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Secretion type Pichi strain and its construction method

A Pichia pastoris, secretion-type technology, applied in the field of genetically engineered bacteria and microbial fermentation, can solve problems such as complex condition control, and achieve the effect of exempting the cell breaking step

Inactive Publication Date: 2007-10-10
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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Problems solved by technology

[0004] Methanol-inducible strains of Pichia pastoris can express DAO from Trichomonas (Yu J, Yang S, Yuan ZY.J.Mol.Catal B-Enzym.18:291-297, 2002), but inducible In the fermentation process, it is necessary to add methanol to induce, and the condition control in the fermentation process is more complicated; and the extraction of the product needs to break the cells first, so it is necessary to further improve

Method used

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  • Secretion type Pichi strain and its construction method

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Experimental program
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Effect test

Embodiment 1

[0018] Embodiment 1, plasmid construction

[0019] 1. Materials:

[0020] Plasmid pGEM-T: a product of Promega Company, with a size of 3.0 kb, containing an ampicillin resistance gene.

[0021] Plasmid pGAPZαB: a product of Invitrogen Company, with a size of 3.1 kb, containing a Zeocin resistance gene, and a promoter of glyceraldehyde triphosphate dehydrogenase gene.

[0022] Plasmid pLHB-3: constructed according to the method of Liu HB. et al. (Liu HB., Jiang WH., Yang YL. Cloning, Sequencing and Expression of D-amino acid oxidase gene. Chinese Journal of Biotechnology 15:337-342, 1999), The size is 6.4kb, it contains DAO gene (HDAO) with histidine purification tag, kanamycin resistance gene, the promoter is T7lac, and it has histidine purification tag.

[0023] 2. Method:

[0024] As shown in Figure 1, using the plasmid pLHB-3 as a template, a pair of primers for amplifying the HDAO gene were designed as follows:

[0025] Upstream primer: 5'-AA CA ATGGCTAAAATCGTTGTT-...

Embodiment 2

[0030] Embodiment 2, plasmid amplification:

[0031] The plasmid pMMWY01 obtained in Example 1 was transformed into Escherichia coli host strain DH5α (Takara) using the conventional chemical transformation method "Reference Molecular Cloning", and a single transformant was picked and inoculated into LB liquid medium (1% peptone, 0.5% yeast extract, 1% sodium chloride), the pMMWY01 plasmid was cultured and amplified at 37°C.

Embodiment 3

[0032] Embodiment 3, plasmid transformation

[0033] After the recombinant Escherichia coli was cultured overnight in LB medium, the recombinant plasmid pMMWY01 was extracted from the Escherichia coli by alkaline lysis according to "Molecular Cloning". The plasmid was about 10 μg, and then the linearized plasmid was mixed with Pichia pastoris host strain GS115 (his - mutate + ) 80 μl of competent cells were mixed and placed in an electroporation cuvette, and a copy of competent cells without linearized plasmid pMMZY03 was prepared as a negative control, and placed in an ice bath for 5 minutes. A Bio-rad electroporation instrument was used, the electroporation parameters were set to 1.5KV, 25μF, 200Ω, and the electroporation time was 4.6ms. Immediately after electroporation, 1ml of sorbitol in an ice bath was added. Electroporation transformation products were coated with 100 μg / ml Zeocin TM YPDS (2% peptone, 1% yeast extract, 2% glucose, 1M sorbitol, 2% agar powder) plate, ...

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Abstract

A secretion-type Pichia yeast expression carrier for expressing the recombinant D-amino acid oxidase with His label is configured. The recombinant D-amino acid oxidase can be secreted in the procedure of culturing its engineering bacteria. The target protein can be easily purified by linking 6 His to the terminal N of DAO gene and using the metallic chelating and affinity chromatography.

Description

technical field [0001] The invention relates to the fields of genetic engineering bacteria and microbial fermentation, in particular to a secreted Pichia yeast strain expressing D-amino acid oxidase and a construction method thereof. Background technique [0002] D-Amino Acid Oxidase (DAO, EC 1.4.3.3) is a typical flavoproteinase with flavin adenine dinucleotide (FAD) as the prosthetic group, which catalyzes the oxidation of D-amino acid The deamination reaction generates the corresponding keto acid and ammonia, and is accompanied by the reduction of a molecule of oxygen to release hydrogen peroxide. In the two-step enzymatic production of 7-amino acid cephalosporanic acid (7-ACA), DAO is used in the first step reaction of converting cephalosporin C (CPC). [0003] Pichia pastoris has become a major eukaryotic microbial expression system (Cregg JM., Vedvick TS., Raschke WC. Bio / Technology. 11:905-910, 1993). Pichia pastoris can ferment at high density, and the promoter com...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/63C12N15/53C12N15/81C12N15/65C12R1/84
Inventor 杨晟郑华宝王筱兰陈军杨蕴刘姜卫红
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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