Methods of synthesizing cell-free protein

A technology for protein synthesis and synthesis method, which is applied in the field of protein synthesis and can solve the problems of low strength of membrane materials, decreased membrane function, and difficulty in protein production.

Inactive Publication Date: 2003-10-15
CELLFREE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still problems to be solved in the continuous method using these filtration membranes: ①The strength of the membrane material used is low; ②The function of the membrane is reduced due to the clogging of the mesh; ③The skilled technology is required due to the complicated operation, etc.
[0008] In addition, the manual continuous cell-free protein synthesis method can be used to synthesize proteins from a small number of genes by using the outer filter membrane method and the dialysis membrane method, but it is necessary to efficiently produce proteins from a large number of genes. very difficult

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  • Methods of synthesizing cell-free protein
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  • Methods of synthesizing cell-free protein

Examples

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Effect test

Embodiment 1

[0046] As an example of a continuous diffusion patched cell-free protein synthesis method, a method consisting of figure 1 The cascaded sequential diffusion repair approach shown implements protein synthesis using wheat germ extract.

[0047] Wheat germ extract is obtained according to the method described in the current publication [Madin K. et al., Proc, Natl. Acad. Sci. USA (2000), 97, 559-564], (WO00 / 68412 publication) .

[0048] Furthermore, in order to synthesize the mRNA constituting the decoded shape in the wheat germ cell-free protein synthesis reaction, the general-purpose plasmid pEU1 ( Figure 6 ) (WO01 / 27260 communiqué). As the gene encoding the target protein, a jellyfish green fluorescent protein (GFP) gene (gfp gene) was used, and inserted into the above-mentioned plasmid according to the usual method. The resulting plasmid was cut with HindIII and processed into a linear form; it was made into a replicating form, and mRNA was synthesized by a usual method. ...

Embodiment 2

[0057] As an example of the method of dilute repair cell-free protein synthesis, the protein synthesis solution containing the wheat germ extract prepared in Example 1 and encoded by GFP was carried out at 26°C according to the existing repair formula. After pre-cultivation for 15 minutes, adding 5 times the volume of the diluted solution, further culturing at 26° C. for 3, 6, and 9 hours under static conditions, the protein was synthesized. As the diluted solution, a solution having the same composition as that of the supply solution prepared in Example 1 was used. The amount of the synthesized protein was measured in the same manner as in Example 1, and the results are shown in FIG. 2(A)(■-■) and FIG. 2(B).

[0058] As shown in Fig. 2(A), compared with the conventional modified formula (O-○) in which the reaction is synthesized within 1 hour, if the cell-free protein synthesis is carried out in the dilution formula, until 6 hours after the start of the reaction, the A linea...

Embodiment 3

[0065] In the sequential repair cell-free protein synthesis method, it was confirmed that in addition to the GTP synthesis performed in Example 1, dihydrofolate reductase (DHFR) produced by Escherichia coli could also be synthesized. This approach is also effective in the synthesis of molecules that are proteins in general.

[0066] The same protein synthesis as in Example 1 was carried out using the protein synthesis reaction solution with the same composition as in Example 1, except that the mRNA was mRNA encoding DHFR. The results are shown in FIG. 3 . The amount of synthesized protein was measured by taking the inhalation amount of radioactive isotope trichloroacetic acid insoluble part chromatography as an index according to the conventional method, and the detection of synthesized protein was separated by SDS-polyacrylamide gel electrophoresis and Coomassie bright Blue (CBB) staining [Endo, Y. et al., (1992) J. Biotech., 25, 221-230], [Proc. Natl. Acad. Sci. USA (2000), ...

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Abstract

The invention provides a synthetic method of a cell-free protein, which connects synthesis reaction solution (reaction phase) containing organism extract to substrate and energy source supplying solution (supplying phase) directly, and free diffuse substrate and energy source atoms of supplying phase by connecting surface, continuously supplying to the reaction phase; and discharge the by-products of reaction phase to the supplying phase to prolong the duration of the reaction. The invention prolongs the duration of the reaction by adding dilution solution to the solution containing wheat germ extracts. The invention re-supply amino acids, ATP, GTP and fiber phosphoric acid for protein synthesis by gel filtration column and translucent film in the solution after synthesis reaction, and discontinuously discharge the by-products of the reaction.

Description

technical field [0001] The present invention relates to a protein synthesis method utilizing a cell-free system. Background technique [0002] The genome project is coming to an end, and the center of its subject rapidly expands from the analysis of genetic structure to the analysis of genetic function. It is generally believed that proteins in cells cannot function alone, and their functions are discovered through the coordination and interaction of various protein genes, nucleic acids, low-molecular species, and cell membrane components. On the other hand, the sum of its interactions functions in the field of biology. [0003] As a central task of the post-genome project, it is to analyze the relationship between the aggregate structure and function of various protein genes. The results obtained in the research include the research of basic biology such as structural biology and biochemistry, and the relationship between the results of genetic decoding and the cause of d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02
CPCC12P21/02
Inventor 远藤弥重太泽崎达也小笠原富夫
Owner CELLFREE SCI
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