Pichia pastoris strain capable of utilizing non methyl alcohol carbon source induced AOX1 promoter to express outside source protein

A Pichia pastoris and exogenous protein technology, applied in the field of Pichia pastoris strains, can solve problems such as increased cost, troublesome industrial production, and higher requirements for equipment cooling capacity.

Inactive Publication Date: 2010-10-13
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1. When inducing the expression of exogenous recombinant proteins, methanol must be used as a carbon source. Methanol is toxic and flammable. In industrial production, the workshop needs to be designed for riot prevention, resulting in increased costs;
[0006] 2. Methanol fermentation consumes a lot of oxygen. Generally, it is difficult to meet the demand for oxygen by increasing the ventilation volume and speed of the air, and pure oxygen is needed. The more methanol consumed, the more pure oxygen is needed, which brings great harm to the actual industrial production. In addition, the more methanol consumed, the greater the heat produced, and the higher the cooling capacity requirements of the required equipment;
[0007] 3. As a petrochemical product, methanol is not suitable for the production of food or additives;
[0008] 4. Methanol metabolism produces H 2 o 2 , leading to hydrolysis of the expressed protein;
[0009] 5. The high-efficiency transcription of AOX1 is inseparable from methanol, and suffers from carbon source repression in other carbon sources

Method used

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  • Pichia pastoris strain capable of utilizing non methyl alcohol carbon source induced AOX1 promoter to express outside source protein
  • Pichia pastoris strain capable of utilizing non methyl alcohol carbon source induced AOX1 promoter to express outside source protein
  • Pichia pastoris strain capable of utilizing non methyl alcohol carbon source induced AOX1 promoter to express outside source protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Glucose or fructose can induce the expression of the Pichia yeast strain of AOX1 promoter

[0049] 1.1 Construction of PpHXT1 deletion fragment in vitro

[0050] The PpHXT1 gene is 1614bp long (SEQ ID NO: 1), encoding 537 amino acids (SEQ ID NO: 2). Adopt PCR, the method of restriction endonuclease ligation, construct a segment of PpHXT1 gene deletion fragment in vitro with pUC18 as carrier (such as figure 1 shown), consisting of 200 bp upstream of the PpHXT1 gene, 1517 bp of the G418 resistance gene, and 200 bp downstream of the PpHXT1 gene. The resulting PpHXT1 gene deletion fragment lacks the entire open reading frame of the gene.

[0051] 1.2 Screening of electroporated Pichia pastoris and Δhxt1 deletion strain

[0052] The PpHXT1 deletion fragment was electrotransformed into Pichia pastoris strain GS115, spread on 4 YPD plates added with G418, and cultured in a 30°C incubator for 48-72 hours. Pick the monoclonal grown on the plate into 10ml YPD+G418 l...

Embodiment 2

[0074] Example 2 Glycerol can induce the expression of the AOX1 promoter Pichia strain

[0075]2.1 Construction of in vitro deletion fragments of PpMIG1 and PpMIG2

[0076] The PpMIG gene of Pichia pastoris encodes a transcriptional repressor. The PpMIG1 gene is 1335bp long (SEQ ID NO: 3), encoding 444 amino acids (SEQ ID NO: 4); 454 amino acids (SEQ ID NO: 6).

[0077] Using PCR, restriction endonuclease ligation method, a section of PpMIG1 gene deletion fragment was constructed in vitro with pUC18 as vector (eg Figure 8 shown), consisted of 728 bp upstream of PpMIG1 gene, 1321 bp of Zeocin resistance gene, 3' end of PpMIG1 gene and 1011 bp downstream of PpMIG1 gene, respectively. The obtained PpMIG1 gene deletion fragment has deleted the first 315 amino acids of the gene.

[0078] Using the same method to construct the PpMIG2 gene deletion fragment (such as Figure 9 shown), the difference is that the Zeocin resistance gene was replaced by the G418 resistance gene KAN, ...

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Abstract

The invention provides a plurality of Pichia pastoris strains capable of utilizing non methyl alcohol carbon source induced AOX1 promoter to express outside source protein. PpHXT1 gene in the Pichia pastoris strain mutates, thus protein can not be coded or the coded protein has no activity, and autophagy gene PpATG30 gene also mutates, thus protein can not be coded or the coded protein has no activity; or PpMIG1 gene in the Pichia pastoris strain mutates, thus protein can not be coded or the coded protein has no activity, and PpMIG2 gene in the Pichia pastoris strain mutates, thus protein can not be coded or the coded protein has no activity. The Pichia pastoris strain of the invention not only has the advantages of the existing Pichia pastoris strain but also can utilize non methyl alcohol carbon source induced AOX1 promoter to express outside source protein, is safe and environmentally friendly, has simple operation and lower cost, also can be applied to production of food or additive and is applicable to large-scale popularization and application.

Description

technical field [0001] The present invention relates to the technical field of bioengineering and biopharmaceuticals, more specifically, to the technical field of expression strains, in particular to a Pichia yeast strain that can use non-methanol carbon sources to induce the expression of foreign proteins from the AOX1 promoter. Background technique [0002] As of 2006, more than 500 foreign proteins have been expressed in Pichia pastoris, and these proteins are derived from the entire biological kingdom, including viruses, bacteria, fungi, plants, insects and vertebrates. The promoter used by Pichia pastoris to express foreign protein is the promoter of AOX1 (Alcohol oxidase 1, alcohol oxidase gene). Fructose, glycerol, etc.) inhibit the expression of AOX1 promoter. [0003] The reason why so many exogenous proteins are expressed using the Pichia pastoris expression system is that it has incomparable advantages over other expression systems, such as the simple operation o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N1/16C12R1/84
Inventor 周祥山张元兴张平张文文柏鹏
Owner EAST CHINA UNIV OF SCI & TECH
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