol/1, a GSSG
concentration of 0.01 to 3, preferably 0.05 to 1 mmol/1, and
with a non-denaturing concentration of the denaturing agent.
In a particular embodiment the activating is carried
out without prior separation of denaturing agent and reducing
agent employed in the solubilizing; in such case the reaction
mixture after the solubilizing is diluted with an activating
buffer and the GSSG concentration exceeds the residual concentra-
tion of reducing agent, for example DTE, in the subsequent
activating.
The denaturing agent used can usually be a denaturing
agent generally used for activating under oxidizing conditions,
or arginine; of known denaturing agents, preference is given to
guanidine hydrochloride, urea or derivatives thereof. Arginine
has also been found suitable. Mixtures of these denaturing
agents may also be used. The arginine and guanidine hydro-
chloride may be employed in an amount of 0.1 to 1.0, prefer-
ably 0.25 to 0.8 mol/l. This activating stage is also prefer-
ably carried out in the presence of an extraneous protein; as
~1 ~9
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-- 6 --
a rule, any extraneous protein is suitable as long as it is not
proteolytically active; in this connection preference is given
to bovine serum albumin (BSA), in an amount of 1 to 3 mg/ml.
The addition of BSA produces a slight increase in yield and
stabilization of the protein (probably by protection of sur-
face denaturing and/or proteolytic decomposition).
The remaining conditions of the method may correspond
to the usual conditions known from the state of the art for re-
activating stages. The activating (incubating) period is pre-
ferably between 20 and 48 hours at room temperature. The halfvalue period of activation, in the presence of 0.5 mmol/l of
' reduced (GSH) and oxidized (GSSG) glutathione, is about lO to 15
hours at 20C. With a longer incubating period t48 hours),
under re-oxidizing conditions, stimulatability with CNBr-FFP
usually decreases. The activating stage is preferably carried
out in the presence of EDTA, the most desirable concentration
being about 1 mmolll of EDTA.
The steps of the method (re-oxidizing/activating)
preceding and following the activating stage, for example, cell --~
decomposition, solubilizing (solubilizing/reducing), and
possibly one or more of the purifying operations, preceding and/
or following the activating stage, may be carried out according
to the conventional methods known from the state of the art, for
example European Published Patent Specifications 0114506 and
0093619; however, if optimal yield and activation are to be
obtained, it may be desirable to carry out some or all steps of
the method in accordance with one or more of the procedures
,,1 ~
7 i 1 329 1 ~7
~ explained herein. For instance, it is also possible to carry,j out the activating stage according to the invention, in the
'J, mixture obtained after the decomposition, without prior de-
naturing and/or reducing, but with a lower yield. Expression
..
is carried outinto procaryotes, preferably into P. putida,
~, more particularly into E. coli. However, the method according
to the invention is just as suitable if the expression is into
other procaryotes (e.g. bacilli).
. . .
` Cell decomposition may be carried out by conventional
~ 10 methods, for example, ultrasonics, high pressure dispersion, or
-~ lysozyme; it is preferably carried out in a suspension medium
:;
in the form of a buffer solution adapted to establish a neutral
,~ to slighly acidic p~l value, for example in 0.1 mol/l oF tris/HC1.
', After decomposition of the cell, the insoluble constituents
' ("refractile bodies") are separated in some suitable manner,preferably by centrifuging at a high g-number and over a long
~ period, or by filtration. After washing with agents which doa~ not affect t-PA but which dissolve foreign cell proteins as much
as possible, for example, water, a phosphate buffer solution,
possibly with the addition of a mild detergent, for example
Triton (trade mark), the precipitate (pellet) is subjected to
solubilizing (solubilizing/reducing). Solubilizing is ~re-
ferably carried out in the alkaline pH range, more particularly
at a pH of ~.6 + 0.4 and in the presence of a reducing agent
from the mercapto group and a denaturing agent.
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Conventional state of the art denaturing agents may be
used for solubilizakion, for example those mentioned in European
Published Patent Specification 0114506, more particularly
guanidine hydrochloride or urea. The concentration of guanidine
hydrochloride is about 6 mol/l, that of urea about 8 mol/l.
Compounds of the general formula I may also be used:
R2-C0-NRR1 (I)
in which R and R1 are the same or different and are selected
from H and alkyl of 1 to 4 carbon atoms, and R2 is H, alkyl of
1 to 3 carbon atoms or NHR, where R1 is as defined above. Suit-
ably the compound (I) is employed in an amount of 0.5 to 4, pre-
ferably 1 to 3.5 mol/l.
Reduced glutathione (GSH) or 2-mercaptoethanol, for
example, may be used as mercaptan group reducing agents, in a
concentration of about 50 to 400 mmol/l o~ dithiothreitol and/or,
in particular, DTE (dithioerythritol) or D~T (dithiothreitol),
for example in a concentration of about ~0 to 400 mmol/l.
Solubilizing is preferably carried out at room temperature,
~ - with an incubating period of one to several hours, preferably
1 20 two hours. In order to prevent oxidizing of the reducing agent
by atmospheric oxygen, it may be desirable to add EDTA. In
addition to solubilizing/reducing, the solubilizing stage also
has a purifying effect, since a large part of the material
3 which does not cross react immunologically with t-PA (extraneous
proteins) does not go into solution.
~ Known conventional purifying stages may be insertedi after solubilizing and before the activating stage; purifying
ls
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1 32q 1 57
. g
.,
methods used are, for example, steric exclusion chromatography
(SEC), in the presence oF guanidine chloride or urea; or ion-
exchangers, in the presence of urea or derivatives thereof; non-
specific re-oxidizing may be prevented by the addition of a
reducing agent (for example 2-mercaptoethanol) or by pH values <
.5 (cf. e.g. RUDOLPH, Biochem. Soc. Transactions 13 (1985) 308
to 311). If DTE was used in the preceding solubilzing stage,
this must be separated in a purifying stage. Purifying may be
carried out, for example, by SEC with Sephadex G 100 (trade mark)
in the presence of guanidine hydrochloride and a reducing agent,
for example GSH at a pH of between 1 and 4 (a large amount of
extraneous protein may be separated in this step); or by separat-
ing the denaturing/reducing agent by desalination with Sephadex
G 25 (trade mark) in 0.01 mol/l of HC1 or 0.1 mol/l oF acetic
acid. Alternatively, separation of the denaturing/reducing agent
may be carried out by dialysis against similar solutions.
The reactivating stage may be followed by a further
purifying step; such purification is generally carried out by
dialysis, or by subsequent isolating of the activated t-PA, for
example by affinity chromatography, for example with lys-
Sepharose.
Qnother embodiment of the invention is based upon the
formation of mixed disulphides of gene technologically produced,
heterologous eucaryotic proteins, containing disulphide bridges,
and glutathione (hereinafter abbreviated to t-PASSG). This can
facilitate both the separation of extraneous proteins in the
denatured condition and further purification of the negative
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protein. Purification after modification of the thiol groups
has the advantage that the protein is protected against atmos-
pheric oxygen and is therefore stable over a wider pH range, a
change in the net charge facilitating the purification. More
particularly, separation from unmodified protein may be carried
out advantageously by ion-exchange treatment.
In order to form the mixed disulphides, dialyzed,
reduced protein, freed from denaturing and reducing proteins,
was incubated with a dilute, for example 0.2 M, solution of
GSSG containing a denaturing agent. Activation was effected
after separating the denaturing and oxidizing agents at a pH
value of 7 to 10.5 of a GSH concentration of 0.5 to 5 mmol/l
and with a non-denaturing concentration of the denaturing agent.
In all other steps of the reaction, activation of the
protein by formation of mixed disulphides with G5SG corresponds
to those in the activation of the previously mentioned part of
the invention. In this embodiment, the pH optimum is 8.5, the
yield is about doubled, and the activated protein is stable in
the renaturing buffer for a longer period of time.
According to the invention, it is possible to activate
t-PA from procaryotes in such a manner that not only is activa-
tion of the normal biological activity achieved, but stimula-
tability~ in the sense defined hereinbefore, is also achieved.
This stimulatability exceeds by far that of the native t-PA and
,~
1 is greater than a factor of 10. It may even exceed a factor of
.i
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Another eucaryotic protein, which can be activated
according to the invention after expression in procaryotes,
is ~-interferon.
The following examples illustrate the invention in
greater detall, but do not restrict it. Unless otherwise
indicated, percentages are percentages by weight and tempera-
tures are in degrees Celsius.
EXAMPLE 1
a) Preparation of "refractile bodies".
100 g of E. coli moist cell mass, taken up in 1.5 1,
0.1 mol/l of tris/HCl (pH 6.5) and 20 mmol/l of EDTA, were homo-
genized (Ultra-Turrax (trade mark), 10 sec.) and 0.25 mg/ml of
lysozyme were added. After 30 min. of incubation at room
temperature, the mass was again homogenized and then cooled to
3C. Cell decomposition was effected by high pressure disper-
sion (550 kg/cm2). This was followed by rinsing with 300 ml of
0.1 mol/l tris/HCl and 20 mmol/l of EDTA. After centrifuging
(SorYail (trade mark) GSA, 2 hours at 13.000 r.p.m., 27.000 g,
4C), the pellet was taken up in 1.3 1, 0.1 mol/l of tris/HCl
(pH 6.5j, 20 mmol/l of EDTA and 0.5% of Triton-x-lO0 and was
homogenized. After renewed centrifuging (Sorvall*GSA, 30 min.
at 13.000 r.p.m., 27.000 g, 4C), the pellet was taken up in
1.3 1, 0.1 mol/l tris/HCl (pH 6.5), 20 mmol/l of EDTA and 0.5%
of Triton-x-100 and homogenized. Alternate centrifuging (Sorvall*
GSA, 30 min. at 13.000 r.p.m., 27.000 g, 4C) and homogenizing of
the pellet in 1 1, 0.1 mol/l tris/HCl (pH 6.5) and 20 mmol/l of
EDTA, was repeated three more times.
*trade mark
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- 12 ' l 329 1 57
The t-PA content of the "refractile bodies" preparation
was quantified by SDS-PAGE, identification of the t-PA bands by
"Western blotting" and densitometrical analysis. By SDS-PAGE and
- "Western blotting", the "refractile bodies" show heavy t-PA bands
with a molecular weight of about 60 kDa. The t-PA proportion of
the total protein content of the "refractile bodies" amounts to
about 21%.
b) Solubilizing/reducing of "refractile bodies".
, "Refractile bodies" with a protein concentration of 1
to 5 mg/ml were incubated in 0.1 mol/l of tris/HC1 (pH 8.6),
6 mol/l of guanidine hydrochloride, 0.15 to 0.4 mol/l of DTE and
1 mmol/l of EDTA, for 2 to 3 h at room temperature. ThereaFter,
the insoluble material (cell wall fragments, etc.) was centri-
fuged out (e.g. Sorvall*SS 34, 30 min. at 15.000 to 20.000 r.p.m.,
35.000 to 50.000 g, 4C~. The pH value oF the remainder was
adjusted to a pH of 3 with conc. HC1. Denaturing and reducing
agents were then separated by dialysis against 0.01 mol/l of
! HC1 at 4 C.
c) Re-oxidizing/activating.
, 20 Re-oxidizing/activating was effected by 1:50 to 1:200
dilution in 0.1 mol/1 of tris/HC1 (pH 10.5), 1 mmol/l of EDTA,
~ 1 mg/ml of BSA, 0.5 mol/l of L-arginine, 2 mmol/l of GSH, 0.2
.j !
j mmol/l of GSSG. After 17 to 24 h of activation at about 20C,
;~ the activity was determined and also the yield, in comparison
, with the activity of native glycosilated t-PA from eucaryontes.
. Yield in relation to total protein content of
:!, "refractile bodies": 2.5 +/- 0.5%
*trade mark
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1 329 1 57
- 13 -
~ Stimulatability: 10 +/- 5
'~ Yield in relation to t-PA proportion of
- "refractile bodies": about 12%
d) Re-oxidizing/activating without separation of
denaturing/reducing agents.
"Refractile bodies" were incubated, at a protein con-
centration of 1.25 mg/ml, in 0.1 mol/l of tris HC1 (pH 8.6),
6 mol/l of guanidine hydrochloride, 0.2 mol/l of DTE and 1 mmol/l
of EDTA, for 2 h at room temperature. Immediately thereafter
re-oxidizing was effected by 1:100 dilution in 0.1 mol/l of
tris/HC1 (pH 10.5), 1 mmol/l of EDTA, 1 mg/ml of BSA, 0.3 mol/l
of L-arginine, and the amounts of GSSG indicated in Table I
below. Furthermore there was a residual concentration in the
activating charge of 0.06 mol/l of guanidine hydrochloride and
, 2 mmol/l of DTE.
;~ Table I
Relationship between activating yield and GSSG concentration
upon activating without separation of denaturing/reducing agents.
`~ GSSG Yield' Stimulatability
20 (mmol/l) %(Factor)
~ 0.2 0
-~ 1 0.134.0
1.491.4
6 1.285.4
~, 7 1.045.8
i 9 0.985.2
1.77 10.0 -~
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- 14 -
'= yield of active t-PA in relation to total
protein content of the "refractile bodies".
EXAMPLE 2
An RB ("refractile bodies") preparation (450 OD550/ml)
was incubated, in 1 ml 0.1 mol/l of tris/HC1 (pH 8.6), 6 mol/l
of guanidine hydrochloride and 0 15 - 0.2 mol/l of DTE, for 2
to 3 h at room temperature. Insoluble material (cell wall
fragments, etc.) was thereafter separated by centrifugi~g
(20 min. at 17.000 r.p.m.). Renaturing and reducing agents
10 were removed by gel filtration with Sephadex*G 25 ("superfine")
~ in 0.01 mol/l of HC1, th~ sample being diluted by a factor of
s about 5 to 10. The reduced material was stored in 0.01 mol/l
of HC1 at -20C.
EXAMPLE 3
The following tables show the effect of various para-
meters according to the invention upon activation and stimulat-
ability of t-PA. For these re-oxidizing experiments, the
l solubilized, reduced proteins according to Example I were not
i 20 additionaly pre-purified.
The reduced protein (in 0.01 mol of HC1) was acti-
vated by dilution oF 1:100 to 1:500 in a "re-oxidizing buffer".
Activation was determined after 22 to 48 h of incubation at room
temperature. The activity of the re-oxidized protein rellates
to a "Standard Re-oxidation" (= 100%) in:
0.1 mol/1 Tris/HC1 (pH = 10.5) + 1 mmol/1 EDTA
0.5 mol/1 L-Arginine
, -~ 1 mg/ml BSA
+ 0.5 mmol/1 GSH (reduced gluthathione)
30 + 0.5 mmol/1 GSSG (Gluthathione disulphide).
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i 1329157
- 15 -
Stimulatability is calculated from E+cNBrFFp/E-cNBrFFp
(cf. W. NIEUWENHUIZEN et al, Biochimica et Biophysica ACTA 755
~1983) 531 to 533). The activity, as a percentage, and the
stimulatability factor were determined according to J.H. VERHEIJEN
Thromb. Haemostas 48(3), 266-269 (1982).
~, The following results were obtained:
- 1. Relationship between the activating yield and the addition
of L-arginine, guanidine hydrochloride
i, Re-oxidation in 0.1 mol/1 Tris/HC1 (pH 10.5)
+ 1 mmol/1 EDTA
+ 1 Mg/ml BSA
+ 0.5 mmol/1 GSH
+ 0.5 mmol/1 GSSG
a~ L-Arginine
.1
. L-Arginine Activity Stimulatability
(mol/1) (%) (Factor)
..
4 2.5
, 0.25 98 7.5
0.5 100 21.9
0.75 27 16.3
, 1.0 23 3.5
,
` In this experiment it is to be borne in mind that t-PA
is inhibited by L-arginine. The drop in activating yield with
3 increased L-arginine concentrations must therefore be corrected
~ for this inhibition.
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b) Guanidine Hydrochloride ~Gdn.HC1)
(Gdn.HC1) Activity
(mol/1)(%)
11
0.25 22
0.5 53
0.75 58
1.0 12
, 2. Relationship between activating yield and the addition of
1 10 urea and urea derivatives
Re-oxidation in 0.1 mol/1 Tris (pH 10.5), 1 mmol/l EDTA,
1 mg/ml BSA, 5 mmol/1 GSH, 0.2 mmol/1 GSSG
a) Urea
Urea Activity
(mol /1 ) ( %)
o
0 5 20
1 59
1.5 . 126
2 162
1 2.5 141
i 3 72
12
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- 17 - i1 329 1 57
b) Methyl urea
i
i Methyl Urea Activity
(mol/1) (%)
. 0.5 22
1 174
1.5 313
~, 2 375
2.5 332
3 215
4 12
c) Ethyl Urea
Ethyl Urea Activity
I (mol/1) (~)
:1 o 5 46
1 212
1.5 323
2 300
2.5 107
1 20 3 19
,j 4 0
1l 5 - 0
:,
! d) Dimethyl Urea
,j _ .
Dimethyl UreaActivity Stimulatability
, (mol/l) ~%) (Factor)
~ 0.5 167 8.8
; 1 256 8.9
.,,
1.5 283 9.4
f 2 177 7.7
2.5 78 8.9
i 3 23 9.9
' 4 4 8.6
, 5 2 3.5
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- 18 -~ ~ 329 1 57
, . .
3. Relationship between activating yield and the addition of
fatty acid amides.
Re-oxidation in 0.1 mol/l Tris (pH 10.5), 1 mmol/1 EDTA,
1 mg/ml BSA, 5 mmol/1 GSH, 0.2 mmol/1 GSS~
a) Formamide
, .
Formamide Activity
~i (mol/l) (%)
~i 0 42
0.5 59
1 175 :~
1.5 . 245
` 2 325
, 2.5 423
', 3 444
4 416
;Z 5 341
~ b) Methylformamide
.~
' Methylformamide Activity
(mol/l) (%)
'~ 0.5 100
.;1 20 1 135
1.5 304
l 2 389
.~ 2.5 466
~ 3 452
:~. 4 425
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c) Acetamide
Acetamide Activity
(mol/l) (%)
0.5 72
1 134
1.5 207
2 261
2.5 204
3 237
~ 10 4 198
i 5 141
I d) Propionamide
i Propionamide Activity
! (mol/l) (%)
0.5 95
1 . 99
1.5 197
2 150
1 2.5 101
,: 20 3 39
4 2
-0
i
e) Butyramide
Butyramide Activity
~ ~mo1/1) (%)
'j 0.5 55
1 52
1.5 17
~i 2 0
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- 20 ~ l 329 1 57
4. Relationship between activating yield and pH value
Re-oxidationinO.1 mol/1 Tris/HC1 t 1 mmol/1 EDTA
+ 0.5 mol/1 L-arginine
` + 1 mg/ml BSA
+ 0.5 mmol/1 GSH
+ 0.5 mmol/l GSSG
pH ActivityStimulatability
(%) (Factor)
8 22 3.0
9 89 13.~
105 20.3
11 95 21.3
5. Relationship between activating yield and GSH/GSSG
'~ concentration.
Re-oxidation in 0.1 mol/1 Tris/HC1, pH 10.5
+ 1 mmol/1 EDTA
+ 0.5 mol/1 L-arginine
+ 1 mg/ml BSA
~, 20a) + 1 mmol/1 6SH
~, ~
] (GSSG) ActivityStimulatability .:
(mmol/1) (%) (Factor)
0.1 239 14.9
0.2 273 15.3
' 0.5 193 13.3
1 198 12.5
17 2.1
0
0
":
., ~ .
:~ :
~; - 21 - ~ 1 329 1 57
: b) + 0.2 mmol/1 GSSG
,;,
.` (GSH) ActivityStimulatability
:, (mmol/1) (%) (Factor)
.~ 0.05 15 2.2
0.1 40 3.8
.i 0.2 112 6.8
.i 0.5 142 7.4
i 1 273 6.8
i 5 260 7.9
-J~; 10 10 143 6.3
5.1
6. Relationship between activity yield and protein
~j concentration during re-oxidation (dilution 1:20 - 1:500)
;, Re-oxidation in 0.1 mol/l Tris/HC1 (pH 10.5)
',; + 1 mmol/l EDTA
~'~, + 0.5 mol/l L-arginine
:. + 1 mg/ml BSA
.c` + 0.5 mmol/1 GSH
+ 0.5 mmol/l GSSG
~,
. .,
Dilution ActivityStimulatability
(%) (Factor)
1:10 29 15.3
1:20 45 25.4
1:50 69 37.9 1 :
~, 1:100 100 37.9
1:200 79 52.7
1:500 29 28.7
~;
;l
,1,
. ~ .
,c~ .
.j ' ~ ,
.. ,~
; , ~;, ~ .
. ~: ;;, . ..
,: : , . ~ , , -
- 22 - l 329 1 57
7. Relationship between activating yield and the addition of BSA.
Re-oxidation in 0.1 mol/1 Tris/HC1 (pH 10.5)
` + 1 mmol/1 EDTA
+ 0.5 mol L-arginine
+ 0.5 mmol/1 GSH
+ 0.5 mmol/1 GSSG
BSA Activity
(mg/ml) (y)
0 47
0.5 83
` 1 100
3 102
52
With reference to the drawings, Figs. 1 and 2 show
i:. activity with and without CNBr-FFP, in the standard test,
after 17 h of re-oxidation at room temperature in 0.1 mol/1
of tristHC1 (pH 10.5) + 1 mmol/1 of EDTA +0.5 mol/L-arginine
+ 1 mg/ml of BSA + 0.5 mmol/1 of GSH + 0.5 mmol/l of GSSG.
In Figs. 1 and 2, curves (A) indicate activity in
the presence of CNBr-FFP, while curves (B) indicate activity
:'
without CNBr-FFP.
EXAMPLE 4
Activating t-PA with mixed disulphides of t-PA and GSSG.
i~ The "refractile bodies" used were obtained in accordance
with the preceding examples. Reduction of the "refractile bodies"
was carried out by 2 h of incubation at room temperature in 0.1
mol/1 of tris/HC1, (pH 8.6), 1 mmol/1 of EDTA, 6 mol/1 of
.,
, .
,'' . ~: '
~ 329 ~ 57
- 23 -
guanadine hydrochloride, 0.2 mol/1 of DTE, at a protein concentra-
i tion of about 1 mg/ml.
The reduced protein, dialyzed against 0.01 mol/1 of H01
was diluted, in a 1:1 ratio, with 0.1 mol/1 of tris (pH 9.3), 9
mol/1 of urea and 0.2 mol/1 of GSSG, and was incubated for 5 h
at room temperature.
After acidification to pH 3 with conc. HC1, dialysis
was carried out against 0.1 mol/1 of HC1 at 4C. After dialysis,
the total protein concentration amounted to 0.33 mg/ml. Optimal
~ 10 reactivating conditions were determined with the t-PASSG thus
'7 prepared.
' a) pH Optimum for activating t-PASSG.
In this case, as in the following optimizing experi-
~, ments: (1) no GSSG was used, and (2) activating was determined
of 17 h of incubation at room temperature. Activation was
effected by 1:100 dilution in 0.1 mol/1 of tris, 1 mmol/1 oF EDTA,
0.5 mol/1 of L-arginine, 1 mg/ml of BSA and 2 mmol/1 of GSH with
~ variations of the pH value.
:~ pH Yield ~%) Stimulatability
6 0.04 3.3
' 6.5 0.37 9.5
7 1.35 11.4 ! :
7.5 5.66 7.1
-~ 8 7.32 8.2
8.5 8.65 7.0
9 8.59 8.7
9.5 8.32 11.7
$ 10 6.15 12.5
', 10.5 3.07 11.2
. I .
The yield was determined as a percentage of active t-PA
in relation to the amount of protein used.
,,
- 24 ~ ~ 329 1 57
b) Reproducibility of the results of activating t-PASSG.
. Under identical activation conditions, different
` yields were observed in the different series of tests. These
are caused by fluctuations of the standard t-PA. In order to
. clarify this range of errors, all activating data are shown
after 1:100 and 1:200 dilution in 0.1 mol/1 of tris/HC1 (pH
8.5), 1 mmol/1 of EDTA, 0.5 mol/1 of L-arginine, 1 mg/ml of
BSA and 2 mmol/1 of GSH.
,
r~ 10 Test Yield (%) Stimulatability
1 8.65 7.0
2 4.47 9.3
3 4.49 9.7
4 8.50 6.5
3.45 17.2
6 4.32 8.3
7 3.29 14.0
8 3.54 13.4
9 5.07 16.4
20Average Value 5.1 +/- 1.9 11.3 t/- 3.8
:j
c) Stability of the activated protein.
Activation was effected in the examples by 1:200 dilu-
tion in 0.1 mmol/1 of tris/HC1, 1 mmol/1 of EDTA, 0.5 mol/1 of
L-arginine, 1 mg/ml of BSA and 2 mmol/1 of GSH.
EXAMPLE 5
Activating gene technologically produced interferon-
~
"Refractile bodies" were produced according to theaforesaid methods. Reducing/solubility of the "refractile bodies"
was carried out as follows: the pellet was incubated for 3 h at
' '
"
'' ~ ~ : . ' '
., .
,
î 329 ~ 57
- 25 -
25C in 10 ml 9.1 mol tris/HC1 (pH 8.6), 6 mol/1 of guanidine
hydrochloride, 1 mmol/1 of EDTA and 0.2 mol/1 of DTE. After 30
min. of centrifuging at 4C and 48.000 g, the pH of the remainder
was adjusted to about 3 with concentrated HC1. Gel filtration with
' Sephadex G 25 F in 0.01 mol/1 of HC1 was then carried out.
The eluate was tested by transmission (280 nm) for
conductivity, protein concentration and ability to be reactivated.
Standard activating (100%) was carried out in 0.1 mol/1
~ 10 of tris/HC1 (pH 10.5), 1 mmol/1 of EDTA, 5 mmol/1 o-f GSH, 0.5
f mmol/1 of GsSG and 0.25 mol/1 of L-arginine.
a) Relationship between activating and time.
The eluate was diluted 1:50 in 0.1 mol/1 of tris/tlC1
(pH 8.5), 1 mmol/1 of EDTA, 5 mmol/1 of GSH, 0.5 mmol/1 of GSSG
and 0.25 mol/1 of L-arginine.
I' Activating Time (h) Activity 0C
t 1 15
3 15
b) Relationship between activating yield and the addition
of L-arginine.
The eluate was diluted 1:50 with 0.1 mol/1 of tris HC1
(pH 8.5), 1 mmol/1 of EDTA, 5 mmol/1 of GSH and was activated
for 20 h at 0C.
` L-arginine dependency of activation.
*trade mark
~ `y'i~,
- 26-t32ql57
L-Arginine ~M) Activity (%)
0 8
0.25 8
0.5 15
0.75 15
; ~
~, c) Relatiorship between activating yield and the addition
of urea.
. The activating solution corresponded to that at point b)
' but activation was carried on for 17 h at O~C.
Urea dependency of activation.
. . .
,
Urea (M) Activity ~%)
0 13
100
-; 1 200
.'`~3. 1.5 100
, I
d) Relationship between activating yield and the addition
of formamide.
Activating as in b); the test pieces were checked after
17 h of activating at 0C.
Formamide dependency of activation.
, ~, I
i~ Formamide (M) Activity (%)
,~
:J o 13
,~
1 13
' 2 13
0
.,,
~ 4 0
,i
, .
;. .:
... . .
1 32~ 1 57
- 27 -
e) Relationship between activating yield and the redox buffer.
The eluate was diluted 1:50 in 0.1 M of tris/HC1 (pH 8.5),
1 mM of EDTA and 0.25 M of L-arginine and the test pieces were
checked after 17 h of activation at 0C.
GSH/GSSG dependency of activation.
i,,
GSH (mM) GSSG (mM)Activity (%)
1 0.5 6
0.5 13
0.5 25
0.5 25
0.1 13
0.5 13
1.0 13
~ 5 5 6
f) Relationship between activating yield and protein
, concentration.
j The eluate was diluted, between 1:10 and 1:100, in 0.1
¦ M of tris/HC1 (pH 8.5), 1 mM of EDTA, 5 mM of GSH, 0.5 mM of GSSG :
and 0.25 M of L-arginine; it was checked after 17 h of activa-
20tion at 0C.
cp dependency of activation.
cp (mg/ml) Activity (%) !
i~ 0.018 13
0.036 13
0.072 13
0.108 ~ 8
0.180 10
~i .
, .
- 28 ~ l 329 1 57
g) Relationship between activating yield and the addition
of BSA.
The eluate was diluted 1:50 in 0.1 M of tris/HC1 (pH
8.5),1 mM o-f EDTA, 5 mM of GSH, 0.5 mM of GSSG and 0.25 M of
L-arginine; it was checked after 17 h of activation at 0C.
BSA dependency of activation.
BSA (mg/ml) Activity (%)
13
1 13
2 25
h) Relationship between activating yield and pH.
. The eluate was diluted 1:50 in 0.1 M of tris/HC1,1 mM
', of EDTA, 5 mMof GSH, 0.5 mM of GSSG and 0.25 M of L-arginine; it
was checked after 17 h of activation at 0C. m
, pH dependency of activation.
, .
, pH Activity (%)
6.5 0
.5 6
8.5 13
9.5 50
10.5 100
.. . .
., .
j
;. ~ . . . .. ~ ~
, .,
; ..... . . :
!, : .
.'': '
