U.S. patent application number 10/307122 was filed with the patent office on 2004-05-06 for method of preparing polypeptides in cell-free system and device for its realization.
Invention is credited to Biryukov, Sergey Vladimirovich, Mayorov, Sergey Gennadievich, Shirokov, Vladimir Anatolievich, Simonenko, Alena, Simonenko, Peter Nikolayevich, Spirin, Alexander Sergeyevich.
Application Number | 20040086959 10/307122 |
Document ID | / |
Family ID | 20203746 |
Filed Date | 2004-05-06 |
United States Patent
Application |
20040086959 |
Kind Code |
A1 |
Biryukov, Sergey Vladimirovich ;
et al. |
May 6, 2004 |
Method of preparing polypeptides in cell-free system and device for
its realization
Abstract
The present invention provides a method for synthesis of
polypeptides in a cell-free system. In this method, products of
synthesis are separated into a low molecular weight fraction and a
high molecular weight fraction, with the high molecular weight
fraction containing the target polypeptide. The method calls for
the removal of at least a portion of the low molecular weight
fraction via a porous barrier.
Inventors: |
Biryukov, Sergey Vladimirovich;
(Moscow Region, RU) ; Simonenko, Peter Nikolayevich;
(Moscow Region, CA) ; Simonenko, Alena; (Moscow
Region, CA) ; Shirokov, Vladimir Anatolievich;
(Moscow Region, RU) ; Mayorov, Sergey Gennadievich;
(Moscow Region, RU) ; Spirin, Alexander Sergeyevich;
(Moscow Region, RU) |
Correspondence
Address: |
Roche Diagnostics Corporation
9115 Hague Road
PO Box 50457
Indianapolis
IN
46250-0457
US
|
Family ID: |
20203746 |
Appl. No.: |
10/307122 |
Filed: |
November 27, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10307122 |
Nov 27, 2002 |
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09647316 |
Apr 23, 2001 |
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6518058 |
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09647316 |
Apr 23, 2001 |
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PCT/EP99/02124 |
Mar 27, 1999 |
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Current U.S.
Class: |
435/68.1 ;
435/297.2 |
Current CPC
Class: |
C12M 29/04 20130101;
C12P 21/02 20130101; C12M 29/16 20130101; C12M 23/02 20130101; C12M
23/34 20130101; C12M 25/16 20130101; C12M 29/18 20130101 |
Class at
Publication: |
435/068.1 ;
435/297.2 |
International
Class: |
C12P 021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 1998 |
RU |
98105294 |
Claims
What is claimed:
1. A method for obtaining polypeptides in a cell-free translation
system by which the reaction mixture, the feed solution and
expendable components of the high molecular weight fraction are
prepared, at least two porous barriers are placed inside the
reactor module, the reaction mixture is introduced to the reactor
volume and incubated at given conditions maintaining the synthesis,
the target polypeptide is removed or retained in the reactor
volume, wherein products of synthesis are branched in a low
molecular weight fraction and a fraction which contains high
molecular weight components with the target polypeptide, the main
part of the low molecular weight fraction is removed via at least
one part of the second porous barrier, the ratio of the volume of
the fractions of feed solution and expendable components to the
volume of the fraction containing the target polypeptide is chosen,
modes of supply of the feed solution and expendable components of
the fractions are realized.
2. The method according to claim 1 wherein in the mode of removal
of the target polypeptide from the reactor volume the fraction
containing high molecular weight products with the target
polypeptide is removed via the first porous barrier which has pore
sizes up to 100 kD, while the low molecular weight fraction with
components of the reaction mixture and products of synthesis is
removed via at least one part of the second porous barrier which
has pore sizes up to 30 kD.
3. The method according to claim 1 wherein in the mode when the
target product is not removed from the reaction mixture, the
fraction of low molecular weight components is removed from the
reactor simultaneously via the first and second barriers which have
pore sizes up to 30 kD.
4. The method according to claim 1 wherein the feed solution is
supplied to the reactor volume either (i) via the inlet of the
reactor volume, or (ii) via the pores of another part of the second
barrier, or (iii) both via the inlet and the pores.
5. The method according to claim 2 wherein the fraction of high
molecular weight components with the target polypeptide is removed
from the reactor continuously or recurrently.
6. The method according to claim 1 wherein the fraction of
expendable high molecular weight components is added to the
reaction mixture either (i) before the synthesis, or (ii) once
during the synthesis, or (iii) recurrently during the synthesis, or
(iv) is supplied continuously during the synthesis.
7. The method according to claim 6 wherein the expendable high
molecular weight components are chosen from (i) ribosome fraction,
(ii) cell-free system extracts (S30, S100 and their modification),
or (iii) polymerases, plasmids, tRNAs that are supplied to the
reactor mixed with the feed solution, each of them taken separately
or combined with the others.
8. The methods according to claim 1 wherein the ratio of the feed
solution volume, that is supplied to the reactor, to the volume of
the fraction containing high molecular weight components with
target polypeptides, that are removed from the reaction mixture, is
from 1 to 100.
9. A method for obtaining polypeptides in a cell-free translation
system by which the reaction mixture the feed solution and
expendable components of the high molecular weight fraction are
prepared, at least two porous barriers are placed inside the
reactor module, the reaction mixture is introduced to the reactor
volume and incubated at given conditions maintaining the synthesis,
the target polypeptide is retained in the reactor, wherein the low
molecular weight fraction which consists of removed components
including low molecular weight components of the reaction mixture
and low molecular weight components of the synthesis is withdrawn
from the reaction volume via at least one the second porous barrier
only, the mode of supply of fractions of the feed solution and
expendable components is realized.
10. The method according to claim 9 wherein low molecular weight
components of the feed solution are supplied to the reactor volume
(i) via the first porous barrier, or (ii) part of the feed solution
is supplied to the reactor volume via the inlet while the other
part is supplied via the first porous barrier.
11. The method according to claim 9 wherein expendable high
molecular weight components are supplied either (i) via the reactor
volume inlet, or (ii) via the first porous barrier, or (iii) part
of the expendable components is supplied via the reactor volume
inlet while the other part is supplied via the first porous
barrier.
12. The method according to claim 9 wherein the first porous
barrier has pore sizes up to 1000 kD and the second porous barrier
has pore sizes up to 30 kD.
13. The method according to claim 9 wherein the fraction of
expendable high molecular weight components is added to the
reaction mixture either (i) before the synthesis, or (ii) once
during the synthesis, or (iii) recurrently during the synthesis, or
(iv) is supplied continuously during the synthesis.
14. The method according to claim 9 wherein expendable high
molecular weight components are chosen from (i) ribosome fraction,
(ii) cell-free system extracts (S30, S100 or their modifications)
and (iii) polymerases, plasmids and tRNAs that are supplied to the
reactor mixed with the low molecular weight feed solution, either
taken separately or combined with the others.
15. A method for obtaining polypeptides in a cell-free translation
system by which the reaction mixture the feed solution fraction and
expendable components of the high molecular weight fraction are
prepared, at least two porous barriers are placed inside the
reactor module, the reaction mixture is introduced to the reactor
volume and incubated at given conditions maintaining the synthesis,
the target polypeptide is removed or retained in the reactor volume
wherein during the synthesis N cycles are formed, everyone of which
consists of at least two steps, at the first step low molecular
weight components of the feed solution are supplied to the reactor
via the first porous barrier and the low molecular weight fraction
with products of synthesis and components of the reaction mixture
is removed via the second porous barrier, at the second step the
supply and removal channels are switched and low molecular weight
components of the feed solution are supplied via the second porous
barrier, the low molecular weight fraction or the high molecular
wieght fraction containing products of synthesis and components of
the reaction mixture is removed via the first porous barrier, the
mode of supply of of fractions of the feed solution and expendable
high molecular weight components is realized
16. The method according to claim 15 wherein in the mode when the
target polypeptide is removed from reaction volume the first porous
barrier has pore sizes up to 100 kD and the second porous barrier
has pore sizes up to 30 kD.
17. The method according to claim 16 wherein in the mode when the
target polypeptide is removed the ratio of the feed solution volume
supplied to the reactor during N cycles to the fraction volume
including the target polypeptide removed from the reaction mixture
is from 2 to 100.
18. The method according to claim 15 wherein in the mode when the
target polypeptide is retained in the reaction volume the first and
second barriers have pore sizes up to 30 kD.
19. The method according to claim 15 wherein the fraction of
expendable high molecular weight components is added to the
reaction mixture either (i) before the synthesis, or (ii) once
during the synthesis, or (iii) recurrently during the synthesis, or
(iv) is supplied continuously during the synthesis.
20. The method according to claim 15 wherein expendable high
molecular weight components are chosen from (i) ribosome fraction,
(ii) cell-free system extracts (S30, S100 or their modifications),
or (iii) polymerases, plasmids and tRNAs that are supplied to the
reactor, each taken separately or combined with the others.
21. The method according to claim 15 wherein the duration of the
first and second steps in every of the N cycles is adjusted
depending on the conditions of synthesis and the changes in its
parameters.
Description
FIELD OF INVENTION
[0001] The present invention concerns to molecular biology and
biotechnology, namely to methods and devices for synthesis of
polypeptides in cell-free translation system.
BACKGROUND OF THE INVENTION
[0002] Several methods of polypeptide synthesis in cell-free
translation system are known. For elimination of restrictions
connected with a lower output of target polypeptides and short-term
operation of cell-free translation systems a method was suggested
which is widely used now (Spirin et al., 1988). This method is
based on the principle of continuous removal from a reaction
mixture of reaction products and continuous restoration of the
initial concentration of low molecular weight components during
synthesis. This method underlies several inventions connected with
its improvement for increasing the synthesized product output
(Alakhov et al., 1991; Baranov et al., 1993; Alakhov et al.,
1995).
[0003] By the input of feeding solutions and removal of products of
synthesis the known methods can be divided as follows: (a) methods
in which dialysis is used to add feed solution components to the
reaction mixture and to remove low molecular weight components from
the reaction mixture through the dialysis membrane or for to
simultaneously remove low and high molecular weight components from
the reaction mixture; (b) methods in which continuous
ultrafiltration is used for a simultaneous removing of low and high
molecular weight components of products through the membrane and a
simultaneous input of feeding solutions directly into the reaction
mixture volume or through the membrane; c) methods in which
periodic input of a feed solution into the reaction mixture and
subsequent removing of low and high molecular weight components
through the membrane are used. Input and output of the flows is
realized by changing the direction of liquid flows at the expense
of consecutive creation of pulses of positive or negative
pressure.
[0004] The method (Mozayeny, 1995) is known in which the removal of
products with large molecular weight is improved by increasing the
area of a ultrafiltration membrane in relation to the reaction
mixture volume. One of the main disadvantages of the given
invention is that during removal of high molecular weight
components through the large area of the membrane with the pore
size of 70 kD to 100 kD, together with the final product useful
working components of molecular weight up to 100 kD are lost. This
is a limiting factor for the operating time of the cell-free
system. The larger is the membrane area, the greater is the amount
of high molecular components of the cell-free system washed-off
from the reactor at a high flow rate. Another disadvantage is the
necessity to use an external loop for creation of a tangential flow
of the reaction mixture along the membrane surface. During passage
of the reaction mixture via liquid communications three factors
influence the work of the cell-free system: (1) when the reaction
mixture passes via the loop the feed solution is not added in the
part of external volume of the reaction mixture, (2) low weight
products which are inhibited the cell-free system are not removed
from the external volume, (3) the liquid communications and pumps
are not thermostable and the reaction mixture changes its
temperature depending on the environment. This leads to
irreproducibility of results and limits the life time of the
cell-free system.
[0005] The method in which authors offer to apply repeated pulse
for input of the feeding solution in the reactor and removal of low
and high molecular weight products of synthesis from the reactor
via a membrane is known (Fischer et al., 1990). This is realized by
changing the direction of the flow through the membrane. One of the
main disadvantages of the given invention is that low molecular
weight components of synthesis which inhibit operation of the
system are not removed from the reactor during a long period. The
time during which the feed solution passes repeatedly via the
membrane is equal to period when a total volume of feed solution
passage via the membrane is equal to the complete volume of the
reaction mixture. For this purpose N cycles are formed to create
positive and negative pressure. Due to the pressure modulation the
inhibiting products come back in the reactor together with a
regular portion of the feed solution. Another disadvantage of this
method is that upon formation of N cycles high molecular weight
components of the cell-free system required for prolonged synthesis
are intensively washed off the reaction mixture. Thus, repeated
returning into the reactor of low molecular weight components
inhibiting operation of the system and removing from the reactor of
high molecular weight components providing effective synthesis
impose restrictions on operation of the system.
[0006] The method (Choi, 1997) is known by which synthesis of
polypeptides is carried out with removal of a target product in a
dialysis mode of operation. For this purpose a membrane divides the
reactor in two parts. The reaction mixture is placed on one side of
the membrane and the feed solution on the other side. The reaction
mixture is fastly circulating along the membrane surface in
tangential direction. A disadvantage of the method is that due to a
large pore size components of the system are removed together with
the target products. Moreover, in spite of the fact that the
dialysis process is quite effective because of the large pore size,
its extent is not enough for operation of highly efficient
cell-free systems.
[0007] The method (Alakhov et al., 1991) is known in which amino
acids, ATP, GTP in an aqueous buffer are added to reactor during
functioning of the system and low weight components such as AMP,
GDP, Pi formed during synthesis and inhibiting the system are
removed through a membrane. To provide a more economical operation
of the system, low molecular weight products are regenerated and
come back into the reactor via the membrane. However from the
description and the given figure it is not quite clear how low and
high molecular weight components of the synthesis are removed from
the reactor and in what way the buffer solution is regenerated
after removal of the polypeptide. Taking into account the
description of examples, low and high molecular weight components
are removed from the reactor via the ultrafiltration membrane. The
use of an ultrafiltration membrane is described in a number of
publications (Spirin et al., 1988; Takanori et al., 1991; Spirin,
1992; Erdmann et al., 1994). A disadvantage of this method is the
use of large sizes of the membrane cutoff. In this case high
molecular weight components of systems necessary for synthesis are
removed from the reactor together with target products. The volume
of low molecular weight components is equal to that of the removed
components which results in fast closing of the ultrafiltration
membrane pores.
[0008] Methods of adding feed solution to the reaction zone and
removing from it of reaction products for different types of
membrane reactors are known in which the reaction zone is placed
between two membranes (Matson et al., 1988; Wrasidlo et al., 1990;
Dziewulski et al., 1992).
[0009] The method described in the patent (Alakhov et al., 1995) is
the prototype of the method proposed herein. For synthesis of
polypeptides in this invention the reaction mixture is placed
between two flat membranes. The membranes differentiate flows of
low molecular weight and high molecular weight components and
divide the reactor into three zones: zone for input of feed
solution, reaction zone, zone of product removal. The first rather
weak flow is formed in the reaction zone. It ensures the reaction
mixture movement along the internal part of porous barriers,
through which high molecular weight components (including
synthesized polypeptides) are removed. The second fast flow is
formed in the zone of feed solution input. It ensures penetration
of of low molecular components via the membrane in the reaction
system. The fast flow of low molecular weight components and the
slow flow of high molecular components are achieved by creating a
tangential flow along the external surface of the first porous
barrier and dialysis process for adding feed solution in the zone
of synthesis. If high molecular weight components are removed from
the reactor, the size of cutoff is chosen from 50 to 100 kD (which
is corroborated by example 7).
[0010] The speed of penetration of feed solution components to the
reactor determined to a greater extent by the dialysis process is
insufficient for maintenance of prolonged operation of highly
effective cell-free systems.
[0011] Requirements to devices for scientific researches and for
synthesis of polypeptides in preparative amounts are different. For
synthesis of small amounts of polypeptides, from 100 to 200 .mu.g,
it is necessary to have a simple and cheap reactor which can ensure
synthesis during 20-50 hours without application of expensive
equipment and provides an opportunity to choose hand-operated or
controlled speeds of flows.
[0012] During synthesis of polypeptides in preparative amounts, the
device should control the process: operate speeds of flows inside
the reactor and provide an opportunity for a prolonged (more than
50 hours) operation at the expense of active mixing or other action
protecting from closing of membranes or hollow fibers, provide
effective addition of the feed solution and expendable high
molecular weight components to the reaction mixture and effective
removal of low molecular weight components from the reactor which
inhibit the synthesis.
[0013] The device (Mozayeny, 1995) is known which operates in the
mode of continuous synthesis of peptides and is controlled by
computer. The system includes a complicated and expensive equipment
(an automatic sampler etc.).
[0014] Devices with one reactor from 1.0 ml. (Spirin et al., 1988)
to 100.0 ml (Spirin, 1992) are known. Using the principle of
dividing the entire reactor volume in several reactors of smaller
volume it is possible to apply identical decisions to devices
intended for synthesis of polypeptides in laboratory conditions and
for preparative synthesis. In this case routine technologies of
synthesis of polypeptides in small volumes of the reaction mixture
from 50 .mu.l up to 1-5 ml can be used in working with the volume
up to 100-200 ml by scaling and increasing the amount of modules
operating in parallel.
[0015] Devices for maintenance of synthesis in cells (Puchinger et
al., 1980; Gebhard et al., 1997; Hu et al., 1997) using the
principle of scaling the modules are known. In these devices inputs
for supplying a feed solution and outputs for removal of products
are connected in parallel for all N reactors. The known devices are
designed for maintenance of cell growth and cannot be applied for
synthesis of polypeptides, as each of N reaction modules serves to
maintain speed, pressure and other parameters of flows of feed
solutions and gases necessary for normal functioning of cells.
[0016] Known modules for bioreactors on the basis of hollow fibers
(Gebhard et al., 1997; Hu et al., 1997) do not take into account
the specifity of working with cell-free system. Fibers used in the
reactors have the same size of a cutoff and their form reminds a
beam placed in a cylinder. Therefore a significant part of the
surfaces of hollow fibers contacts each other and reduces the
working surface.
[0017] The device (Yagihashi et al., 1996) is known whose
construction represents two layers of hollow fibers. Each layer
consists of glued hollow fibers placed in parallel. Both layers of
hollow fibers have the same size of a cutoff and a significant part
of their surface is in contact.
[0018] The device (Pedersen et al., 1994) is known in which
separate modules are single-layer constructions from hollow fibers
with the same size of a cutoff.
[0019] However this technical decision has been developed for
filtration of liquids and cannot be used in reactors for cell-free
systems without essential medication in the design since it is
intended for working with large volumes of liquid flows.
[0020] A large number of designs constructed on the basis of flat
membranes is known. They also have disadvantages being intended
basically for filtration or dialysis. The device for synthesis of
polypeptides in cell-free system is known (Mozayeny, 1995) whose
structure includes two flat membranes. Originally this device
(OMEGA.TM.) was intended for filtration and has a large void volume
in the zones of product selection. The feed solution and high
molecular wheight components are added to the reactor through one
input. This device is not intended for assembly in a general
construction consisting of N modules.
[0021] The prototype of the proposed device for synthesis of
polypeptides is the device described in the patent (Alakhov et al.,
1995). For synthesis of polypeptides in a mode of product removal
the device contains two porous barriers. These barriers can be
executed as flat membranes or hollow fibers. The reaction mixture
can be placed both from the external and internal sides of hollow
fibers.
[0022] A disadvantage of the given device is that it contains
porous barriers with the same size of a cutoff and provides for
removal from the reactor of one flow consisting either of low
molecular weight (the cutoff size of 7.5 kD) or high molecular
weight (the cutoff size up to 100 kD) fractions.
SUMMARY OF THE INVENTION
[0023] The presence invention provides a methods for synthesis
polypeptides in a cell-free system by which products of synthesis
are branched in a low molecular weight fraction and a fraction
which contains high molecular weight components with the target
polypeptide, the main part of the low molecular weight fraction is
removed via at least one part of the second porous barrier, the
ratio of the volume of the fractions of feed solution and
expendable components to the volume of the fraction containing the
target polypeptide is chosen, modes of supply of the feed solution
and expendable components of the fractions are realized.
[0024] It is futher object of the present invention to describe a
methods for synthesis polypeptides in a cell-free system by which
the low molecular weight fraction which consists of removed
components including low molecular weight components of the
reaction mixture and low molecular weight components of the
synthesis is withdrawn from the reaction volume via at least one
the second porous barrier only, the mode of supply of fractions of
the feed solution and expendable components is realized,
[0025] It is therefor, also, an object of present invention to
provide the method for obtaining polypeptides in which during the
synthesis N cycles are formed, everyone of which consists of at
least two steps, at the first step low molecular weight components
of the feed solution are supplied to the reactor via the first
porous barrier and the low molecular weight fraction with products
of synthesis and components of the reaction mixture is removed via
the second porous barrier, at the second step the supply and
removal channels are switched and low molecular weight components
of the feed solution are supplied via the second porous barrier,
the low molecular weight fraction or the high molecular wieght
fraction containing products of synthesis and components of the
reaction mixture is removed via the first porous barrier, the mode
of supply of of fractions of the feed solution and expendable high
molecular weight components is realized.
[0026] It is a futher object of the present invention to describe a
reactor which comprises at least one reactor volume, whose external
surface contacts the external surface of the first and second
porous barriers, the internal surface of the second porous barrier
is connected to the zone of the inlet or outlet of low molecular
weight flows, the internal side of the first porous barrier is
connected to the zone of the inlet and outlet of low molecular
weight flows and flows containing high molecular weight components
with the target polypeptide.
BREEF DESCRIPTION OF DRAWINGS
[0027] The invention is explained by examples of performance with
the references to the following figures.
[0028] FIGS. 1(a-c) shows a scheme of principles of flow
distribution for different modes of operations.
[0029] FIGS. 2(a-f) shows block diagrams of modules based on the
declared principle.
[0030] FIG. 3 shows a block diagram of the device for synthesis in
a mode without removal of the target polypeptide.
[0031] FIG. 4 shows a block diagram of the device for synthesis in
a mode of removal of the target polypeptide.
[0032] FIG. 5 shows a block diagram of the device for synthesis in
a mode of removal of the target polypeptide with a three porous
barriers in module.
[0033] FIG. 6 shows a block diagram of the device for synthesis in
a mode with periodic removal of the target polypeptide.
[0034] FIG. 7 shows a histogram reflecting the amount of GFP in the
fractions in accordance with the description in Example 1.
[0035] FIG. 8 shows the dependence of the change in the
concentration of synthesized GFP on the duration of synthesis in a
control aliquot according to the data of Example 1.
[0036] FIG. 9 is a photo of SDS electrophoresis according to the
data of Example 1.
[0037] FIG. 10 shows a diagram of dependence of the change in the
concentration of synthesized GFP on the duration of synthesis in a
control aliquot according to the data of Example 2.
[0038] FIG. 11 is a photo of SDS electrophoresis according to the
data of Example 2.
THE LIST OF ABBREVIATIONS
[0039] F1 is the feed solution flow.
[0040] F10-F20 are flows of low molecular weight components of the
reaction mixture.
[0041] Ps are synthesized target polypeptides.
[0042] Pc are high molecular weight components of the system.
[0043] Rm is the reaction mixture.
[0044] Positions 100 are constructive elements of the reactor.
[0045] Positions 200 are liquid communications of the device.
[0046] Positions 300 are separate elements of the device: valves,
pumps, compartments for the eluate and compartments for storage of
feed solution.
[0047] Position 400 is the controller block.
DETAILED DESCRIPTION OF INVENTION
[0048] Generally the process can contain all of the following
steps:
[0049] 1. Prepare a reaction mixture on the basis of cell-free
prokaryotic or eukaryotic lysates;
[0050] 2. Prepare a feed solution and exendable components of high
molecular weight fraction;
[0051] 3. Determine the mode of operation of reactor, choose a type
of the reactor module with the given types of porous barriers and
install a device for synthesis whose structure includes, at least,
one reactor module;
[0052] 4. Place the reaction mixture in each of the reaction
modules;
[0053] 5. Depending on the mode of operation determine and set
rates of the feed solution flow and the flow of expendable high
molecular weight components through the reaction mixture and speeds
of removal of low and high molecular weight products;
[0054] 6. Carry out the synthesis providing the selected modes of
operation, during the synthesis continuously or recurrentlly remove
through a porous barrier the high molecular weight components or
leave them in the reactor volume;
[0055] 7. During the synthesis analyze the yield of the synthesized
product, correct the parameters determining rates of feed solution
flow and of the synthesized product, and supply or not supply
expendable high molecular weight components.
[0056] FIGS. 1(a-e) shows block diagrams explaining the principle
of flow branching in a low molecular weight fraction and a fraction
which contains high molecular weight components with target
polypeptide for different modes of operations.
[0057] FIG. 1a shows a block diagram of flow branching inside the
module in a mode of continuous removal of the target polypeptide.
The reaction module contains case 100, within which three zones are
formed using the first porous barrier 110 and second porous barrier
120. The reaction mixture Rm is supplied to reaction zone 130
before the beginning of the synthesis. The flow of feed solution F1
passes through input 151 in reaction zone 130 and displace high and
low molecular weight components of the synthesis from the reaction
mixture through the first and second porous barriers. The basic
part of low molecular weight fraction passes through the second
porous barrier 120 with a small size of a cutoff, forming flow F20
removed from the reactor volume through output 142.
[0058] Simultaneously high molecular weight components of synthesis
Ps, components of cell-free system Pc and part of lower molecular
weight components F10 which form the total flow Pc+Ps+F10 are
displaced from the reaction mixture through barrier 110 with a
large size of a cutoff and through output 162. The ratio between
the total volume of feed solution supplied to the reactor and the
volume of the total flow Pc+Ps+F10 is chosen from 2 to 100. It is
preferable that this ratio is from 10 to 50. A decrease in the rate
of flow through the first barrier 110 diminishes formation of a
layer of high molecular weight components above the surface of
porous barrier 110 and diminishes the process of closing of
membrane pores by high and low molecular weight components. The
concentration of synthesized polypeptides Ps in the reaction
mixture increases, thus promoting the next step of its clearing.
The size of the cutoff of the first barrier 110 is chosen depending
on the molecular weight of the synthesized polypeptide and its
spatial organization.
[0059] FIG. 1b shows a block diagram of flow branching of low and
high molecular weight fractions inside the module in a mode of
periodic removal of the target polypeptide. The reaction module
contains case 100, inside which three zones are formed using the
first porous barrier 110 and second porous barrier 120. Before the
beginning of synthesis reaction mixture Rm is supplied inside
reaction volume 130 through input 151. During the synthesis N
cycles of flow input/removal through porous barriers 110 and 120
are formed. Each cycle consists of two steps. During the first step
via input 161 the flow of feed solution F1 penetrates through the
first barrier 110 inside inside reaction volume 130. The feed
solution displaces low molecular weight components of reaction
mixture and low molecular weight products of synthesis which form
flow F20 from reaction mixture through porous barrier 120 and
output 142. During the second step the direction of flow
input/removal through porous barriers 110, 120 is changed. Through
input 141 and second barrier 120 the flow of feed solution F2
penetrates inside zone 130. The pressure of feed solution forms a
flow of low and high molecular weight components of the reaction
mixture which passes through porous barrier 110 and output 162, and
flow Pc+Ps+F10 is formed. Upon termination of the second step, the
cycle comes to its end, the next cycle begins, or synthesis is
terminated. The ratio between the total volume of the feed
solution, supplied to the reactor during N cycles, to the volume of
total flow Pc+Ps+F10 is selected from 2 to 100. It is preferable
that this ratio varies from 10 to 50.
[0060] A decrease in the rate of flow through the first barrier 110
diminishes formation of a layer of high molecular weight components
above the surface of porous barrier 110 and diminishes the process
of closing of membrane pores by high and low molecular weight
components. The concentration of synthesized polypeptides Ps in the
reaction mixture increases, thus promoting the next step of its
clearing. The size of the cutoff of the first barrier 110 is chosen
depending on the molecular weight of the synthesized polypeptide
and its spatial organization. The first and second porous barriers
are cleaned by changing directions of flows through porous
barriers. Duration of a cycle and the temporary ratios between
duration of the first and second steps are maintained constant
during all synthesis, or are changed depending on conditions of the
synthesis. If the synthesis goes actively and the concentration of
polypeptides in the reaction mixture quickly grows, to prevent
closing of the porous membrane the duration of each cycle is
reduced which automatically results in more frequent clearing of
the pores.
[0061] FIG. 1c shows a scheme of flow directions in the mode of
operation without product selection. During the synthesis all high
molecular weight components remain inside the reaction mixture. The
feed solution flow F1 enters through the first porous barrier 110,
which has pore size up to 1000 kD. The flow of low molecular weight
products F10, which inhibiting the cell-free system, is removed via
the second porous barrier 120. The first porous barrier is an
allocator flow F1 and provides uniform input of the feed solution
to all points of the reaction mixture.
[0062] FIG. 1d shows a scheme directions of flow in the mode of
operation without product selection for reactor module which have
at least one the first porous barrier and two parts of second
porous barrier. During the synthesis feed solution flow F1 and/or
part of expenable components with high molecular weight penetrate
through porous of the first barrier 110, which has pore size up to
1000 kD This porous barrier is an allocator flow F1 and provides
uniform input of the feed solution to all points of the reaction
mixture. The flow of low molecular weight products F10 which
inhibiting the cell-free system is removed via the two parts of
second porous barrier 121-122, which has pore size up to 30 kD.
[0063] FIG. 1e shows a scheme directions of flow in the mode of
operation with product selection for reactor module which have at
least one the first porous barrier and two parts of second porous
barrier. During the synthesis all high molecular weight components
including target polypeptide is removed from reaction mixture via
the first porous barrier 110, which has pore size up to 100 kD The
feed solution flow F1 enters through the one part of the second
porous barrier 121, which has pore size up to 30 kD. This porous
barrier is an allocator flow F1 and provides uniform input of the
feed solution to all points of the reaction mixture. The flow of
low molecular weight products F10 which inhibiting the cell-free
system is removed via the another part of second porous barrier
122, which has pore size up to 30 kD.
[0064] For realization of the considered method several
constructions of the reactor module are offered. The device can
provide synthesis of products in different modes. The module should
allow for several conditions:
[0065] 1. In each point of the reactor at least two processes
should be carried out simultaneously: (a) input of feed solution,
(b) removal of low molecular weight components which inhibit the
synthesis.
[0066] 2. Input of feed solution and removal of low molecular
weight components of products should be carried out in the time
during which the synthesis drops below the admissible
magnitude.
[0067] 3. The device should allow effective clearing of the porous
membranes or hollow fibers and contain a minimum of void volumes in
the liquid communications for input/removal of lower/higher
molecular weight components/products of synthesis.
[0068] For realization of the first and second conditions, a
reactor module in which whatever thin layers of the reaction
mixture can be formed is most preferable. The thickness of a layer
is chosen from 0.1 mm up to 10 mm provided that at the given areas
of the first and second porous barriers and chosen sizes of their
cutoff the average speed of feed solution input in the reactor
ensures input of the feed solution components in the most remote
points of the reaction mixture in time during which the feed
solution concentration in the remote points drops to the admissible
level, and the concentration of low molecular weight components
inhibiting the synthesis does not exceed this level.
[0069] With devices working in the mode in which the feed solution
or/and high molecular weight components move directly to the
reactor through at least one input it is preferable to use
additional mixing in the reactor. Such mixing can be performed by a
rotating magnetic stir bar or transfer of the reaction mixture via
the external closed loop or other methods.
[0070] For devices in which a thin layer of the reaction mixture is
formed, a module with a separator between layers of porous barriers
is most preferable. A different type of separator can be used and
may be selected from the group of a single or multiple layer,
capillary materials, a combination of layers of capillary materials
and porous particles, a single or multiple thin layer sheets from
organic, synthetic or ceramic material, metals or their
compositions and porous particle between layers. Another type of
separators may be of hollow-fibers. Filters widely used for
filtration and made of different type of cellulose or synthetic
polymeric materials, metals and ceramics may be also used as
separators. The role of layered capillary materials is not only to
divide two porous barriers, but also to increase the area on which
binding of molecules occurs that increases the speed of reactions
connected with the synthesis (Alberts et al., 1986).
[0071] It is more expedient to use immobilized porous particles
plated on the surface of layered capillary structures. The diameter
of fibers is taken from 0.1 up to 0.001 depending on the diameter
of hollow fibers. Particles of porous material from 10 microns to
0.1 mm are layered on the surface of fibers. In case hollow fibers
are used as the first and/or second porous barriers, the fibers of
layered materials are accommodated either along hollow fibers
occupying the space between them, or at an angle to the central
axes of hollow fibers not exceeding 90 degrees (i.e. across the
layers of hollow fibers). The materials of which porous particles
can be made include (Choi et al., 1997): polymeric materials (
cellulose, gelatin, collagen), metal compounds and inorganic oxides
(aluminium, silica, titanium, zirconium, molybden, vanadium,
cobalt) and various zeolites. Porous particles can be used as
granules (Ovodov et al., 1995) on the basis of negatively charged
polysaccharides and positively charged polymers which form
polyelectrolyte complexes with polysaccharides. Such complexes can
be formed, for example, by sodium alginate and poly-L-lysine,
sodium alginate and chitosane, pectin and polyimine, pectin and
chitosane. The materials of which the porous particles are made may
include sorbents used in chromatography, affinity sorbents can be
also used to create a porous medium in reaction volume and to
isolate the target polypeptide from the reaction system after
synthesis. Chemical activity and possibility to inhibit the
synthesis is restricted for some porous material.
[0072] When a cell-free system is immobilized in granules (Ovodov
et al., 1995; Alakhov et al., 1995), the most preferable design is
the reactor module allowing to plate several layers of microgranule
with immobilized cell-free system on the surface of the first
porous barrier so that they fill the whole reactor. In this case a
rather thin layer of microgranules is formed determined by the
height of the reactor, which allows to supply feed solution to the
zone of synthesis and to remove the low and high molecular weight
components at the optimal speed.
[0073] FIGS. 2a-e shows variants of thin layer formation inside the
reactor module when flat semipermeable membranes and hollow fibers
or their combinations are used as porous barriers.
[0074] FIG. 2a shows the case when thin layers of the reaction
mixture are formed between the flat surface of membrane 120 and
cylindrical surfaces of hollow fibers that play the role of the
first barrier 110 and has the form of at least one layer of
parallel hollow fibers. The amount of hollow fibers depends on the
cutoff size of the first and second barriers as well as on the
diameter of hollow fibers determining their area. If the ratio of
the cutoff sizes of the first and second barriers is from 3 up to
10, the area of the first barrier is taken from 0.2 to 1.0 of that
of the second barrier.
[0075] FIG. 2b shows the case when the reaction layer is formed
between two layers of hollow fibers functioning as the first
barrier 110 and second barrier 120. In every layer hollow fibers
are placed parallel to each other. The central axes of hollow
fibers placed in different layers are either parallel or are at an
angle from 70 degrees to 110 degrees. The ratio of the amount of
hollow fibers in the first layer to the amount of hollow fibers in
the second layer is taken to be from 1 to 0.1 depending on the
ratio of the cutoff sizes. Most preferable is such a design of this
module when the total volume of reactors is assembled from several
modules. In this case the modules are located so that layers of
hollow fiber from the first and second barriers are alternated,
thus allowing to distribute uniformly the flows of low and high
molecular weight components via the whole volume of the
reactor.
[0076] When the synthesis is carried out with cell-free systems
whose efficiency is from 2 to 4 times higher than the known level
in 100-200 Mg (for example if concentrate reaction mixture is
used), it is necessary to improve the feed solution input to the
reactor and decrease the influence of closing of membrane pores by
products of synthesis. To ensure uniform distribution of the feed
solution over the entire volume of the reaction mixture and to
lower the closing of the cutoff, a module with three porous
barriers is used. In this case a layer of hollow fibers parallel to
each other is used as the first porous barrier. This layer is
placed in the middle part of the module between two porous
barriers, which function as the second porous barrier with a small
size of the cutoff. In this variant the area of the second porous
barrier is doubled, that positively influences the removal of low
molecular weight products inhibiting the synthesis. This permits to
lower the pressure upon the formation of the flow of removed low
molecular weight products.
[0077] FIG. 2c shows a reactor module in which the reaction layer
is placed between two flat membranes with a layer of hollow fibers
between the latter. This design of the module is more preferable
when one reactor unit is formed from several modules. In this case
zones for removal of low molecular weight components are joined
together and the number and length of liquid communications is
reduced.
[0078] FIG. 2d shows a design of the module from three layers of
hollow fibers. Hollow fibers in each layer are placed parallel to
each other. The middle layer is placed in such a manner that the
central axes of its hollow fibers are at an angle from 70 up to 110
degrees relative to the central axes of the other layers or are
parallel. It is preferable to use perpendicular allocation of the
axes. In this case the area of contacting zones between the
surfaces of the first, second and third layers of hollow fibers is
reduced if no grids or porous layered materials are placed between
the layers. The ratio of the amount of hollow fibers in the first
and third layers to the amount of hollow fibers in the second layer
is accepted from 1 up to 0.1 depending on the ratio of the cutoff
sizes of the hollow fibers.
[0079] In the above versions of the reactor modules the shape of
sheet membranes and sheets consisting of one-layer hollow fibres
can be either square or round. FIG. 2e and FIG. 2f show reactor
modules having the form of cylinders in which the reaction volume
has the shape of (i) a helix or (ii) a cylinder.
[0080] A difference from the known designs is the formation of a
two-layer construction with different cutoff sizes and diameters of
hollow fibers used. FIG. 2e represents a module in which the
central element made of a hollow fiber plays the role of the first
porous barrier with a cutoff up to 100 kD. It is coated by fibrous
material to prevent direct contact of the first and second porous
barriers. Then hollow fibers of the second porous barrier are
placed around the central element. The amount of hollow fibers in
the second porous barrier and the area of their surface should
exceed those of the first barrier by no less than 5-10 times. Then
a beam of hollow fibers is placed in a cover which has an input and
output for the reaction mixture and its end faces are glued as
described by Yagihashi et al. (1996), forming an output for one
porous barrier from one side of the cylindrical cover and an output
for the other porous barrier from the opposite side. FIG. 2f shows
a two-layer design in which porous or layered material is placed
between the two layers. Then preliminarily prepared sheets of their
single-layer constructions playing the role of the first and second
barriers are curled and placed inside the cylindrical cover with
input 151 to the reactor. Internal outputs of each hollow fiber of
the first barrier are united in a common output 161 from one part
of the cylinder, and internal outputs of each hollow fiber of the
second barrier are united in a common output on the opposite part
of the cylinder.
[0081] The form of the reaction module is chosen depending on
conditions providing for the following: washing the reaction zone,
accessible input of the reaction mixture to the reactiony zone
without formation of air zones, maintenance of minimal volumes in
the zones of input/removal of feed solution and low and high
molecular weight components of synthesis. Of great importance is
the simplicity of assembly and disassembly of the module for
clearing-the cutoffs of membranes and hollow fibers. An assembly
construction (when at least one module is used) is formed by
installation of membranes, hermetic layers, top and bottom covers
into the frame of the module. It is preferable that one porous
barrier is pasted to the frame and the other barrier is removable.
This provides an opportunity of easy access to the reaction zone
and enables to determine defects of membranes or hollow fibers that
can appear during operation.
[0082] It is preferable to use mould materials which can perform
two functions, i.e. be a support for porous barriers and
hermetically seal the layers. Such materials can be silicon
hermetic or other synthetic materials with good adhesion to
polymeric materials from which the membrane and hollow fibers are
made. The properties of these materials should provide repeated
restoration of the form after elimination of squeezing effort.
[0083] Porous barriers can be made of different materials.
Nevertheless most preferable are those which can allow regeneration
and purification of pores after termination of synthesis without
disassembly of the reactor or at its disassembly and subsequent
assembly. Clearing of many types of membranes and hollow fibers are
described in detail in technical catalogues of firms (Operating
Guide, 1997).
[0084] Cutoffs of the first porous barrier 110 are chosen from 30
up to 300 kD. For most polypeptides with molecular weight of 20-40
kD it is more preferable to use cutoffs of 50-100 kD. Cutoffs of
the second barrier 120 are taken from 1 up to 30 kD depending on
the molecular weight of synthesized polypeptides and conditions of
passage through this barrier of the given flow of low molecular
weight components. For synthesis of polypeptides with molecular
weight of 20-40 kD it is preferable to use cutoffs of barrier 120
from 10 to 12 kD.
[0085] FIGS. 3-6 show examples of schemes of devices for different
modes of operation. The most simple device for synthesis of
polypeptides (FIG. 3) uses a mode without removal of high molecular
weight components from the reaction mixture. It consists of at
least one reaction module 100, pump 312 and capacities for the feed
solution 315 and waste compatment 316 for low molecular weight
components. The reaction mixture enters the reactor through input
151. By pump 312 the feed solution is supplied through input 161
and first barrier 110 to the reaction mixture. Low molecular weight
components inhibiting operation of the system, are removed from the
reactor via second porous barrier 120 and output 142 and come to
waste compartmemt 316.
[0086] In another example when the experimenter chooses a mode of
continuous removal of the product, the flows are distributed as
follows. At the first step installation of the system for
polypeptide synthesis (FIG. 4) is done using reactor 100 which
consists of at least one reaction module with two porous barriers,
pumps 312 and 314, capacities for storage of feed solution 315,
waste fraction 316 and high molecular weight (with the targed
polypeptide) fraction 317. The device works in the following way.
The reaction mixture enters the reaction zone of the module via
liquid communication input 150, the feed solution from capacity 315
is supplied to the next input 151 of this module through liquid
communications 210 and pump 312 and is distributed over the entire
volume of the reactor, thus displacing products of synthesis. High
molecular weight products of synthesis, including synthesized
polypeptides, penetrate through the first porous barrier 110 and
are removed from the reactor via pump 314 with a given speed. Due
to the pressure created by pump 312 low molecular weight components
are removed through the second porous barrier 120 and output 142,
which is connected directly to waste compartment 316. The size of
the cutoff of the first and second porous barriers as well as the
speed of pumps depend on conditions of the synthesis. The design of
the module used for this mode requires the presence of at least two
porous barriers in the form of two flat membranes (FIG. 1a) or two
layers of hollow fibers (FIGS. 2b,e,f), or a combination when one
of barriers is a membrane and the other consists of hollow fibers
(FIG. 2a).
[0087] When conditions of synthesis require a uniform input of the
feed solution in all points of the reaction zone, a device with
reactor modules consisting of three porous barriers is used. Such a
device is shown in FIG. 5. The reaction mixture enters the reactor
through input 151. The flow of feed solution 210 enters the reactor
from capacity 315 via liquid communications 230, input 142 and
porous barrier 121. The type of a porous barrier depends on
conditions of synthesis. It is more expedient that the first
barrier 110 is made from hollow fibers and two second barriers 121
and 122 are formed from flat membranes or hollow fibers. Through
porous barrier 121 the feed solution penetrates into the reactor on
the thin layer of the reaction mixture due to negative pressure in
the second part of porous barrier 122. The negative pressure is
created by pump 312 whose input is connected to output 143
connected with internal surfaces of the second porous barrier
122.The output of pump 312 is connected to waste compartment 316
for low molecular weight components. High molecular weight
components of products are removed through the first barrier 110
which is made from hollow fibers placed in regular intervals over
the thin layer of the reaction mixture. The internal part of hollow
fibers is incorporated in a common output 161, which is connected
to the input of pump 314, whose output is connected to collector
317 for high molecular weight components.
[0088] When it is required to carry out preparative synthesis and
the reaction mixture volume is from 1 ml up to 500 ml, a parallel
connection of N reaction modules is used. The scheme of a device
using a parallel connection of N modules is shown on FIG. 6. This
example is based on periodic removal of the product when high
molecular weight components of target products are removed
periodically at the end of each cycle with simultaneous automated
clearing of pores of the first and second porous barriers. Reactor
100 is filled with the reaction mixture through input 151. In the
initial condition valves V3 and V4 are open whereas valves V1 and
V2 are closed. At the first step of a cycle the feed solution moves
from capacity 315 through pump 312 and valve V3 simultaneously to
all N inputs 161.
[0089] Then the feed solution passes through internal apertures of
hollow fibers or across the internal surface of the membrane from
which the first porous barrier 110 is formed. Through pores of the
first porous barrier 110 the feed solution penetrates readily to
volume with reaction system. Low molecular weight components of the
synthesis pass through the second porous barrier 120 with a small
cutoff and are removed through N outputs 142 and open valve V4 in
waste compartment 316 for the low molecular weight components. On
termination of the first step of a cycle and beginning of the
second step valves V3 and V4 are closed and valves V1 and V2 open.
Pump 313 creates negative pressure in the channel connected to
valve V2. Under negative pressure, high molecular weight components
of synthesis including target polypeptide leave the reactor through
the first barrier 110 and come to fraction collector 317 via output
161, valve V2 and pump 313. At the same time the feed solution
comes to the reactor from capacity 315, input 141 and second
barrier 120 under the action of negative pressure created by pump
313. The flow of the feed solution via,the second porous barrier
120 at the second step of the cycle has a different direction than
that low molecular weight components formed at the first step of
cycles. Therefore pores of the second barrier 120 which could be
closed at the first step of a cycle open during the second step of
the cycle and hydraulic resistance of second barrier 120 is
restored. After termination of the second step of a cycle its first
step is formed. The flow of hihg molecular weight components via
the first porous barrier 110 is stopped. At the first step of a
cycle the flow via first porous barriers changes its direction and
pores of the first barrier 110 which could be closed at the second
step of a cycle by high molecular weight components open and
hydraulic resistance of the first barrier 110 is restored. A cycle
terminates with the end of the second step, and the control device
monitors switching of valves thus giving impetus for the formation
of the first step of the cycle. The ratio of the feed solution
volume entering the reactor and the volume of the fraction of high
molecular weight components removed from the reactor is changed by
varying temporary ratios of the times of the first and second steps
of a cycle and changing its total duration.
[0090] It is known (Kim et al., 1996) that by raising the
concentration of lysate it is possible to increase the yield of
synthesized polypeptides. The data reported in the cited art relate
to a batch type reaction, when the products inhibiting the
cell-free system are not removed from the reaction mixture. This
reduces the yield of the synthesized product. It is possible to
raise the yield of polypeptides by removing low molecular weight
components of products from the reactor and supplying high
molecular weight components which lose their activity during
synthesis. Experiments have shown that on removal of low and high
molecular weight components of products from the reactor it is
possible to add in the reactor not only the feed solution but such
components of lysate as ribosomes fraction, extracts (S30, S100 and
others), polymerases (T7, T5, SP6 and others), plasmids, tRNA,
enzymes or their combinations.
[0091] The proposed construction of a reactor module allows to add
the feed solutution and lysate components to the reactor in the
following ways: (a) the whole volume of feed solution is supplied
via the reactor input; (b) the whole volume of feed solution is
supplied via the first barrier; (c) part of the feed solution is
supplied via the first barrier and the other part via the reactor
input; (d) part of the feed solution is supplied via the first
barrier during the first step of a cycle and the other part is
supplied via the second barrier during the second step of the
cycle; (e) the whole volume of lysate components is supplied via
the reactor input together with the whole volume of the feed
solution or its part; (f) the biggest components of lysate
(ribosomes and others) and part of the feed solution are supplied
via the input of the reaction zone while small components of lysate
and part of the feed solution are supplied in the reaction zone via
the pores of at least one barrier. As an example FIG. 5 shows the
case when pump 314 can supply high molecular weight components of
lysate from capacity 318 as monitored by the controller.
[0092] Reactor modules are thermostated from 20.degree. C. to
40.degree. C. (the usual range is from 25.degree. C. to 37.degree.
C.). It is preferable if the feed solution temperature is from
+2.degree. C. to +7.degree. C. The pumps are hand-operated or
monitored by controller block 400. This block should provide
programming of modes of operation. The automated systems on the
basis of computers developed by Roche Diagnostic Boehringer
Mannheim have been shown to yield very good (Simonenko, 1998). The
controller block allows to adjust the duration of cycles and the
ratio of two steps in a cycle.
[0093] At installation, for example, of pressure gauges it is
possible to trace the change of pressure in the liquid circuits by
the level of closing of the cutoff and to change conditions of the
process in due time.
[0094] The proposed method of flow branching provides synthesis of
polypeptide in a cell-free prokaryotic and eukaryotic extracts with
high speed during tens of hours with removal of functionally active
products. As an example the synthesis of fibrous GFP is given. The
synthesis was carried out with the help of a device whose block
diagram is given in FIG. 4. Ultrafiltration membrane with the
cutoff from 50 kD to 10 kD were used as the first and second porous
barriers in the reactor module. The volume of the reaction zone was
400 .mu.l.
[0095] In the given example the method of preparative synthesis of
polypeptides in the conjugated system of transcription/translation
is used (Baranov et al., 1989). Most frequently estimation of the
efficiency of cell-free systems is made by measuring the amount of
radioactive amino acid contained in the synthesized polypeptides
(Alakhov et al., 1991). As an alternative method, specific
properties of polypeptides such as fluorescence (Kolb et al., 1996;
Crameri et al., 1996) are employed.
[0096] The S30 extract from E. coli is prepared by a modified
method (Zubay, 1973) as follows.
[0097] E. coli A19 cells are grown to optical density of the
culture--0.8 at the wavelength of 582 nm. The cells are collected
by centrifugation. The obtained biomass is washed twice being
resuspended in buffer A: 10 mM Tris-Ac pH 8.2, 14 mM Mg(OAc).sub.2,
60 mM KCl, 1 mM dithiothreitol by centrifugation for 30 min at
10,000 g. The washed biomass is resuspended in buffer A with the
ratio 4 volumes of the buffer to 1 volume of cells. The obtained
suspension is destroyed by the French-press at the pressure drop of
1600 bars. Destruction is carried out at +4.degree. C. Commercial
protease inhibitors in concentrations recommended by their
manufacturers are added to the obtained cell extract, and the
mixture is centrifuged for 30 min at 30,000 g. Eliminating
agitation, 2/3 of the supernatant volume is collected. The volume
collected is centrifuged once again for 30 min at 30,000 g.
Eliminating agitation, 2/3 of the supernatant volume is collected.
All procedures are carried out at +4.degree. C. Buffer B containing
750 mM Tris-Ac pH 8.2, 21 mM Mg (OAc).sub.2, 7.5 mM dithiothreitol,
6 mM ATP, 500 mM acetylphosphate, 500 .mu.M of each of 20 amino
acids. The obtained solution is incubated at 37.degree. C. during
80 min. After termination of incubation the extract is dialyzed
against buffer C: 10 mM Tris-Ac pH 8.2, 14 mM Mg (OAc).sub.2, 60 mM
KOAc, 0.5 mM dithiothreitol during 16 h. The dialysis is carried
out at +4.degree. C. in a 500-fold volume of buffer C with two
changes. After termination of dialysis the resulting volume is
centrifuged for 30 min at 10,000 g, selected in aliquots and frozen
in liquid nitrogen for subsequent storage at -70.degree. C.
[0098] The coupled system of transcription/translation is prepared
as follows.
[0099] 1 ml of the reaction mixture contains 200-400 .mu.l of the
S30 extract from E. coli, 0.1-0.5 mg of the total tRNA from E.
coli, 0.01-0.03 of the plasmid superhelical DNA, 2000-3000 U
DNA-dependent RNA from polymerase of bacteriophage T7, 10-50 U of
ribonuclease inhibitor of human placenta in buffer D: 50-100 mM
HEPES-KOH, or 50-100 mM Tris-Ac, or 50-100 mM TES-KOH, or 50-100 mM
MOPS-KOH, or 50-100 mM BES-KOH, pH 7.0-7.6. To the same reaction
mixture we add low molecular weight compounds containing 10-20 mM
Mg (OAc).sub.2 or MgCl.sub.2, 120-230 mM KOAc or K-L-glutamate,
1.0-2.0 mM ATP, 1.0-2.0 mM GTP, 0.8-1.5 mM CTP, 0.8-1.5 mM UTP,
25-40 mM acetylphosphate, 40 .mu.g/ml leucovorin, 1 mM
dithiothreitol, 4% glycerol, 0.02% NaN.sub.3 and 150-250 .mu.M of
each of 19 amino acids excepting the one with which control
synthesis in an aliquot of a fixed volume is carried out.
[0100] The prepared transcription/translation system is divided in
two unequal volumes. The smaller volume (30-50 .mu.l) is placed in
a microtube and the amino acid containing a radioactive label is
added to it. Then aliquots of 5-10 .mu.l are taken from the
microtube to estimate the kinetics of the synthesis in a fixed
volume. The amount of synthesized polypeptides is determined by
aliquot precipitation on a glass fibrous filter with trichloracetic
acid, with particles irradiated by the isotope being counted in a
liquid scintillator. Then the lacking amino acid in the
concentration of 150-250 .mu.M is added to the remaining volume and
after slight and cautious stirring is placed in the reaction cell.
Synthesis is carried out at 26 to 37.degree. C. passing the feed
solution through the reaction mixture to the reactor module.
[0101] The feed solution is prepared as follows.
[0102] 1 ml of the feed solution contains the following low
molecular weight substances: 10-20 mM Mg (OAc).sub.2 or MgCl.sub.2,
120-230 mM KOAc or K-L-glutamate, 1.0-2.0 mM ATP, 1.0-2.0 mM GTP,
0.8-1.5 mM CTP, 0.8-1.5 mM UTP, 25-40 mM acetylphosphate, 40
.mu.g/ml leucovorin, 1 mM dithiothreitol, 4% glycerol, 0.02%
NaN.sub.3 and 150-250 .mu.M of each of 20 amino acids in buffer D.
Buffer D contains 50-100 mM HEPES-KOH, or 50-100 mM TES-KOH, or
50-100 mM MOPS-KOH, or 50-100 mM BES-KOH, pH 7.0-7.6. To the same
solution we add 200-400 .mu.l of buffer C containing 10 mM Tris-Ac
pH 8.2. 14 mM Mg (OAc).sub.2, 60 mM KOAc, 0.5 mM
dithiothreitol.
EXAMPLE 1
[0103] The coupled transcription/translation of green fluorescent
protein (GFP) of coelenterate bacterium Aequoria victoria from a
DNA template containing a GFP gene, in continuous cell-free system
from E. coli, with branched flows.
[0104] A plasmid DNA containing a mutant gene of GFP and nucleotide
sequences of a ribosome-binding site and a promotor of the
DNA-dependent RNA-polymerase of bacteriophage T7 is used as a
template.
[0105] The coupled transcription/translation system is prepared as
follows.
[0106] 1 ml of the reaction mixture contains 200-400 .mu.l of the
S30 extract from E. coli, 0.1-0.5 ml of the total E. coli tRNA
preparation, 0.01-0.03 mg of plasmid superhelical DNA, 2000-3000 U
of DNA-dependent RNA polymerase of bacteriophage T7, 10-50 U
ribonuclease of human placenta inhibitor in buffer E (100 mM
HEPES-KOH, pH 7.6). To the same reaction mixture we add low
molecular weight substances containing 12 mM Mg(OAc).sub.2, 220 mM
KOAc, 1.2 mM ATP, 1.0 mM GTP, 0.8 mM CTP, 0.8 mM UTP, 30 mM
acetylphosphate, 40 .mu.g/ml of leucovorin, 1 m dithiothreitol, 4%
glycerol, 0.02% NaN.sub.3 and 160 .mu.M of each of 19 amino acids
excepting leucine.
[0107] The prepared transcription/translation system is divided in
two unequal volumes. The smaller volume (30-50 .mu.l) is placed in
a microtube and [.sup.14C]-L-leucine with specific radio-activity
38 mCu/mmol and 100 .mu.M concentration is added to it. Then
aliquots of 5-10 .mu.l are selected from the microtube to estimate
the kinetics of synthesis in a fixed volume. The amount of the
synthesized polypeptide is determined by aliquot precipitation on
the glass fibrous filter by trichloroacetic acid with subsequent
estimation of irradiated particles in a liquid scintillation
counter. Leucine in 160 .mu.M concentration is added to the
remainder volume and after slight and cautious mixing the
preparation is placed in a reaction cell.
[0108] The synthesis is carried out at 26.degree. C. in the
reaction cell. The feed solution is supplied to the reaction cell
at a rate equal to its 1.5-2 internal volumes per 1 hour. The
product is removed at a rate equal to {fraction (1/20)}-1/8 of the
internal volume of the cell per 1 hour. During the entire synthesis
the specific fluorescence of the product removed through a membrane
with the cutoff of 50 kD is recorded. The efficiency of the system
is estimated by fluorescence of all assembled volumes. FIG. 7
represents a histogram showing the amount of GFP in fractions
collected during synthesis. The amount of GFP in the fractions is
estimated by the calibration curve plotted using the data of
specific fluorescence measurements of the purified GFP preparation
versus its concentration. FIG. 8 shows a diagram of dependence of a
change in the concentration of synthesized GFP on the time of
synthesis in a control aliquot incubated in a constant volume. The
concentration is determined by incorporation of radioactive amino
acid in the polypeptide. In addition the newly synthesized product
is controlled by registration of fluorescence after electrophoresis
of all assembled volumes in polyacrylamide gel. FIG. 9 shows a
photo of gel electrophoresis with aliquots selected from fractions
collected during synthesis. A purified GFP preparation is used as
control. The amount of layered protein is 0.3 mg.
[0109] A portion of 150-200 .mu.g of a functionally active product
is obtained during 48 h per 1 ml of the coupled
transcription/translation system.
EXAMPLE 2
[0110] The coupled transcription/translation of green fluorescent
protein (GFP) of coelenterate bacterium Aequoria victoria from a
DNA template containing a GFP gene in a continuous cell-free system
from E. coli with one flow.
[0111] The DNA is obtained and the coupled
transcription/trnaslation system is prepared as described in
Example 1. The synthesis is carried out using a device whose block
diagram is given in FIG. 3. Hollow fibers of 1 mm in diameter and
the cutoff size of 100 kD were used as the first porous barrier in
the reactor module. An ultrafiltration membrane with the cutoff
size of 10 kD and the area of 4 cm.sup.2 was used as the second
porous barrier. The reactor volume was 400 .mu.l.
[0112] The feed solution flow is supllied to the reaction cell at a
rate equal to its 1.5-2 internal volumes per 1 hour. The product is
not removed.
[0113] The efficiency of the system is estimated from the
fluorescence of the solution removed upon termination of synthesis
from the reaction cell with a subsequent determination of the
amount of the polypeptides synthesized. Then it is compared to, the
efficiency of control synthesis in a fixed volume. FIG. 10 shows a
diagram of dependence of a change in the concentration of
synthesized GFP on the duration of synthesis in the control aliquot
incubated in a constant volume. The concentration is determined by
incorporation of radioactive amino acids in the polypeptide. In
addition the newly synthesized product is controlled by recording
fluorescence after electrophoresis of all collected volumes in
polyacrylamide gel. FIG. 11 represents a photo of gel
electrophoresis with aliquots selected from the coupled
transcription/translation system in a fixed volume and from the
reaction cell after termination of synthesis. A purified
preparation of GFP is used as control. The amount of layered
protein is 0.3 mg.
[0114] During synthesis 60 .mu.g of a functionally active product
are obtained for 24 h per 1 ml of the coupled
transcription/translation system.
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* * * * *