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Method for secretory production of protein

A protein and heterologous protein technology, applied in the field of coryneform bacteria, can solve problems such as unclear and reduced penicillin-binding protein relationships

Active Publication Date: 2014-07-23
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the relationship between reduced activity of penicillin-binding proteins and / or reduced activity of cell surface proteins and secretion of heterologous proteins is unclear

Method used

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  • Method for secretory production of protein

Examples

Experimental program
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Effect test

Embodiment 1

[0225] Example 1: Construction of Corynebacterium glutamicum lacking penicillin-binding proteins PBP1a and PBP1b (1) Construction of vector pBSΔCgl0278 for deletion of PBP1a encoding gene Cgl0278

[0226] The genome sequence of Corynebacterium glutamicum ATCC13032 and the sequence of the Cgl0278 gene encoding the penicillin-binding protein PBP1a have been determined (Genbank Accession No. BA000036, NCBI Gene Entry NCgl0274). Referring to this sequence, primers shown as SEQ ID NO: 01, 02, 03, and 04 were synthesized. By PCR, using the chromosomal DNA of the Corynebacterium glutamicum ATCC13869 strain prepared in a conventional manner (the method of Saito and Miura [Biochim. Biophys. Acta, 72, 619 (1963)]) as a template, and primer SEQ ID NO:01 and 02, and SEQ ID NO:03 and 04 respectively amplified the about 1 kbp 5' side upstream region and about 1 kbp 3' side downstream region of the Cgl0278 gene encoding PBP1a. Then, by using the two amplified DNA fragments as templates and ...

Embodiment 2

[0230] Then, the Corynebacterium glutamicum YDK010 strain described in WO2004 / 029254 was transformed with the constructed pBSΔCgl0278 and pBSΔCgl2986, respectively. Corynebacterium glutamicum YDK010 strain is a cell surface protein PS2-deficient strain of Corynebacterium glutamicum AJ12036 strain (FERM BP-734) (WO2004 / 029254). According to the methods described in WO2005 / 113744 and WO2006 / 057450, strains were screened from the obtained transformants to obtain Cgl0278 gene-deficient YDK010ΔPBP1a strain and Cgl2986 gene-deficient YDK010ΔPBP1b strain. Example 2: Using penicillin-binding protein PBP1a and PBP1b deficient Corynebacterium glutamicum strains to secrete and express the H chain region of antibody trastuzumab (trastuzumab) Fab fragment

[0231] (1) Construction of a plasmid for secreting and expressing the H chain region of the antibody trastuzumab Fab fragment

[0232] The gene sequence of the variable region of the H chain of the breast cancer cell-specific antibody ...

Embodiment 3

[0239] Example 3: Use of penicillin-binding protein PBP1a-deficient Corynebacterium glutamicum to secrete and express the L chain region of the antibody trastuzumab Fab fragment

[0240] (1) Construction of a plasmid for secreting and expressing the L chain region of the antibody trastuzumab Fab fragment

[0241] The gene sequence of the variable region of the L chain of the breast cancer cell-specific antibody trastuzumab has been determined (Genbank Accession No. AY513485). Referring to this sequence and the sequence of the non-variable region of the L chain of the universal antibody, considering the codon usage frequency of Corynebacterium glutamicum, DNAs shown as SEQ ID NO: 55-70 were synthesized. Using the above-mentioned DNA as a template, and using the DNA sequences synthesized respectively as shown in SEQ ID NO:71 and 72 as primers, the full-length L chain region of trastuzumab was amplified by PCR to obtain a DNA sequence as shown in SEQ ID NO: A DNA fragment of abo...

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Abstract

A method for secretory production of a heterologous protein is provided by developing a novel technique for improving ability of a coryneform bacterium to produce a heterologous protein by secretory production. By utilizing a coryneform bacterium having an ability to produce a heterologous protein by secretory production which has been modified so that activity of a penicillin-binding protein is reduced and in which activity of a cell surface layer protein has been reduced as an expression host, a heterologous protein is produced by secretory production.

Description

technical field [0001] The present invention relates to a coryneform bacterium that efficiently secretes and produces heterologous proteins, and a method for secreting and producing heterologous proteins. Background technique [0002] Regarding the production of heterologous proteins secreted by microorganisms, the use of Bacillus bacterium (Non-Patent Document 1), methanol-assimilating yeast, Pichia pastoris (Non-Patent Document 1) has been reported so far. 2) Filamentous fungi of the genus Aspergillus (Non-Patent Documents 3 and 4), etc. secrete and produce heterologous proteins. [0003] Attempts have also been made to produce heterologous proteins secreted by coryneform bacteria. Regarding the production of heterologous proteins by secretion of coryneform bacteria, it has been reported that Corynebacterium glutamicum (Corynebacterium glutamicum, hereinafter also abbreviated as C. glutamicum) secretes nucleases and esterases (Patent Document 1, Non-Patent Document 5), S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P21/00C12R1/15C12R1/13
CPCC07K14/34C12N15/77C07K16/00
Inventor 松田吉彦板屋宽菊池庆实别府春树J.A.V.乔曼塔斯E.A.库塔科瓦
Owner AJINOMOTO CO INC
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