Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1992-04-03
2002-06-04
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S069100, C536S023100
Reexamination Certificate
active
06399323
ABSTRACT:
FIELD OF THE ART
This invention relates to molecular biology and bio-engineering, and more particularly to methods of preparing polypeptides in a cell-free translation system.
Said polypeptides are widely used in medicine as regulators of biological processes. Known in the prior art are, e.g., polypeptides activating the immune system, polypeptides which are neuromediators and transmitters, polypeptides regulating salt metabolism, etc. Polypeptides are also used in agriculture as biological stimulants, e.g., growth hormones. They are also used in bioelectronics, e.g., as rodopsin films.
STATE OF THE ART
Known in the art is a method for a preparative expression of cell genetic material by the method of genetic engineering based on the introduction of a foreign DNA into a live cell, the genetic material of said foreign DNA being expressed by the apparatus of the host cell. This method is widely employed in commercial production of proteins.
However, the method has a limited application. This is associated with the complexity of isolation of the products of gene expression by the transformed cells, lethality of some specific products for the host cell, elimination of the transformed plasmids from the cell, proteolytic degradation or aggregation of the product of expression of the foreign gene.
It follows from the foregoing that the method of genetic engineering does not provide for possibilities of a preparative expression of all genes.
Known is another method of expression of genes based on the use of a cell-free system of continuous conjugated transcription/translation (Gene, 1989, v. 84, p. 463). This system is free of limitations imposed by a cell and ensures expression of substantially any gene in the form of a DNA molecule engineered in the required manner.
However, this method cannot be applied for cell-free eukaryotic systems. The problem is that upon expression of genes by the said method the use is made of endogenous RNA-polymerases of the cells employed for preparation of the cell-free system of conjugated transcription/translation. This requires the use of special methods for isolation of the cell extract which ensure the maintenance of the activity of endogenous RNA-polymerases. Moreover, in eukaryotic cells the transcription and translation processes are, as a rule, dispersed in space and time: the transcription takes place in the cell nucleus, while the translation occurs in the cell cytoplasm after relevant modifications of mRNA. Therefore up to the present all attempts to obtain a reliable system of conjugated transcription/translation based on eukaryotic cell extracts have been unsuccessful. The only method providing a reliable preparation of such extracts is based on the preparation of the S30 extract from bacterial cells of
Escherichia coli
. Besides, the plasmid containing the gene coding for the specific product has a selection gene (a gene providing resistance to the action of an antibiotic) which is also controlled by an promoter of the RNA-polymerase of
E. coli
and is expressed as efficiently as the gene coding for the specific product. As a result, in addition to the specific product, a side product is synthesized upon functioning of the system.
Known in the art is one more method of preparative synthesis of polypeptides based on the use of continuous cell-free translation system containing a template RNA as a nucleic acid. This method consists in preparing polypeptides on ribsomes in a cell free translation system containing ATP, GTP and amino acids as substrates accompanied by the formation of translation products in the system which include the specific product, AMP, GDP, pyrophosphate and inorganic phosphate. In the process of translation, translation products, including AMP, GDP, pyrophosphate, inorganic phosphate and the specific product, are removed from the system as substrates are converted to products with a simultaneous delivery in the system of substrates in the form of amino acids, ATP, GTP to maintain their initial concentration unchanged (Science, 1988, v. 242, p. 1162).
Said method makes it possible to carry out preparative synthesis of substantially any polypeptides in cell-free translation systems prepared from cells of any organisms.
However, the application of this method makes impossible the expression of the genetic material as DNA molecules. A template RNA is used in this method. This means that to realize the method, it is necessary to carry out an additional synthesis of template RNAs. As known, a template RNA is obtained from DNA molecules using transcription by RNA-polymerases. This is a labor-consuming and expensive process. Thus, at present the available methods do not permit to synthesize polypeptides using DNA molecules in any cell-free systems.
DISCLOSURE OF THE INVENTION
The object of the invention is to develop such a method of preparation of polypeptides in cell-free systems which would ensure preparation of polypeptides with the use of DNA molecules in any cell-free system based on both prokaryotic and eukaryotic extracts.
This object is accomplished by provision of the method for preparation of polypeptides in the cell-free translation system containing a nucleic acid and ATP, GTP, and amino acids as substrates, with formation of translation products including the specific polypeptide, AMP, GDP, pyrophosphate and inorganic phosphate which are removed from the system as substrates are consumed with a simultaneous delivery of ATP, GDP and amino acids as substrates for maintenance of their initial concentration. According to the invention, the system also contains an exogenous RNA-polymerase as well as DNA molecules comprising protein-coding genes with promoter sites specific to the above polymerase, CTP and UPD as substrates and, in addition, CDP and UDP as products.
Prokaryotic and eukaryotic cell-free translation systems are used as cell-free translation systems according to the invention. E.g., systems based on
E. coli
extracts can be used as prokaryotic cell-free systems, and systems based on extracts from wheat embryos or on lysates from rabbit reticulocytes can be used as eukaryotic cell-free systems. The ratio of the components in the reaction mixture, ion and temperature conditions of the synthesis are optimal for the organisms from which cell-free systems and exogenous RNA-polymerases are prepared. The range of these conditions is rather wide.
The method implies the use of an exogenous phage RNA-polymerase, e.g., phage T7 RNA-polymerase or phage SP6 RNA-polymerase, as an exogenous RNA-polymerase.
As said, in some cases it is expedient to use a prokaryotic cell-free system based on
E. coli
extracts. Such a translation system contains an endogenous RNA-polymerase. To prevent the formation of additional translation products, an additional delivery of an inhibitor of the endogenous RNA-polymerase should be used. E.g., rifampicin is used as an inhibitor of the prokaryotic endogenous RNA-polymerase.
The nucleic acid employed in the system in represented by the protein-coding in the form of DNA molecules with promoter sites specific to an exogenous RNA-polymerase. Such protein-coding genes can be contained in, DNA molecules obtained by amplification of a DNA fragment or plasmid DNA.
The proposed method has no disadvantages of the genetic engineering method and known methods of preparative synthesis of polypeptides in continuous cell-free translation systems. It provides preparation of polypeptides within various cell-free systems without a preliminary synthesis of template RNA molecules. Due to the choice of the components, the synthesis of a template RNA proceeds directly in the cell-free system.
The proposed method ensures the preparative synthesis of polypeptides at a constant rate during tens of hours with a yield of the functionally active product (polypeptide) of 1 to 10 nmol per 1 ml of the reaction mixture and can be employed in commercial production of preparing polypeptides in any cell-free systems.
REFERENCES:
Baranov Et Al. (1989), Gene 84: 463-466.*
Krieg Et Al. (1984), Nucl. Acids Res. 12(18):7
Baranov Vladimir Ivanovich
Ryabova Ljubov Anatolievna
Spirin Alexandr Sergeevich
Yarchuk Oleg Bronislavovich
Arent Fox Kintner Plotkin & Kahn
Institute for Protein Research
McKelvey Terry
Sandals William
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