Note: Descriptions are shown in the official language in which they were submitted.
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IIV VITRO PROTEIN SYNTHESIS USING GLYCOLYTIC INTERMEDIATES AS AN ENERGY
SOURCE
INTRODUCTION
The directed synthesis of proteins and other biological macromolecules is one
of the great
achievements of biochemistry. The development of recombinant DNA techniques
has allowed the
characterization and synthesis of highly purified coding sequences, which in
turn can be used to
produce highly purified proteins, even though in native cells the protein may
be available only in
trace amounts. Polypeptide chains can be synthesized by chemical or biological
processes. The
biological synthesis may be performed within the environment of a cell, or
using cellular extracts
and coding sequences to synthesize proteins in vitro.
For several decades, in vitro protein synthesis has served as an effective
tool for lab-scale
expression of cloned or synthesized genetic materials. In recent years, in
vitro protein synthesis
system has been considered as an alternative to conventional recombinant DNA
technology,
because of disadvantages associated with cellular expression. In vivo,
proteins can be degraded
or modified by several enzymes synthesized with the growth of the cell, and
after synthesis may
be modified by post-translational processing, such as glycosylation,
deamination or oxidation. In
addition, many products inhibit metabolic processes and their synthesis must
compete with other
cellular processes required to reproduce the cell and to protect its genetic
information.
Because it is essentially free from cellular regulation of gene expression, in
vitro protein
synthesis has advantages in the production of cytotoxic, unstable, or
insoluble proteins. The over-
production of protein beyond a predetermined concentration can be difficult to
obtain in vivo,
because the expression levels are regulated by the concentration of product.
The concentration of
protein accumulated in the cell generally affects the viability of the cell,
so that over-production of
the desired protein is difficult to obtain. In an isolation and purification
process, many kinds of
protein are insoluble or unstable, and are either degraded by intracellular
proteases or aggregate
in inclusion bodies, so that the loss rate is high.
In vitro synthesis circumvents many of these problems. Also, through
simultaneous and
rapid expression of various proteins in a multiplexed configuration, this
technology can provide a
valuable toot for development of combinatorial arrays for research, and for
screening of proteins.
In addition, various kinds of unnatural amino acids can be efficiently
incorporated into proteins for
specific purposes (Noren et al. (1989) Science 244:182-188). However, despite
all its promising
aspects, the in vitro system has not been widely accepted as a practical
alternative, mainly due to
the short reaction period, which causes a poor yield of protein synthesis, and
to the high cost of
the reaction components.
The development of a continuous flow in vitro protein synthesis system by
Spirin et al.
(1988) Science 242:1162-1164 proved that the reaction could be extended up to
several hours.
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Since then, numerous groups have reproduced and improved this system (Kigawa
et al. (1991) J.
Biochem. 110:166-168; Endo et al. (1992) J. Biotechnol. 25:221-230. Recently,
Kim and Choi
(1996) Biotechnol. Prog. 12: 645-649, reported that the merits of batch and
continuous flow
systems could be combined by adopting a 'semicontinuous operation' using a
simple dialysis
membrane reactor. They were able to reproduce the extended reaction period of
the continuous
flow system while maintaining the initial rate of a conventional batch system.
However, both the
continuous and semi-continuous approaches require quantities of expensive
reagents, which must
be increased by a significantly greater factor than the increase in product
yield.
Several improvements have been made in the conventional batch system (Kim et
al.
(1996) Eur. J. Biochem. 239: 881-886; Kuldlicki et al. (1992) Anal. Biochem.
206:389-393;
Kawarasaki et al. (1995) Anal. Biochem. 226: 320-324). Although the
semicontinuous system
maintains the initial rate of protein synthesis over extended periods, the
conventional batch system
still offers several advantages, e.g. convenience of operation, easy scale-up,
lower reagent costs
and excellent reproducibility. Also, the batch system can be readily conducted
in multiplexed
formats to express various genetic materials simultaneously.
Recently, Patnaik and Swartz (1998) Biotechniques 24:862-868 reported that the
initial
specific rate of protein synthesis could be enhanced to a level similar to
that of in vivo expression
through extensive optimization of reaction conditions. It is notable that they
achieved such a high
rate of protein synthesis using the conventional cell extract prepared without
any condensation
steps (Nakano et al. (1996) J. Biotechnol. 46:275-282; Kim et al. (1996) Eur.
J. Biochem. 239:881-
886). Kigawa et al. (1999) FEBS Lett 442:15-19 report high levels of protein
synthesis using
condensed extracts and creatine phosphate as an energy source. Their result
implies that further
improvement of the batch system, especially in terms of the longevity of the
protein synthesis
reaction, would substantially increase the productivity for batch in vitro
protein synthesis.
However, the reason for the early halt of protein synthesis in the
conventional batch system has
remained unclear. Kim and Swartz (1999) Biotechnol.Bioeng. 66(3): 180-188
describe a novel
ATP regeneration system.
As shown from the above, both protein productivity and production amount are
still low,
which is an obstacle in implementing the industrialization of cell-free
protein synthesis. Therefore,
improvements are greatly required in terms of the total productivity.of the
protein by increasing the
specific production rate and the length of system operation. Optimizing these
conditions of great
interest for development of commercial processes.
SUMMARY OF THE INVENTION
Compositions and methods are provided for the enhanced in vitro synthesis of
protein
molecules. Glycolytic intermediates or glucose are used as an energy source,
in combination with
NADH or NAD+ added in catalytic quantities. Coenzyme A may also be included in
the reaction
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mix. In addition, inhibition of enzymes catalyzing undesirable reactions is
achieved by: addition of
inhibitory compounds to the reaction mix; modification of the reaction mixture
to decrease or
eliminate the responsible enzyme activities; or a combination of the two.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph illustrating the synthesis of chloramphenicol acetyl
transferase. 33 mM
sodium pyruvate, 0.33 mM NAD and 0.27 mM CoA were added in the indicated
combinations to
15 wL reaction mixtures to regenerate ATP during the synthesis reaction.
Reactions were carried
out for 2 hours and TCA-insoluble radioactivities were measured. In the
control reaction, 33 mM
PEP was used instead of pyruvate and cofactors.
Figure 2. Proposed mechanism of ATP regeneration with pyruvate.
Figures 3A and 3B. Time course of protein synthesis and ATP concentration. 120
wL
standard reaction mixtures with 33 mM PEP were prepared and incubated in the
presence of 0.33
mM NAD, 0.27 mM CoA, and 2.7 mM sodium oxalate. In order to measure the ATP
concentrations (A), 10 ~.L samples were withdrawn, mixed with the same volume
of 10 % TCA
solution, and centrifuged for 10 min. 10 wL of the supernatant was used for
ATP analysis as
described in the Materials and Methods. At the given time points, 5 w1 samples
were taken and
TCA-insoluble radioactivities were counted to measure protein synthesis (B).
Open circles, control
reaction; filled circles, with NAD and CoA; asterisks, with NAD, CoA and
oxalate; filled squares,
with NAD, CoA, oxalate, and 2 mM amino acids. At the end of each reaction, 5
~L samples were
taken to run a 16% SDS-PAGE gel. The gel was stained with Coomassie Blue
following the
standard procedures (inset of Figure 3A). Lanes M, standard molecuiarweight
markers; C, control
reaction without the template plasmid; 1, standard reaction; 2, reaction with
0.33 mM NAD and
0.27 mM CoA; 3, reaction with 0.33 mM NAD, 0.27 mM CoA, and 2.7 mM sodium
oxalate; 4,
reaction with 0.33 mM NAD, 0.27 mM CoA, 2.7 mM sodium oxalate, and 2 mM amino
acids.
Figures 4A and 4B. Supplementation of PEP, amino acids, and magnesium during
protein
synthesis. A synthesis reaction was carried out in the presence of 2 mM amino
acids, 33 mM
PEP, 0.33 mM NAD, 0.27 mM CoA, and 2.7 mM sodium oxalate in a 120,1 volume.
During the
incubation period, the initial concentrations of PEP, amino acids, and
magnesium acetate were
added to the reaction every hour. 5~1 samples were taken at the given time
points to measure the
concentration of ATP (Figure 4A) and the yield of CAT synthesis (Figure 4B).
The same volumes
of water were added to the single batch reaction. Open circles, single batch
reaction; filled circles,
reaction with the additions. Samples taken at the end of each reaction were
analyzed on a SDS-
polyacrylamide gel followed by Coomassie Blue staining (inset of panel B). M,
standard molecular
weight markers; C, control reaction without template plasmid; B, sirigle batch
reaction; FB, reaction
with the additions of PEP, amino acids, and magnesium acetate. The arrow
indicates the
expressed CAT.
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Figures 5A and 5B. Expression of CAT using glucose-6-phosphate as the
secondary
energy source. 33 mM glucose-6-phosphate, 0.33 mM NAD and 0.27 mM CoA were
added to a
120 pL synthesis reaction. 5 wL and 10 p,L samples were taken to determine
protein synthesis and
ATP concentration respectively and were assayed as in Figure 3. Open circles,
conventional
reaction using PEP; closed circles, reaction using glucose-6-phosphate as the
energy source.
Figure 5A, time course of CAT synthesis; Figure 5B, time course of ATP
concentration.
DETAILED DESCRIPTION OF THE EMBODIMENTS
Compositions and methods are provided for the enhanced in vitro synthesis of
protein
molecules, by the use of glycolytic pathways in the generation of ATP to drive
the reaction. In
order to maintain activity of fhe glycolytic pathway in the reaction mix,
NAD+/NADH is added to the
reaction. Exemplary is the use of glucose in combination with the enzyme
hexokinase; pyruvate;
or phosphoenol pyruvate (PEP) as the energy source. In a preferred embodiment,
acetyl CoA is
also included in the reaction mixture. The phosphate that is hydrolyzed from
ATP is recycled
during the glucose or pyruvate oxidation, thereby preventing a net
accumulation of free phosphate,
which can have an inhibitory effect on synthetic reactions.
DEFINITIONS
It is to be understood that this invention is not limited to the particular
methodology,
protocols, cell lines, animal species or genera, and reagents described, as
such may vary. It is
also to be understood that the terminology used herein is for the purpose of
describing particular
embodiments only, and is not intended to limit the scope of the present
invention which will be
limited only by the appended claims.
As used herein the singular forms "a", "and", and "the" include plural
referents unless the
context clearly dictates otherwise. Thus, for example, reference to "a cell"
includes a plurality of
such cells and reference to "the protein" includes reference to one or more
proteins and
equivalents thereof known to those skilled in the art, and so forth. All
technical and scientific terms
used herein have the same meaning as commonly understood to one of ordinary
skill in the art to
which this invention belongs unless clearly indicated otherwise.
Glucose or glycolytic intermediate energy source, as used herein, refers to
compounds that
provide energy for the synthesis of ATP from ADP, and which are part of the
glycolytic pathway.
These energy sources include glucose, glucose-1-phosphate, glucose-6-
phosphate, fructose-6-
phosphate, fructose-1,6-diphosphate, triose phosphate, 3-phosphoglycerate, 2-
phosphoglycerate,
phosphoenol pyruvate (PEP) and pyruvate. Preferred energy sources are PEP,
pyruvate, and
glucose-6-phosphate.
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The energy sources may also be homeostatic with respect to phosphate, that is
they do not
result in the accumulation of inorganic phosphate, Such secondary sources of
energy recycle the
free phosphate generated by ATP hydrolysis. Instead of exogenous addition of a
source of high
energy phosphate bonds, the required high energy phosphate bonds are generated
in situ, e.g.
through coupling with an oxidation reaction. A homeostatic energy source will
typically lack high
energy phosphate bonds itself, and will therefore utilize free phosphate
present in the reaction mix
during ATP regeneration. Since inorganic phosphate can be an inhibitory by-
product of synthesis,
the period of time when synthesis is maintained in vitro can be extended. A
homeostatic energy
source may be provided in combination with an enzyme that catalyzes the
creation of high energy
phosphate bonds.
Exemplary glycolytic intermediates that are homeostatic for phosphate
metabolism are
pyruvate and glucose. When glucose is used, it is desirable to include the
enzyme hexokinase if
not already present in the cell extract. However, it has been found that in
the presence of NADH,
it is not necessary to include a regenerative enzyme, such as pyruvate
oxidase.
The energy source may be supplied as a suitable biologically acceptable salt
or as the free
acid, e.g. pyruvic acid, where applicable. The final concentration of energy
source at initiation of
synthesis will usually be at least about 1 mM, more usually at least about 10
mM, and not more
than about 1000 mM, usually not more than about 100 mM. Additional amounts may
be added to
the reaction mix during the course of synthesis to provide for longer reaction
times.
Cofactors: exogenous cofactor NADH or NAD+ (p-nicotinamide adenine
dinucleotide) is
added to the reaction mixture at a concentration of at least about 0.1 mM,
preferably 0.2 to 1 mM,
and usually not more than about 10 mM.
Optionally, acetyl CoA (acetyl coenzyme A) or coenzyme A is also included in
the reaction
mixture. Although not required for the use of glucose or glycolytic
intermediates as an energy
source, it has been found to enhance the reaction. The useful concentrations
are of least about
0.05 mM, usually at least about 0.1 mM, and not more than about 1 mM, usually
not more than
about 0.5 mM.
Use of Glucose: Where the homeostatic energy source is glucose, an enzyme will
be
included in the reaction mixture to catalyze the formation of glucose-6-
phosphate from glucose.
Hexokinase, EC 2.7.1.1, is generally used for this purpose. Hexokinase is
widely available
commercially, and has been isolated and cloned from a number of species.
Examples include the
enzymes corresponding to SwissProt P27595, HXK1 BOVIN; P19367, HXK1 HUMAN;
P17710,
HXK1 MOUSE; P05708, HXK1 RAT; Q09756, HXK1 SCHPO; P04806, HXKA YEAST; Q42525,
HXK_ARATH; P50506, HXK_DEBOC; P80581, HXK_EMENI; P33284, HXl4 KLULA; Q02155,
HXK_PLAFA; Q26609, HXK_SCHMA.
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Where glucose is the energy source, the reaction mix will comprise a
concentration of
hexokinase sufficient to maintain the ATP pool, usually at least about 0.1
U/ml, more usually at
least about 1 U/ml, and preferably at least about 10 U/ml, where the unit
definition is that 1 unit
reduces 1 pmole of NAD per minute in a coupled assay system with glucose-6-
phosphate
dehydrogenase at 30°C, pH 8Ø It will be understood by one of skill in
the art that higher
concentrations may be present, although generally at less than about 1000
U/ml.
The hexokinase may be provided in the reaction mix in a variety of ways.
Purified or semi-
purified enzyme may be added to the reaction mix. Commercial preparations are
available, or the
enzyme may be purified from natural or recombinant sources according to
conventional methods.
The genetic sequences of hexokinases may be used as a source of recombinant
forms of the
enzyme, for example S. cerevisiae hexokinase PII gene, accession number
M14410; or
hexokinase PII, accession number M14411, both described in Kopetzki et al.
(1985) Gene 39:95-
102, etc.
The enzyme may also be included in the extracts used for synthesis. For
example,
extracts can be derived from E. coli for protein synthesis. The E. coli used
for production of the
extracts may be genetically modified to encode a suitable hexokinase.
Alternatively, where the
synthetic reactions are protein synthesis, a template, e.g. mRNA encoding
hexokinase, plasmid
comprising a suitable expression construct of hexokinase, etc. may be spiked
into the reaction
mix, such that a suitable amount of hexokinase is produced during synthesis.
Use of Pyruvate and PEP: Aspartic acid and asparagine are formed from
phosphoenol
pyruvate. The enzyme phosphoenol pyruvate synthetase (pps) converts pyruvate
into PEP and
consumes 2 equivalents of high-energy phosphate bonds (as ATP is converted to
AMP) per
molecule of PEP synthesized. When pyruvate is being used as an energy source,
this enzyme
therefore has the potential to waste both pyruvate and ATP, thereby robbing
the protein synthesis
reaction of its energy supply.
Addition of oxalic acid, which has been reported to inhibit pps
(Narindrasorasak and
Bridger (1978) Can. J. Biochem. 56: 816-9), was able to extend the reaction
period both in the
PEP and pyruvate systems. With both pyruvate and PEP as energy sources,
inhibiting pps with
oxalic acid decreased the rate of asp/asn production and increased the protein
yield. Oxalic acid
is added at a concentration of at least about 0.5 mM, and not more than about
100 mM, usually at
least about 1 mM, and preferably at a concentration of about 3 mM.
For efficient use of energy source in both the PEP and the pyruvate system,
the genes for
E. coli pyruvate oxidase, which converts pyruvate into acetate consuming
oxygen, and/or
phosphoenol pyruvate synthetase (pps) can be disrupted or otherwise
inactivated. The coding
sequence for E. coli phosphoenol pyruvate synthetase may be accessed in
Genbank, no. X59381;
and is also published in Niersbach et al. (1992) Mol. Gen. Genet. 231:332-336.
The coding
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sequence for E. coli pyruvate oxidase may be accessed in Genbank, no. X04105;
and is also
published in Grabau and Cronan (1986) Nucleic Acids Res. 14:5449-5460.
In vitro synthesis: as used herein refers to the cell-free synthesis of
polypeptides in a
reaction mix comprising biological extracts andlor defined reagents. The
reaction mix will
comprise at least ATP, an energy source; a template for production of the
macromolecule, e.g.
DNA, mRNA, etc.; amino acids, nucleotides and such co-factors, enzymes and
other reagents that
are necessary for the synthesis, e.g. ribosomes, tRNA, polymerases,
transcriptional factors, etc.
Such synthetic reaction systems are well-known in the art, and have been
described in the
literature. The cell free synthesis reaction may be performed as batch,
continuous flow, or semi-
continuous flow, as known in the arfi.
Reaction mix; as used herein refers to a reaction mixture capable of
catalyzing the
synthesis of polypeptides from a nucleic acid template. The mixture may
comprise metabolic
inhibitors that decrease undesirable enzymatic reactions. Alternatively, or in
combination, the
enhanced reaction mix will be engineered through genetic or other processes to
decrease the
enzymatic activity responsible for undesirable side-reactions, that result in
amino acid depletion or
accumulation.
In a preferred embodiment of the invention, the reaction mixture comprises
extracts from
bacterial cells, e.g. E. coli S30 extracts, as is known in the art. For
convenience, the organism
used as a source of extracts may be referred to as the source organism. While
such extracts are
a useful source of ribosomes and other factors necessary for protein
synthesis, they can also
contain small amounts of endogenous enzymes responsible for undesirable side-
reactions that are
unrelated to protein synthesis, but which deplete ATP, pyruvate or other
reagents.
As used herein, the term endogenous is used to refer to enzymes, factors, etc.
present in
the extracts. Exogenous components are those that are introduced into the
extracts through
addition, and may be added at the time of synthesis, or may be added through
genetic or other
manipulation of the cells used as the starting material for extracts. For
example, plasmids
encoding an exogenous enzyme of interest may be added to the bacterial cells
priorto preparation
of the extracts.
Methods for producing active extracts are known in the art, for example they
may be found
in Pratt (1984), coupled transcription-translation in prokaryotic cell-free
systems, p. 179-209, in
Hames, B. D. and Higgins, S. J. (ed.), Transcription and Translation: a
practical approach, IRL
Press, New York. Kudlicki et al. (1992) Anal Biochem 206(2):389-93 modify the
S30 E. coli cell-
free extract by collecting the ribosome fraction from the S30 by
ultracentrifugation.
The extracts may be optimized for expression of genes under control of a
specific
promoter, (for example see Nevin and Pratt (1991) FEBS Lett 291(2):259-63,
which system
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consists of an E, coli crude extract (prepared from cells containing
endogenous T7 RNA
polymerase) and rifampicin (an E. coli RNA polymerase inhibitor)). Kim et al.
(1996) Eur. J.
Biochem. 239: 881-886 further enhance protein production by optimizing reagent
concentrations.
The reaction mix may comprise metabolic inhibitors of the undesirable enzyme
activity.
Frequently such inhibitors will be end-products of the reaction, that then
inhibit by a feedback
mechanism. The specific inhibitors are determined based on the metabolic
pathways of the
source organism. These pathways are well-known in the art for many bacterial
and eukaryotic
species, e.g. E. coli, S, cerevisiae, H, sapiens, etc. The inhibitor is added
at a concentration
sufficient to inhibit the undesirable enzymatic activity while increasing
protein synthesis. Pathways
of particular interest relate to the metabolism of pyruvate in E, coli cells,
including the synthesis of
aspartate from oxalacetate.
In an alternative embodiment to adding metabolic inhibitors, the undesirable
enzymes may
be removed or otherwise deleted from the reaction mix. In one embodiment of
the invention, the
coding sequence for the enzyme is "knocked-out" or otherwise inactivated in
the chromosome of
the source organism, by deletion of all or a part of the coding sequence;
frame-shift insertion;
dominant negative mutations, etc. The genomes of a number of organisms,
including E, coli, have
been completely sequenced, thereby facilitating the genetic modifications. For
example, a
markerless knockout strategy method is described by Arigoni et al. (1998) Nat
Biotechnol
16(9):851-6.
A preferred method for inactivating targeted genes is described by Hoang et
al. (1998)
Gene 212:77-86. In this method, gene replacement vectors are employed that
contain a
tetracycline resistance gene and a gene encoding levan sucrase (sacB) as
selection markers for
recombination. The target gene is first cloned and mutagenized, preferably by
deleting a
significant portion of the gene. This gene is then inserted by ligation into a
vector designed for
facilitating chromosomal gene replacement. The E. coli cells are then
transformed with those
vectors. Cells that have incorporated the plasmid into the chromosome at the
site of the target
gene are selected, then the plasmid is forced to leave the chromosome by
growing the cells on
sucrose. Sucrose is toxic when the sacB gene resides in the chromosome. The
properly mutated
strain is selected based on its phenotype of tetracycline sensitivity and
sucrose resistance. PCR
analysis or DNA sequencing then confirms the desired genetic change.
However, in some cases the enzyme reducing the duration and yield of the
protein
synthesis reaction may be essential for the growth of the source organism. In
those cases, a
conditional knock-out may be used. For example, anti-sense sequences
corresponding to the
targeted gene are introduced into the source organism on an inducible
promoter. The cells are
grown for a period. of time, and then the anti-sense construct induced, in
order to deplete the cell of
the targeted enzyme.
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The enzyme can be removed from the cell extract after cell disruption and
before use. Any
of the several means known in the art of protein purification may be used,
including affinity
purification techniques such as the use of antibodies or antibody fragments
with specific affinity for
the target enzymes; use of affinity tags expressed as part of the target
enzymes to facilitate their
removal from the cell extract; and conventional purification methods.
In another embodiment, an antibody or antibody fragment (e.g., Fab or scFv) is
selected for
specific affinity for the target enzyme using phage display or other well
developed techniques.
That antibody or antibody fragment is then immobilized on any of several
purification beads or
resins or membranes using any of several immobilization techniques. The
immobilized antibody is
contacted with the cell extract to bind to the target enzyme, and the
immobilized antibody/enzyme
complex then removed by filtration or gentle centrifugation.
For example, the coding sequence of the targeted protein may be modified to
include a tag,
such as the Flag~ extension (developed by Immunex Corp, and sold by
Stratagene), or a poly-
histidine tail. Many other examples have been published and are known to those
skilled in the art.
The tagged proteins are then removed by passage over the appropriate affinity
matrix or column.
The amino acid extension and binding partner are chosen so that only specific
binding occurs
under conditions compatible with the stability of the cell extract, and
without significantly altering
the chemical composition of the cell extract.
In yet another example, the target enzyme or enzymes are separated by any of
several
methods commonly used for protein purification, such as substrate affinity
chromatography, ion
exchange chromatography, hydrophobic interaction chromatography,
electrophoretic separation,
or other methods practiced in the art of protein purification.
METHODS FOR IN VITRO SYNTHESIS
The subject system is useful for in vitro protein synthesis, which may include
the
transcription of RNA from DNA or RNA templates. The reactions may utilize a
large scale reactor,
small scale, or may be multiplexed to perform a plurality of simultaneous
syntheses. Continuous
reactions will use a feed mechanism to introduce a flow of reagents, and may
isolate the end-
product as part of the process. Batch systems are also of interest, where
additional reagents may
be introduced to prolong the period of time for active synthesis. A reactor
may be run in any mode
such as batch, extended batch, semi-batch, semi-continuous, fed-batch and
continuous, and
which will be selected in accordance with the application purpose.
Of particular interest is the translation of mRNA to produce proteins, which
translation may
be coupled to in vitro synthesis of mRNA from a DNA template. Such a cell-free
system will
contain all factors required for the translation of mRNA, for example
ribosomes, amino acids,
tRNAs, aminoacyl synthetases, elongation factors and initiation factors. Cell-
free systems known
in the art include wheat germ extracts (Roberts et al. (1973) P.N.A.S.
70:2330), reticulocyte
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extracts (Pelham et al. (1976) Eur. J. Biochem. 67:247), E. coli extracts,
etc., which can be treated
with a suitable nuclease to eliminate active endogenous mRNA.
In addition to the above components such as cell-free extract, genetic
template, amino
acids and energy sources, materials specifically required for protein
synthesis may be added to
the reaction. These materials include salt, polymeric compounds, cyclic AMP,
inhibitors for protein
or nucleic acid degrading enzymes, inhibitor or regulator of protein
synthesis, oxidation/reduction
adjuster, non-denaturing surfactant, buffer component, spermine, spermidine,
etc.
The salts preferably include potassium, magnesium, ammonium and manganese salt
of
acetic acid or sulfuric acid, and some of these may have amino acids as a
counter anion. The
polymeric compounds may be polyethylene glycol, dextran, diethyl aminoethyl,
quaternary
aminoethyl and aminoethyl. The oxidation/reduction adjuster may be
dithiothreitol, ascorbic acid,
glutathione andlor their oxides. Also, a non-denaturing surfactant such as
Triton X-100 may be
used at a concentration of 0-0.5 M. Spermine and spermidine may be used for
improving protein
synthetic ability, and cAMP may be used as a gene expression regulator.
When changing the concentration of a particular component of the reaction
medium, that of
another component may be changed accordingly. For example, the concentrations
of several
components such as nucleotides and energy source compounds may be
simultaneously controlled
in accordance with the change in those of other components. Also, the
concentration levels of
components in the reactor may be varied over time.
Preferably, the reaction is maintained in the range of pH 5-10 and a
temperature of 20°-50°
C., and more preferably, in the range of pH 6-9 and a temperature of
25°-40° C.
When using a protein isolating means in a continuous operation mode, the
product output
from the reactor flows through a membrane into the protein isolating means. In
a semi-continuous
operation mode, the outside or outer surtace of the membrane is put into
contact with
predetermined solutions that are cyclically changed in a predetermined order.
These solutions
contain substrates such as amino acids and nucleotides. At this time, the
reactor is operated in
dialysis, diafiltration batch or fed-batch mode. A feed solution may be
supplied to the reactor
through the same membrane or a separate injection unit. Synthesized protein is
accumulated in
the reactor, and then is isolated and purified according to the usual method
for protein purification
after completion of the system operation.
Where there is a flow of reagents, the direction of liquid flow can be
perpendicular and/or
tangential to a membrane. Tangential flow is effective for recycling ATP and
for preventing
membrane plugging and may be superimposed on perpendicular flow. Flow
perpendicular to the
membrane may be caused or effected by a positive pressure pump or a vacuum
suction pump.
The solution in contact with the outside surface of the membrane may be
cyclically changed, and
may be in a steady tangential flow with respect to the membrane. The reactor
may be stirred
internally or externally by proper agitation means.
to
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WO 02/40497 PCT/US00/31449
During protein synthesis in the reactor, the protein isolating means for
selectively isolating
the desired protein may include a unit packed with particles coated with
antibody molecules or
other molecules immobilized with a component for adsorbing the synthesized,
desired protein, and
a membrane with pores of proper sizes. Preferably, the protein isolating means
comprises two
columns for alternating use. Alternately, the protein product may be absorbed
using expanded
bed chromatography, in which case a membrane may or may not be used.
The amount of protein produced in a translation reaction can be measured in
various
fashions. One method relies on the availability of an assay which measures the
activity of the
particular protein being translated. An example of an assay for measuring
protein activity is a
luciferase assay system, or chloramphenical acetyl transferase assay system.
These assays
measure the amount of functionally active protein produced from the
translation reaction. Activity
assays will not measure full length protein that is inactive due to improper
protein folding or lack of
other post translational modifications necessary for protein activity.
Another method of measuring the amount of protein produced in coupled in vitro
transcription and translation reactions is to perform the reactions using a
known quantity of
radiolabeled amino acid such as 35S-methionine or 3H-leucine and subsequently
measuring the
amount of radiolabeled amino acid incorporated into the newly translated
protein. Incorporation
assays will measure the amount of radiolabeled amino acids in all proteins
produced in an in vitro
translation reaction including truncated protein products. The radiolabeled
protein may be further
separated on a protein gel, and by autoradiography confirmed that the product
is the proper size
and that secondary protein products have not been produced.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meaning as commonly understood to one of ordinary skill in the art to which
this invention belongs.
Although any methods, devices and materials similar or equivalent to those
described herein can
be used in the practice or testing of the invention, the preferred methods,
devices and materials
are now described.
All publications mentioned herein are incorporated herein by reference for the
purpose of
describing and disclosing, for example, the cell lines, constructs, and
methodologies that are
described in the publications which might be used in connection with the
presently described
invention. The publications discussed above and throughout the text are
provided solely for their
disclosure prior to the filing date of the present application. Nothing herein
is to be construed as
an admission that the inventors are not entitled to antedate such disclosure
by virtue of prior
invention.
The following examples are put forth so as to provide those of ordinary skill
in the art with a
complete disclosure and description of how to make and use the subject
invention, and are not
intended to limit the scope of what is regarded as the invention. Efforts have
been made to ensure
11
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accuracy with respect to the numbers used (e.g. amounts, temperature,
concentrations, etc.) but
some experimental errors and deviations should be allowed for. Unless
otherwise indicated, parts
are parts by weight, molecular weight is average molecular weight, temperature
is in degrees
centigrade; and pressure is at or near atmospheric.
EXPERIMENTAL
Example 1
Pyruvate, glucose or glycolytic intermediates can provide the energy forATP
regeneration,
even in the absence of any exogenous enzymes. While not limiting to the
subject matter of the
invention, two pathways may be proposed for the mechanism of action, whereby
pyruvate
provides ATP regeneration potential to the synthesis reaction. In the first
pathway, ATP
regeneration is accomplished through an electron transport phosphorylation
reaction. Since the
extract is prepared from a total cell lysate, it is likely that the extract
contains inverted membrane
vesicles with the respiratory chain components properly embedded. Thus, after
its conversion into
acetyl-CoA by the endogenous pyruvate dehydrogenase complete, the pyruvate
enters the TCA
cycle to regenerate NADH, which in turn regenerates ATP using the respiratory
chain and the
F1 FO ATPase.
In a second proposed pathway, the generated acetyl-CoA is converted to acetyl
phosphate
by phosphotransacetylase. The resulting acetyl phosphate is then used for ATP
regeneration. In
either case, the oxidation of pyruvate provides the energy for ATP generation
without
accumulating any harmful by-products, and exogenous enzyme is not required.
Alternatively,
phosphoenol pyruvate can be used as the energy source, combining both the
energy obtained by
glycolysis and energy obtained from in situ ATP generation.
Materials and Methods
Phosphoenolpyruvate (PEP) and L.coli total tRNA mixture were purchased from
Roche
14
Molecular Biochemicals (Indianapolis, IN). ~-[U- C] leucine was from Amersham
Pharmacia
Biotechnology (Uppsala, Sweden). All other reagents were obtained from Sigma
(St.Louis, MO).
T7 RNA polymerase was prepared from E.coli strain BL21 (pAR1219) according to
the procedures
of Davanloo et al. (1984). Plasmid pK7CAT which contains the bacterial
chloramphenicol
acetyltransferase (CAT) sequence between the T7 promoter and T7 terminator was
used as a
template for protein synthesis. The plasmid was purified using the Maxi kit
from Qiagen (Valencia,
CA).
S30 extract was prepared from L.coli K12 (strain A19) as described earlier
(Kim et al.,
1996, supra.; Kim and Swartz (1999), supra.) The standard reaction mixture
consists of the
following components: 57 mM Hepes-KOH (pH7.5), 1.2 mM ATP, 0.85 mM each of
GTP, UTP and
CTP, 1 mM DTT, 0.64 mM cAMP, 200 mM potassium glutamate, 80 mM ammonium
acetate, 12
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mM magnesium acetate, 34 wg/ml folinic acid, 6.7 ~.glml plasmid, 33 pg/ml
T7RNA polymerase,
500 ~M each of 20 unlabeled amino acids, 11 pM [~4C]leucine, 2 % PEG 8000, 32
mM PEP, and
0.24 volume of S30 extract. In reactions where pyruvate or glucose-6-phosphate
was used as the
ATP regenerating compound, 33 mM of the energy source was added along with
0.33 mM NAD
and 0.26 mM CoA. In certain reactions, 2.7 mM sodium oxalate was used to
enhance fhe stabilify
of secondary energy sources. Reactions were run for given time periods in 15
to 120 wL reaction
volumes at 37°C.
The amount of synthesized protein was estimated from the measured TCA-
insoluble
radioactivities as described by Kim et al., 1996, supra.) using a liquid
scintillation counter
(Beckman LS3801). To determine the amount of soluble product, samples were
centrifuged at
12,000 g for 10 min and TCA-precipitable radioactivities in the supernatants
were measured. To
estimate the molecular weight of synthesized protein, samples were loaded on a
16 % SDS-PAGE
gel (Invitrogen, CA) with standard molecular weight markers (See Blue,
Invitrogen, CA). Resulting
gels were stained with Coomassie Brilliant Blue following standard procedures.
The protein
concentrations of cell-extracts were measured following the procedures of
Bradford using a
commercial assay reagent (Pierce, Rockford, IL).
To measure ATP concentration, diluted samples were added to an opaque
microtiter plate
containing luciferase solution (0.1 p,glmL luciferase and 125 pM luciferin)
and the intensity of
luminescence was measured in a plate luminometer (ML 3000, Dynatech
Laboratories, Chantilly,
VA). ATP concentrations in the samples were determined from the calibration
curve obtained with
ATP standards. Enzymatic activity of synthesized CAT was determined by
spectrophotometric
procedures. After diluting a sample by 40-fold in water, 10 wL of the diluted
sample was added to
a cuvette containing 1 mL of prewarmed assay mixture (100 mM Tris-CI(pH 7.8),
0.1 mM acetyl-
CoA, 0.4 mg/mL 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), 0.1 mM
chloramphenicol) and the rate
of increase in absorption at 412 nm was measured. The change in absorbance
units per minute
was divided by 13.6 to give the result in units (1 unit of CAT acetylates 1
wmole of chloramphenicol
per minute).
Results
Use ofpyruvate as a secondary energy source for cell-free protein synthesis.
Previously, it
has been reported that pyruvate can serve as a secondary energy source to
regenerate ATP
during a cell-free protein synthesis reaction. In that system; the enzyme,
pyruvate oxidase,
converts pyruvate into acetyl phosphate which is then used to regenerate ATP
with endogenous
acetate kinase. This pyruvate oxidase-dependent system substantially reduces
the cost for
energy source, leads to a stable maintenance of ATP during protein synthesis
and avoids
phosphate accumulation.
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WO 02/40497 PCT/US00/31449
The conversion of pyruvate into acetyl phosphate, however, requires an
exogenous
enzyme, pyruvate oxidase (E.C.1.2.3.3) from Lactobacillus or Pediococcus sp.
because the E. coli
enzyme (E.C.1.2.2) cannot catalyze the formation of acetyl phosphate (it
converts pyruvate to
acetate instead of acetyl phosphate). Thus, in the absence of exogenous
pyruvate oxidase, ATP
is not regenerated and the level of protein synthesis is negligible. The use
of commercial pyruvate
oxidase, however, offsets some of the economic benefits of the new system
Since this enzyme
requires molecular oxygen for the oxidation of pyruvate into acetyl phosphate,
the synthesis
reaction cannot be easily scaled-up in a simple batch configuration due to the
limitation of oxygen
transfer.
To avoid these limitations, we sought to eliminate the requirements for
exogenous enzyme
and oxygen. For pyruvate to be used for ATP regeneration in the cell-free
system, it first needs to
be converted to acetyl phosphate. In E.coli cells, pyruvate is not directly
converted to acetyl
phosphate. Instead, two different enzymes can convert pyruvate into acetyl-
CoA, which can be
used to produce acetyl phosphate by phosphotransacetylase. First, pyruvate
dehydrogenase
catalyzes the condensation of CoA and pyruvate to make acetyl-CoA in the
presence of NAD as a
cofactor. NAD is reduced to NADH during this reaction. On the other hand,
pyruvate-formate
lyase can also make acetyl-CoA from pyruvate producing formate as the by-
product. All of the
enzymes required for these reactions were assumed to be present in the cell-
extract. We thus
tested the addition of the cofactors, NAD and CoA, to stimulate the
regeneration of ATP from
pyruvate in support of cell-free protein synthesis.
33 mM sodium pyruvate was added to reaction mixtures with or without the
cofactors.
After a 2 hr incubation, significant synthesis was observed (Figure 1) in the
presence of NAD and
CoA. The final yield of protein synthesis was about 70 % of that from the
reaction in which PEP
was used. The use of pyruvate without the cofactors resulted in low protein
synthesis equivalent
to the control reaction without any secondary energy source. This result
indicates that the
conversion of pyruvate to acetyl-CoA is accomplished by pyruvate dehydrogenase
rather than
pyruvate-formate lyase as the latter enzyme does not require cofactors. Since
only a catalytic
amount of NAD (0.33 mM) is required, it seems obvious that NAD is also
regenerated during the
synthesis reaction. It is assumed that the reduced NAD is reoxidized during
the conversion of
pyruvate into lactate. Thus, by coupling the oxidation and reduction reactions
of pyruvate, NAD is
recycled. A catalytic amount of CoA (0.27 mM) was also required for optimal
protein synthesis.
The hypothesized pathway of ATP regeneration is depicted in Figure. 2.
Utilization of pyruvate generated from PEP. Consumption of PEP in the
conventional ATP
regeneration system produces pyruvate as a by-product. Based on the above
results of pyruvate
utilization for ATP regeneration, we tested the addition of NAD and CoA to the
conventional
system to enhance ATP supply through the secondary utilization of pyruvate
generated from PEP.
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WO 02/40497 PCT/US00/31449
Figure 3A shows that the presence of those cofactors does improve ATP supply.
Especially, the
initial decrease in the ATP concentration in the PEP system was substantially
slowed upon the
addition of NAD and CoA.
Oxalate, a potent inhibitor of phosphoenolpyruvate synthetase enhances ATP
concentration in the synthesis reactions with pyruvate or PEP. The effect of
oxalate was still
observed in the presence of NAD/CoA. The yield of CAT synthesis also increased
upon the
addition of 2.7 mM oxalate (Figure 3B). In addition, we discovered that
protein synthesis was
further stimulated by increasing the initial concentrations of amino acids.
When the concentration
of ATP was elevated by the additions of the cofactors and oxalate, both the
rate and duration of
protein synthesis was significantly improved by increasing the initial
concentrations of amino acids
to 2 mM (Figure 3B). Higher concentrations were not as effective. As a result,
the final yield of
CAT synthesis after a 1 hour incubation was as high as 350 ~glmL. Furthermore,
periodic
additions of fresh amino acids, PEP, and magnesium according to published
procedures allowed
the synthesis reaction to continue for over 3 hours resulting in the final
yield of 750 wg/mL (Figure
4). Synthesized CAT gave a single intense band on a SDS-PAGE gel after
Coomassie Blue
staining. Approximately 60 % of expressed CAT was soluble and the measured
specific activity
was comparable to published value of 125 units/mg (Table 1).
Table 1:
CAT yields and specific activities after 3 hr incubations with different ATP
regeneration
systems
Specific activity
Secondary energy Total yield of Yield of soluble (units/mg
sources synthesis (mg/ml) product (mg/ml) % soluble soluble product)
PEP 168 t 12.7 107 X10.4 61 181 t 21.5
Pyruvate 134 t 8.6 75 t 15.4 56 153 t 15.7
PEP + Pyruvate 317 t 53.0 189 t 42.7 60 167 t 14.9
Glucose-6-phosphate 228 t 12.9 143 t 4.5 63 158 t 26.1
To utilize the pyruvate generated from PEP, NAD, CoA, and oxalate were added
to the reaction
mixture as described in Materials and Methods.
Glucose-6-Phospate as an alternative secondary energy source. Encouraged by
the
observation that pyruvate could be used for ATP regeneration, we further
investigated if earlier
glycolytic intermediates could be used to regenerate ATP in our cell-free
system. The use of
glucose-6-phosphate was examined as it is the first intermediate of the
glycolytic pathway. When
CA 02428693 2003-05-13
WO 02/40497 PCT/US00/31449
33 mM glucose-6-phosphate was used underthe same reaction conditions as in the
pyruvate/NAD
system, it did support protein synthesis. In addition, although the initial
rate was substantially
lower than in the reaction with PEP, protein synthesis continued for over 2
hours (Figure 5A). As a
result, approximately 30 % more CAT was produced at the end of incubation.
Most likely, this is
due to the remarkably extended maintenance of ATP concentrations. The time
course of ATP
concentration during protein synthesis with glucose-6-phosphate was
characterized by a period of
relatively stable maintenance of ATP level followed by a slow decrease over
the incubation period
(Figure 5B). Unlike the reactions using PEP or pyruvate, the addition of
sodium oxalate did not
further improve the maintenance of ATP concentration.
The ATP regeneration with glucose-6-phosphate indicates that all of the
glycolytic enzymes
required to convert glucose-6-phosphate into pyruvate are active under the
present reaction
conditions. This provides a great flexibility in choosing a secondary energy
source for protein
synthesis. Any of the glycolytic intermediates between glucose-6-phosphate and
pyruvate can be
used for ATP regeneration.
The specific activity of synthesized CAT was greater than the published data
with all of the
secondary energy sources described here; PEP, pyruvate, PEP/pyruvate, and
glucose-6-
phosphate (125 units/mg) (Table 1).
It is shown herein that regeneration of ATP during cell-free protein synthesis
can be
accomplished by using alternative energy sources such as pyruvate and glucose-
6-phosphate in
the absence of exogenous enzymes. With pyruvate as the secondary energy
source, a synthesis
yield of approximately 70 % was obtained as compared to the reaction using
PEP, the
conventional energy source. This is not surprising since half of the pyruvate
is needed to recycle
NADH to NAD. However, because of the expense of PEP, the use of pyruvate still
improves the
economic efficiency of protein synthesis.
The reactions of ATP regeneration using pyruvate also can be used to improve
the
utilization efficiency of the conventional energy source, PEP. After being
used for ATP
regeneration or being degraded by phosphatase activities of cell-extract, PEP
produces an
equimolar amount of pyruvate. If the cofactors NAD and CoA are present in the
reaction mixture,
half of the newly generated pyruvate is available for ATP regeneration (the
other half is used for
the regeneration of NAD). As a result, through the two-stage utilization of
the energy source, the
overall concentration of ATP is elevated and prolonged and the productivity of
protein synthesis is
improved. In accordance with the previous report, addition of a metabolic
inhibitor of
phosphoenolpyruvate synthetase (sodium oxalate) further enhanced the ATP
concentration.
Again, improved ATP supply led to increased productivity. In addition, with
the enhanced ATP
level, the yield of protein synthesis further increased when the initial
concentrations of amino acids
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WO 02/40497 PCT/US00/31449
were increased. Finally, fed-batch additions of amino acids, PEP, and
magnesium allow the
synthesis reaction to continue for over 3 hours to produce a final yield of
750 wg/mL.
It is also shown that glucose-6-phosphate, the first intermediate of
glycolytic pathway, can
be used as the secondary energy source resulting in a yield higherthan with
the PEP system. As
compared to PEP, the use of glucose-6-phosphate substantially increased the
synthesis yield
mainly by prolonging the reaction period. This seems to be due to the enhanced
supply of ATP,
which can be explained since glucose-6-phosphate offers a greater potential to
regenerate ATP
compared to PEP or pyruvate. While PEP or pyruvate can regenerate, at best,
only the equivalent
number of ATP molecules, 3 molecules of ATP can be generated during the
oxidation of glucose-
6-phosphate into pyruvate. (The two molecules of pyruvate generated from the
glycolytic pathway
are required to regenerate the two NAD molecules that are reduced by
glyceraldehyde-3-
phosphate dehydrogenase). These results strongly imply that we can use any of
the glycolytic
intermediates as a secondary energy source to support cell-free protein
synthesis.
The use of glucose would provide a cell-free system that is highly competitive
with
traditional technologies for protein expression in terms of economic
efficiency. Our initial results
suggest such a possibility. The use of glucose along with hexokinase did
support protein
synthesis. In addition, even though ATP is not regenerated by oxidative
phosphorylation in this
system, the present cell-extract may contain active membrane vesicles, that
could be used in
oxidative phosphorylation to provide an extremely efficient method for ATP
supply to the protein
synthesis by mimicking the function of living cells.
Because each mRNA is used multiple times, we estimate that the translational
demand for
ATP dominates. During protein synthesis, the EF-Tu cycle consumes 2 GTPs and
EF-G requires
another molecule of GTP for the translocation of ribosome. In addition, since
a molecule of ATP is
hydrolyzed to AMP during the aminoacylation of tRNA, we assumed that 5
molecules of ATP are
required to add an amino acid residue. Based on these assumptions, we have
calculated the
efficiencies of ATP utilization (Table 2).
Table 2: Efficiency of ATP utilization in different ATP regeneration systems
Maximum ATP available Amount of synthesized CAT Efficiency of ATP
(~molesl15 ~.L) (pmolesll5 ~,L) utilization (%)
PEP 0.50 99.3 t 7.1 22.6
Pyruvate 0.25 65.9 117.5 29.9
PEP + Pyruvate' 0.75 181.5 t 60.7 27.5
PEP + Pyruvate~ 0.75 312.9 t 44.0 47.4
Glucose-6-phosphate 1.50 116.0 t 10.1 8.8
To utilize the pyruvate generated from PEP, NAD, CoA, and oxalate were added
to the reaction mixture
as described in the Materials and Methods
~ Initial concentrations of amino acids were 2 mM
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WO 02/40497 PCT/US00/31449
In the standard reaction using PEP, the final amount of synthesized CAT in a
15 ~,L
reaction was 99.3 pmole which represents 2.2 x 10-8 moles of peptide bonds.
Thus we can
assume that 0.11 wmole of ATP was used for protein synthesis. Since the total
amount of ATP
that can be generated in the same reaction mixture is 0.5 moles, the
efficiency of ATP utilization
is estimated to be 22 %. On the other hand, we could produce 66 pmoles of CAT
in the reaction
using pyruvate which equals to 1.50 x 10-8 moles of peptide bonds. Since only
half of the pyruvate
can be used for ATP regeneration, 0.25 .moles of ATP can be generated and the
utilization
efficiency becomes 30 %.
By the additions of NAD, CoA, and oxalate to the conventional PEP system, we
could
obtain 182 pmoles of CAT in a 15 ~L reaction. Since the total amount of
available ATP was 0.75
wmoles (0.5 moles from PEP and 0.25 pmoies from the pyruvate produced from
PEP), the
efficiency of ATP utilization was estimated to be 27.0%. However, when the
initial concentrations
of amino acids were raised to 2 mM, the amount of produced CAT increased to
393 pmoles and
the efficiency of ATP utilization reached 47.4 %.
In contrast, the efficiency of ATP utilization with glucose-6-phosphate was
only 8.8 % (note
That 1 molecule of glucose-6-phosphate can generate 3 molecules of ATP). Thus
only a small
fraction of the potential ATP pool is used for protein synthesis. This
suggests either that the
majority of the regenerated ATP is degraded by ATPase activities present in
the current cell-
extract or that side reactions are degrading glycolytic intermediates. In
other words, we can
expect to improve protein synthesis from glucose-6-phosphate by identifying
and removing those
activities, for example by disrupting the genes encoding enzymes that catalyze
non-productive
degradation of ATP. For the removal of enzymatic activities that are essential
for growth, we can
genetically mark those enzymes with affinity tags so that we can remove them
during cell-extract
preparation. The improved utilization efficiency of ATP will also have a great
impact on the
economics of cell-free protein synthesis as ATP regeneration accounts for a
substantial portion of
the reagent cost. Through such a "genetic optimization" of the E.coli strain
combined with the
improved ATP regeneration systems, a highly efficient batch cell-free protein
synthesis system is
provided.
18
