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Method of producing protein

A technology of heterologous proteins and signal peptides, applied in the direction of introducing foreign genetic material using vectors, fermentation, recombinant DNA technology, etc., can solve problems such as unknown functions, unknown protein secretion, and failure to reach a practical level

Active Publication Date: 2007-05-30
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although genes with high homology to genes encoding elements of the Tat system have been shown to also exist in coryneform bacteria, they include tatA (GENEBANK cg103060 1571065-1571382), tatB (GENEBANK cg103060 1167110-1167580), tatC (GENEBANK cg1030601569929- 1570873) and tatE(gi|41223046|emb|CAF18991.1), but their functions are unknown, and it is not known whether the protein is secreted through the Tat system pathway in coryneform bacteria
[0009] In addition, although it has been reported that by introducing plasmids expressing the tatA, tatB and tatC genes encoding the secretory elements of the Tat system into Escherichia coli, it was reported that the amount secreted into the periplasm by the Tat pathway was improved, but the accumulated amount was only 5 to 10mg / L, which has not reached the practical level in industry (Biochem.Biophys.Res.Commun., 304, 279-284(2003))

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 - Secretive expression of isomaltglucanase using the signal sequence of isomaltglucanase from Arthrobacter globosa

[0094] (1-1) Construction of a plasmid for secretory expression of isomaltglucanase using the signal sequence of isomaltglucanase of Corynebacterium glutamicum

[0095] The sequence of the isomaltoglucanase gene derived from Arthrobacter sphaericus strain T6 (EC. 176, 7730-7734 (1994)). With reference to this sequence, primers having the sequences shown in SEQ ID NO.11 (5'-ATGATGAACCTGTCCCGCCG-3') and SEQ ID NO.12 (5'-CGCGGATCCCTGAGGGCGGGAAC-3') were synthesized using the conventional method (Saitoh and Miura The method, (Biochim.Biophys Acta, 72,619 (1963)) prepares the chromosomal DNA of Arthrobacter globosa as template, amplifies the region of encoding isomaltglucanase by PCR.Use Pyrobest DNA polymerase (Takara) For the PCR reaction, the reaction conditions were according to the protocol suggested by the manufacturer. The sequence of SEQ ID...

Embodiment 2

[0114]Example 2 - Expression and secretion of protein-glutaminase with protomer structure using the IMD signal sequence from Arthrobacter globosa

[0115] (2-1) Obtaining protein glutaminase gene from Chryseobacterium proteolyticum

[0116] The protein-glutaminase (EC.3.5.1) gene from Chryseobacterium proteolyticum has been sequenced earlier (Eur. J. Biochem. 268.1410-1421 (2001)). Referring to this sequence, the gene sequence shown in SEQ ID NO. 3 was constructed by converting codons to those highly used in C. glutamicum. This sequence contains the signal sequence (pro-region) encoding protein-glutaminase, the protomer region and the region of mature protein-glutaminase. This complete gene sequence is prepared synthetically.

[0117] According to the constructed gene sequence data of SEQ ID NO.3, primers having sequences shown in SEQ ID NO.17 (5'-CATGAAGAACCTTTTCCTGTC-3') and SEQ ID NO.18 (5'-GTAAAAGGATCCATTAATTAAAATCC-3') were synthesized. The primer shown in SEQ ID NO.17...

Embodiment 3

[0135] Example 3 - Secreted expression of protein-glutaminase using the TorA (trimethylamine-N-oxidoreductase) signal sequence from Escherichia coli

[0136] (3-1) Acquisition of the gene encoding the TorA signal peptide from Escherichia coli

[0137] The TorA gene containing the TorA signal peptide from E. coli has been sequenced earlier (Mol. Microbiol. 11:1169-1179 (1994)). Referring to this sequence, primers of the sequences shown in SEQ ID NO. 22 (5'-ATGAACAATAACGATCTCTTTCAGG-3') and SEQ ID NO. 23 (5'-CCGGATCCTGGTCATGATTTCACCTG-3') were synthesized, using an ordinary method (the method of Saitoh and Miura , (Biochim.Biophys Acta, 72,619 (1963)) the chromosomal DNA of Escherichia coli strain W3110 prepared, amplifies a region by PCR, and said region contains the region of coding TorA and the signal sequence that is positioned at its upstream.Use Pyrobest DNA polymerase (Takara) is used for PCR reaction, and reaction condition is according to manufacturer's suggested schem...

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PUM

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Abstract

The present invention provides a method for efficiently producing an industrially useful protein in coryneform bacteria, and more particularly, a method for efficiently producing a protein for which secretion was difficult with conventional protein secretion pathways. In particular, the present invention provides a method for efficiently producing heterologous proteins comprising: culturing coryneform bacteria containing an genetic construction containing a promoter sequence which functions in coryneform bacteria, a nucleic acid sequence encoding a Tat system-dependent signal peptide region, and a nucleic acid sequence encoding a heterologous protein, in the direction from 5'-end to 3'-end, and secretory producing the heterologous protein by coryneform bacteria.

Description

field of invention [0001] The present invention relates to a method for the secretory production (production and secretion) of heterologous proteins in coryneform bacteria, and more particularly, to the secretory production of heterologous proteins, including industrially useful enzymes and physiologically active proteins, in coryneform bacteria Methods. Background of the invention [0002] Coryneform bacteria are very useful in the fermentation industry as producers of L-amino acids such as L-glutamic acid and L-lysine and nucleic acids. Furthermore, coryneform bacteria inherently secrete very low levels of proteins extracellularly compared to mold, yeast and bacillus bacteria which are considered to be more preferred for secretion of heterologous proteins, which makes the production and secretion of heterologous proteins It is possible to simplify or omit purification steps. Coryneform-type bacteria can also grow rapidly in simple media containing sugars, ammonia, and in...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12N15/77
Inventor 伊达雅代菊池庆实板屋宽中村奈巳
Owner AJINOMOTO CO INC
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