Novel method for co-transformation of pichia pastoris through multiple genes

A Pichia pastoris and multi-gene technology, applied in the field of multi-gene co-transformation of Pichia pastoris, can solve the problems of difficult screening of yeast transformants, difficult selection of yeast transformants, and low expression of products

Inactive Publication Date: 2014-05-28
HUAIHUA UNIV
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Problems solved by technology

However, there are obvious deficiencies in the above three methods: firstly, the different genes are connected by PCR, fused and then constructed into an expression vector.
However, the expression level of the product obtained by this method is often very low
The main reason is that two or more genes are connected in series in the same reading frame, coupled with the IRES sequence, the gene fragments are larger than the gene fragments expressed by fusion, and when only the common expression regulatory elements are regulated, especially It is under the control of the only promoter, which will affect the transcription and translation of the gene, so it is difficult to obtain high-level secretory expression of Pi

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  • Novel method for co-transformation of pichia pastoris through multiple genes
  • Novel method for co-transformation of pichia pastoris through multiple genes
  • Novel method for co-transformation of pichia pastoris through multiple genes

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Embodiment Construction

[0010] In the present invention, Pichia pastoris X-33 strain is selected, and the integrative expression plasmid pPICZαA vector is purchased from Invritrogen Company of the United States. The medium formula used is as follows:

[0011] 1) YPDZ plate: Dissolve 1 g of yeast extract, 2 g of peptone, and 1.8 g of agar powder completely to a volume of 80 mL, autoclave at 121 °C for 15-20 min, and add sterilized medium when the medium cools to 60 °C 10% glucose solution 20 mL, 100 μL 1 mg / mL Zeicin, pour 10 cm plate, pour 15 mL per plate, cool at room temperature in the dark for 1-2 hours, and store in a 4°C refrigerator for later use. 2) LBZ plate: 0.5 g NaCl, 1 g peptone, 0.5 g yeast extract, 1.8 g agar powder, distilled water to 100 L, autoclave, add 25 μL of 1 mg / mL when the medium is cooled to 60 °C Zeicin, pour 10 cm plates, pour 15 mL into each plate, cool at room temperature in the dark for 1-2 hours, and store in a 4°C refrigerator for later use.

[0012] A new method fo...

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Abstract

The invention discloses a novel method for co-transformation of pichia pastoris through multiple genes. By adopting the novel method, the fact that multiple exogenous genes required to be expressed are connected to the same pichia pastoris expression vector is realized, and in the expression vector, each gene is positioned in an independent expression frame; the fact that multiple genes are efficiently, simultaneously and stably integrated into a pichia pastoris genome is achieved by adopting a built coexpression vector to transform the pichia pastoris. The invention provides the efficient novel method provided for achieving that multiple genes are simultaneously and independently expressed in the same pichia pastoris cell.

Description

technical field [0001] The present invention relates to a new method for multi-gene co-transformation of Pichia pastoris, in particular to a method for co-transforming yeast with two or more genes and obtaining multi-gene integration into the same Pichia genome. The source genes have the ability to express independently under the control of the same and independent gene expression regulatory elements. Background technique [0002] The methanolic yeast expression system is a very widely used yeast expression system. The exogenous gene expression system using Pichia pastoris (P. pastoris, Pichia pastoris) as the host has developed the fastest in recent years and is also the most widely used. The reason is that in addition to the characteristics of general yeast, the system also has The following advantages: ① It has a unique strong alcohol oxidase gene AOX1 promoter, and the expression of foreign proteins can be strictly regulated by methanol; ② As a eukaryotic expression sys...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12R1/84
Inventor 李洪波吴东海
Owner HUAIHUA UNIV
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