JPH07203984A - Synthesis of protein - Google Patents

Abstract

PURPOSE:To synthesize a protein by a cell-free protein synthesis system having increased protein synthesis rate. CONSTITUTION:A protein is synthesized from a gene information in a cell-free protein synthesis system by suppressing the activation of an activity inhibiting factor which is a factor induced by cytoclasis and inhibiting the activity of nucleic acid synthesis and/or protein synthesis.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for synthesizing a protein from a cell-free protein synthesis system using a nucleic acid (DNA or RNA) as a template from genetic information.

[0002]

2. Description of the Related Art A cell-free protein synthesis system (Nirenberg, MW, et al., Proc. Natl. Acad. Sci. USA, developed by Nirenberg et al.
47,1588-1602 (1961)) played a major role in elucidating the mechanism of protein synthesis. After that, a cell-free protein synthesis system with high translation efficiency was prepared not only from Escherichia coli but also from wheat germ, rabbit reticulocyte, etc., and is now widely used for identifying gene translation products ( Wu, R., Et
al., (ed) Methods in Enzymolgy, vol.101, P.598, P.616,
P.629, P.635, P.644, P.650, P.674, P.690, Academi
c Press, New York (1983)).

[0003]

However, the amount of protein synthesis in the above-mentioned cell-free protein synthesis system is extremely low, which is 1 / 10,000 to 1 / 10,000 as compared with that of living cells. Has the drawback that it cannot be used as

Recently, the former Soviet Union's Spirin et al.
Aiming to improve the efficiency of the cell-free protein synthesis system, a continuous cell-free protein synthesis system (Continuous Flow Cell-Free Tran
slation System) (Spirin, AS, et al., Sc
ience, 242, 1162-1164 (1988)). This system differs from the conventional cell-free protein synthesis system in that the substrates such as amino acids and energy sources consumed in the synthesis reaction are continuously supplied to the reaction tank, while the translation product is taken out from the system. However, their system also uses a cell extract with low translation activity prepared by a conventional method from wheat germ, rabbit reticulocyte, Escherichia coli, etc. as a protein synthesis reaction system. No improvement in synthesis efficiency can be expected.

[0005]

The present inventor has found that the activity of an activity inhibitor of nucleic acid synthesis and / or protein synthesis, which is activated in association with cell destruction, which has been inevitable and inevitable in the prior art. It was found that the cell-free protein synthesizing system can be prepared with higher efficiency than the conventional ones by suppressing the activation. The present invention is the following invention based on this finding, and its protein synthesis system (composition for protein synthesis)
Is the invention of.

[0006] It is characterized in that a protein is synthesized from gene information in a cell-free protein synthesis system that suppresses activation of an activity inhibitor of nucleic acid synthesis and / or protein synthesis that is induced by cell destruction. Method for synthesizing protein.

The activity inhibitor of nucleic acid synthesis and / or protein synthesis in the present invention is a factor induced by cell destruction, and the factor is usually an enzyme composed of a polypeptide or protein. There is also. This activity-inhibiting factor is induced by the action of the programmed cell death mechanism associated with cell destruction, and is considered to be a factor originally possessed by individual cells to enable the organism or the population to survive. When this factor is activated, it is considered that activation of nucleic acid synthesis and / or protein synthesis is inhibited by inhibiting transcription activity or translation activity, for example.

This activity inhibitor is an enzyme usually composed of various polypeptides or proteins, and specific examples thereof include ribosome inactivating protein which inhibits ribosome activity, ribonuclease, ribonucleotide phosphorylase and the like. As a means for suppressing the activation of the above-mentioned activity inhibitory factor, it is possible to employ not only removing it from the protein synthesis system but also a means for inhibiting the activation in the protein synthesis system. In particular, since it is often difficult to selectively remove this activity inhibitor from the protein synthesis system, it is possible to employ a means of inhibiting its activation, particularly a means of using a specific inhibitor. preferable.

As the specific inhibitor of the above activity inhibitor, an antibody that specifically binds to this activity inhibitor and suppresses its activity is preferable. It is considered that this neutralizing antibody inhibits the activation of the activity inhibitor by neutralizing the enzyme activity of the activity inhibitor. The antibody may be a polyclonal antibody or a monoclonal antibody. The specific inhibitor of the above activity inhibitor is not limited to these antibodies, and various drugs such as antibiotics (protein inhibitors, nucleic acid system inhibitors, etc.) may be used. These inhibitors can be used in combination of two or more including antibodies.

A substance capable of specifically binding to the above activity inhibitor such as an antibody can also be used in a method for selectively removing this activity inhibitor from a protein synthesis system. For example, a substance capable of specifically binding can be immobilized on a suitable polymer substance, and the protein synthesis system can be contacted in the presence of this immobilized carrier to remove the activity inhibitor from the protein synthesis system.

The protein synthesis system in the present invention is a so-called cell-free protein synthesis system which does not substantially contain living cells. This protein synthesis system is obtained by cell disruption and utilizes the protein synthesis ability of the disrupted cells. The protein synthesis system may be the cell disruption solution itself, but is preferably prepared by removing coarse solids. Usually, it is prepared by adding components as needed to the cell extract from which unnecessary components have been removed.

The cell of origin (cell to be destroyed) for preparing the cell-free protein synthesis system is not particularly limited, and may be a nucleated organism,
Any prokaryotic cell can be used. That is, animal cells, plant cells, fungal cells, bacterial cells and the like can be used. Specific examples include mammalian cells, insect cells, higher plant cells, yeast cells, actinomycetes cells, Escherichia coli cells and the like.

The protein synthesis by the cell-free protein synthesis system of the present invention can be carried out in the same manner as in the conventional method except that it is a cell-free protein synthesis system in which the above-mentioned activity inhibitor is inactivated and removed. This method may be a well-known batch method, or may be a continuous method such as the above-mentioned continuous cell-free protein synthesis system of Spirin et al.

The present invention will be further described with reference to specific examples through experiments conducted using a wheat germ system.

The present inventors have previously elucidated the toxicity mechanism of ricin, which is a cytotoxin protein present in castor seeds, at the molecular level (Endo, Y., et al., J. Biol. Chem. 198
7), 262, 5908-5912, Endo, Y., et al., J. Biol. Chem. (198
7), 262, 8128-8130). That is, the lysin A chain is an N- at a specific site in which the structure of a large RNA molecule (26-28S rRNA in higher organisms and 23S rRNA in E. coli) that constitutes ribosomal large subparticles is evolutionarily conserved.
RNA N-glycosidase (RNA N-glycosidase) is a unique enzyme that catalyzes the hydrolysis of glycoside bonds (4324 from the 5'end in rat liver 28rRNA).
oxidase). The ribosome releases one molecule of adenine by this enzymatic activity and completely loses its translation function.

Then, a ribosome inactivating protein (Ribosome-Inactivating-
Protein (hereinafter abbreviated as RIP) was isolated from many plants (Soria, MR, et al., In Genetically Engineered
Toxins (ed.by Frankel, AE), pp.193-212, Marcel Dek
ker, Inc. (1992)). Moreover, many of these RIPs are considered to be involved in the self-defense mechanism of plants because they have antiviral effects, but there are many unclear points regarding their substance.

Since Nirenberg et al. Developed a cell-free protein synthesis system from E. coli approximately 30 years ago, cell-free protein synthesis systems have been prepared from various organisms (Miller, JS, et.
al., in Methods in Enzymology, part C, 101, pp.650-67
4, Academic Press (1983)). Among them, wheat germ,
The cell-free protein synthesis system prepared from rabbit reticulocytes, Escherichia coli or the like has a relatively high protein synthesis activity, and kits thereof are now commercially available. However, in any of the systems, the translation reaction is stopped within 1 to 2 hours and the translation efficiency is extremely low, so that it has a drawback that it cannot be used as a practical means for preparing a protein.

Generally, the amount of protein synthesized in a cell-free protein synthesis system is estimated to be one hundredth to tenth thousandth that of living cells (Mancheter, KL, in Mammal.
ianProtein Metabolism, ed.by Munro, HN, et al., IV,
pp.229-298, Academic Press (1970)) The cause was unknown. The inventor first investigated a high-efficiency strategy of the cell-free protein synthesis system by investigating the mechanism of reduction of protein synthesis activity in the wheat embryo cell-free system.

RIP called tritin exists in wheat germ, and its RNAN-glycosidase activity is known to inactivate plant cell ribosome by the same mechanism as lysin A chain ( Roberts, W.
K., Biochemistry (1979), 18,2615-2621). Based on the results of the studies conducted so far, the inventors considered that the low translation activity in the wheat germ cell-free system may be due to the inactivation of the autoribosome by this endogenous tritin. Therefore, when a cell-free protein of wheat germ was prepared and activation of tricine and inactivation of wheat germ ribosome due to cell destruction were investigated, 28SrRN of wheat germ ribosome was examined.
Deadenin at the N-glycosidase-specific action site of A was observed, and it was identified using an antibody that the N-glycosidase was tritin.

Next, the protein synthesis efficiency of the wheat embryo cell-free protein synthesis system was examined in the presence of the tritin antibody under the above conditions. Dihydrofolate reductase (DHF) as mRNA
When R ¹⁴ ) was used to measure ¹⁴ C-leucine uptake, the reaction time in the antibody-added system was at least 1.5 times longer than that in the non-added system, and the uptake amount was 2.5 times or more.

To summarize the above, (1) Tritin, which is involved in the programmed cell death mechanism inherent in wheat germ, is inevitably mixed in the cell extract with the crushing of the wheat germ and inactivates its own ribosome. (2)
This is because the decrease in protein synthesis in cell-free systems is due to this. (3) By removing and neutralizing the tritin activity by using an anti-tritin antibody, for example, the protein synthesis reaction can be sustained for a long time. It was shown that the efficiency of synthetic activity can be improved.

The experiment described above will be specifically described below as an example. However, the present invention is not limited to this embodiment.

[0023]

【Example】

Example 1 Tritin Activation and Wheat Germ Ribosome Inactivation Induced by Wheat Germ Crushing

[0024] Commercially available wheat germ is used as Erickso
After crushing by the method of n) to obtain a cell extract, a cell-free protein synthesis solution was prepared (Erickson, AH, et al., in Metho
ds in Enzymology (1983), 96, 38-50). Using a mRNA encoding dihydrofolate reductase (DHFR) as a template, a protein synthesis reaction was carried out at 26 ° C. according to the method of Ericsson et al. Endogenous RN of wheat germ ribosome
The deadenination reaction by AN-glycosidase was examined by the method of the present inventors (Endo, Y., et al., J. Biol. C.
hem. (1987), 262, 5908-5912).

RNA was extracted from each of the reaction solutions after 2, 4, and 6 hours of the reaction, and after treatment with aniline under acidic condition, 28 rRNA was separated by gel electrophoresis. Was observed (arrows in lanes 3, 4, and 5 respectively). The production of RNA fragments is not seen before the start of incubation (lane 1). Since this RNA fragment shows the same mobility as the separately prepared RNA fragment marker (lane 2), the dead adenine part of 28S rRNA of wheat germ ribosome is RNA N whose evolutionarily preserved structure. -Specific site of action of glycosidases (4324 of murine liver 28 rRNA)
Corresponding to the second adenine).

The above results indicate that the endogenous RNA N-glycosidase was activated by some mechanism in association with the disruption of wheat germ. Furthermore, this result suggests that in the wheat germ cell-free system, ribosome inactivation occurs due to the action of this RIP during the protein synthesis reaction.

Next, for the purpose of checking whether or not the substance of this RNA N-glycosidase as a protein is tritin, the above-mentioned R was prepared using a tritin antibody prepared in a rabbit.
NAN-glycosidase neutralization experiment was tried. A tritin antibody was added to the same protein synthesis system as above and reacted for 6 hours, and RNA was similarly degraded by gel electrophoresis.

As a result, by adding the tritin antibody,
It was confirmed that the production of RNA fragments was remarkably suppressed (comparing the darkness of the band in the arrow portion between lane 5 and lane 6 in FIG. 1, lane 6 became significantly lighter). This fact is due to the modification reaction (deadening) of the wheat germ ribosome.
Were catalyzed by the endogenous RIP tritin. Furthermore, this result shows that wheat germ tritin, whose physiological function was unknown, acts as a so-called programmed cell death machine mechanism that inactivates ribosomes in injured embryo cells and kills themselves to prevent viral infection. It indicates that

The results of this experiment also show that the activity of tritin caused by cell disruption can be effectively neutralized by using an antibody against the protein. However, it can be seen that the neutralization by the method of adding an antibody to this translation reaction system is not complete (this can be seen from the formation of some specific bands in lane 6). Therefore, for the purpose of removing the tritin activity, the following two methods were used in combination to prepare the wheat germ extract. That is, (1) the extract is prepared in the presence of the tritin antibody, and (2) the extract is passed through a column on which the tritin antibody is immobilized to collect and remove the endogenous tritin.

Using the thus obtained wheat germ extract, a protein synthesis reaction was carried out in the same manner as above, and RNA was analyzed. The results are shown in lane 7 of FIG. As can be seen from the photograph, almost no specific RNA fragment is produced in the reaction for 6 hours (almost no specific band is seen in the arrow portion of lane 7). When non-immunized antibodies were used in the control experiments corresponding to lanes 6 and 7 (lanes 8 and 9), a significant ribosome deadenination reaction was observed as in lane 5.

[Example 2] Improvement of efficiency of wheat germ cell-free protein synthesis system using tritin antibody

It is known that the ribosome deadenified by the action of RNA N-glycosidase freezes on mRNA and the peptide chain elongation reaction is stopped. (Furutani, M., et al., Arch.Biochem.Biophys. (19
92), 293, 140-146). Therefore, DHFR synthesis was measured from the incorporation of ¹⁴ C-leucine into the protein under the conditions shown in Example 1 above. The experimental method used was the ordinary method of Ericsson, AH, et al. The result is shown in FIG.

As shown in FIG. 2, in the conventional wheat embryo cell-free protein synthesis system, the translation reaction proceeds depending on the addition of the template mRNA, but the reaction is completely stopped after 2 hours (● -●). When the tritin antibody was added to the same reaction solution, the translation reaction proceeded almost linearly up to about 3 hours,
Furthermore, it can be seen that the reaction continues after that (□-
□). Furthermore, it was revealed that in the reaction system in which endogenous tritin was removed and a tritin antibody was added during the reaction, the translation reaction proceeded almost linearly over 5 hours or more (Δ-Δ). Even if a similar experiment is performed in the presence of a non-tritin antibody, the effect is not observed at all (■-■).
If the template mRNA is not added, protein synthesis will not be performed (○-○).

These results indicate that the anti-tritin antibody markedly increases the efficiency of protein synthesis in the wheat embryo cell-free protein synthesis system. When the amount of DHFR synthesized per ml of the reaction system was calculated from the ¹⁴ C-leucine uptake radioactivity in FIG. 2, the maximum amount was about 40 μg in the conventional cell-free system, while using the tritin antibody, Approximately 105μ in the system in which endogenous tritin was removed and neutralized
It was g. This yield is the amount of protein synthesized using the continuous cell-free protein synthesis system developed by Spirin et al. [Reaction liquid 1 ml, reaction time 20 hours 97 μg] (End
o, Y., et al., J. Biotechnology (1992), 25, 221-230).

[Example 3] Identification of translation product

In FIG. 2, the incorporation of ¹⁴ C-leucine into the protein was measured.
To confirm that FR was synthesized, after a reaction for 6 hours, a part (2.5 μl) of the reaction solution was taken and separated by protein SDS-gel electrophoresis, and the protein band was separated by Coomassie Brilliant Blue. Was used for staining (Fig. 3).

Lane 1 is for protein synthesis in the absence of DHFR mRNA, and Lane 2 is for protein synthesis in the presence thereof.
Lane 3 shows the reaction solution in which tritin antibody was added to the reaction system, and lane 4 shows the analysis of the reaction solution in which tritin was removed and neutralized using the tritin antibody. A protein band showing the same mobility as DHFR is clearly observed in the arrow portion of lane 4. Further, since the corresponding band could not be confirmed in lane 1, it was concluded that this is a translation product of DHFR mRNA. In the conventional wheat germ cell-free protein synthesis system, DHFR is not observed as a clear band because the amount produced is small.

Furthermore, from the results of scanning the DHFR band using the densitometers in lane 4 and lane 3, the present inventors have already used the conventional cell-free system.
We attempted synthesis and found that this translation product retains enzymatic activity (Endo, Y., et al., J. Biotechnology.
(1992), 25, 221-230), so DHF synthesized in this experiment
It is considered that R also retains its structure as a protein having enzymatic activity.

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, the life of a cell-free protein synthesis system in which the reaction has been stopped in about 1 to 2 hours is 3 to
In addition to extending the reaction time to 5 hours or more, it also has the excellent effect of increasing the reaction efficiency. In particular, when the amount of produced protein reaches 2.5 times or more that of the conventional system, it greatly contributes to the reduction of protein production cost. The effect is recognized. Further, further efficiency can be expected by combining with the continuous protein synthesis system developed by Spirin et al.

[Brief description of drawings]

FIG. 1 is an electrophoretogram showing the experimental results of Example 1.

FIG. 2 is a graph showing the experimental results of Example 2.

FIG. 3 is an electrophoretogram showing the experimental results of Example 3.

Claims (9)

[Claims] 1. A cell-free protein synthesis system that suppresses activation of an activity inhibitor, which is an activity inhibitor that is induced by cell destruction and that inhibits the activity of nucleic acid synthesis and / or protein synthesis. A method for synthesizing a protein, which comprises synthesizing a protein from genetic information. 2. The synthetic method according to claim 1, wherein the cell-free protein synthesis system is a protein synthesis system obtained by destroying cells. 3. The cells are animal cells, plant cells, fungal cells,
Alternatively, the synthetic method according to claim 2, which is a bacterial cell. 4. The synthetic method according to claim 1, wherein the activity inhibitor is a polypeptide or protein. 5. The synthetic method according to claim 1, wherein the activity inhibitor is a nucleic acid. 6. The method according to claim 1, wherein the inhibition of activation of the activity inhibitor is due to the use of an inhibitor of the activity inhibitor. 7. The synthetic method according to claim 6, wherein the inhibitor is an antibody that neutralizes an activity inhibitor. 8. The method according to claim 1, wherein the inhibition of activation of the activity inhibitor is due to removal of the activity inhibitor. 9. A composition for protein synthesis, which is obtained by cell disruption and utilizes the protein synthesizing ability of the disrupted cells, which is an activity inhibitory factor induced by cell disruption A cell-free protein obtained by removing an activity inhibitor, which is a factor that inhibits the activity of synthesis and / or protein synthesis, from the composition for protein synthesis or suppressing the activation in the composition for protein synthesis. Synthetic composition.