Expression method for recombination human apolipoprotein AI Milano variant in pichia pastoris

A technology of apolipoprotein and Pichia pastoris, applied in the field of bioengineering

Inactive Publication Date: 2008-02-20
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Expression method for recombination human apolipoprotein AI Milano variant in pichia pastoris
  • Expression method for recombination human apolipoprotein AI Milano variant in pichia pastoris

Examples

Experimental program
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Embodiment 1

[0025] Example 1 Construction and Identification of Recombinant Expression Plasmids

[0026] The recombinant plasmid pPIC9k-apoAI-milano is obtained by site-directed mutagenesis of the pPIC9k-apoAI plasmid by recombinant PCR method, and the 173rd Arg codon (CGC) of the cDNA is mutated into a Cys codon (TGT). Sequencing verification. The mutated apoAI-milano cDNA was inserted into the Xhol and EcoRI sites of pPIC9k, with a size of about 747bp.

Embodiment 2

[0027] Example 2 Screening and Identification of Yeast Engineering Strains

[0028] Recombinant expression plasmid pPIC9K-apoAI-milano DNA was digested with Bg / ll, mixed with Pichia pastoris GS115 competent cells, transformed by electric shock under the conditions of voltage 1300V, resistance 200Ω, capacitance 25μF, coated RDB plate, and cultured at 30°C for 3 Day, more than 1500 transformants were obtained. The transformants were screened for G418 resistance, and 23 highly resistant transformants that could still grow on the G4184mg / ml plate were obtained. Through shaking flask culture and SDS-PAGE identification, 20 yeast engineering strains capable of secreting and expressing rAIM were screened from G418 highly resistant transformants.

Embodiment 3

[0029] Example 3 Induced Expression of Yeast Engineering Bacteria

[0030] According to the conventional method, the engineered yeast strain was inoculated in a test tube of 3ml BMGY medium, and cultured with shaking at 30°C for 18h. Inoculate 1 ml of the cultured species into 50 ml of BMGY medium, culture with shaking at 30°C for 24 hours, centrifuge at room temperature 6000r / min for 6min, and collect the bacteria. The cells were transferred to 15ml of BMMY medium, cultured with shaking at 30°C for 72h, and methanol was added every 24h. The fermentation supernatant was taken for SDS-PAGE and Western blot identification, and it was found that the engineered bacteria were cultivated and induced by conventional methods, and the expression product rAIM would be partially degraded, and its molecular weight was slightly smaller than that of human plasma ApoAI (28.3KD), about 23KD.

[0031] Further, the present invention also optimizes the BMMY medium and the culture conditions, th...

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Abstract

The invention pertains to the technical field of bio-engineering, in particular to an expression method of a recombinant human apolipoprotein A-I-Milano variant excreted in Pichia yeast. The mutated apoAI-milano cDNA is inserted into expression plasmid pPIC9K, and induced to Pichia yeast GS115 by electric shock, and by the optimization of substrate in a shake flask and culture conditions, the fermented suspentant liquid has obvious rAIM expression with a mocular weight identical to the ApoAi extracted from human blood plasma. The purified rAIM has phospholipid-binding activity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetic engineering expression method of human apolipoprotein I-milano (AIM). Background technique [0002] Human apolipoprotein AI Milan mutant (AIM) is a natural mutant of ApoAI, which was first discovered in some residents of a village in Milan, Italy. ApoAI on HDL in their plasma mutated, that is, Arg at position 173 was mutated into Cys. So far, it has been found that there are more than 40 AIM carriers in this family. Although these residents have low plasma HDL levels and high triglyceride levels, they seldom suffer from cardiovascular diseases and live a long life. Studies have found that HDL containing AIM can improve the efficiency of reverse cholesterol transport (RCT). On HDL, 70% Cys form inter-bond disulfide bonds, and 30% Cys thiols still exist in a free state. AIM can lead to the production of homodimeric apolipoprotein AIM / AIM or heterodim...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N1/19C12R1/84
Inventor 宋大新赵志安张彦钟江
Owner FUDAN UNIV
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