A kind of Pichia pastoris low temperature medium

A culture medium and low-temperature technology, applied in the direction of microorganism-based methods, fungi, microorganisms, etc., to achieve the effect of expanding application conditions, reducing the difficulty of downstream purification treatment, and improving purity

Active Publication Date: 2022-07-19
BEIJING SL PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Similarly, it does not involve the cultivation improvement of Pichia pastoris under low temperature conditions

Method used

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  • A kind of Pichia pastoris low temperature medium
  • A kind of Pichia pastoris low temperature medium
  • A kind of Pichia pastoris low temperature medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The preparation of embodiment 1 culture medium

[0057] In the following embodiments of the present invention, the culture medium used, according to the total volume of the culture medium, is composed of the following components:

[0058] Low temperature modified medium:

[0059]

[0060] Low temperature modified medium trace element solution formula:

[0061]

[0062] On the basis of the above-mentioned medium, the medium was prepared according to the formula in Table 1, and three different improved medium were obtained, which were named as Modified I, Modified II and Modified III.

[0063] Table 1

[0064]

[0065] The preparation process is as follows. Dissolve the ingredients in the table with 80% to 90% of the final volume of purified water. After fully stirring, use purified water to make up the volume to the final volume. After the prepared medium is sterilized by high temperature, add PTM1 solution to detect The pH was adjusted to the required pH wit...

Embodiment 228～32

[0066] Example 2 BSM+PTM at 28~32℃ 1 Fermentation test of culture medium

[0067] Adopt BSM substratum as fermentation substratum, in 1.5T fermentation scale, carry out the cultivation and expression of recombinant insulin analog precursor Pichia pastoris engineering bacteria, comprising the following steps:

[0068] Step 1, take the recombinant insulin analog precursor recombinant Pichia pastoris engineering bacteria, add 1000 μl working strains to a 500 ml Erlenmeyer flask containing 100 ml YPG medium, and shake at 28 to 32 ° C and 220 rpm / min for 52 hours; A 500ml conical flask containing 150ml of YPG medium was added with 750μl of bacterial liquid cultured for 52h, and incubated at 28 to 32°C with shaking at 220rpm / min for 16 hours to obtain 6L of secondary seed liquid.

[0069] Step 2, prepare 54L fermentation medium (containing 60L medium composition), add 6L secondary seed liquid to above-mentioned fermentation medium by 10% inoculation amount, carry out tertiary seed ...

Embodiment 2

[0075] In embodiment 2, in the BSM medium, the growth situation of thalline wet weight sees figure 1 , see the 108h recombinant protein detection chart figure 2 , after induction to 108h, the wet weight of bacteria, the expression of recombinant protein and the proportion of heterogeneous impurities are shown in Table 2.

[0076] Table 2

[0077] Fermentation medium Bacteria wet weight Recombinant protein expression Non-uniform proportion of N-terminal or C-terminal segment BSM 525g / L 5.06g / L 17.677％

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Abstract

The invention discloses a low-temperature type medium of Pichia pastoris, which is helpful for realizing the expression of Pichia pastoris while increasing the expression amount of a unit cell under the condition of low temperature (18-22° C.). High-density fermentation; it can also inhibit various proteases in the fermentation broth, which is conducive to maintaining the stability of the product structure and reducing the generation of product-related impurities; when applied to industrial preparation, it can significantly improve the expression of exogenous proteins yield and reduce the difficulty of downstream purification. The preparation cost and processing difficulty of genetically engineered medicines, enzyme preparations and other products using Pichia pastoris as a host are reduced.

Description

technical field [0001] The invention belongs to the field of biological fermentation, and relates to a medium suitable for culturing Pichia pastoris and expressing exogenous proteins under low temperature (18-22°C) conditions. The composition, pH and culture conditions of the medium are included. [0002] technical background [0003] In recent years, Pichia pastoris has been widely used in biopharmaceutical, enzyme preparation and other industries, and is considered to be one of the most promising heterologous protein production systems. Researchers generally refer to the "Pichia Fermentation Manual" launched by Invitrogen for fermentation and induction of such engineered bacteria. BSM+PTM is given in this manual 1 It is recommended that the culture temperature be controlled at 30°C. Under such high temperature culture conditions, Pichia pastoris can often reach a high density of 500 g / L wet cells, which is conducive to the increase of the total amount of exogenous protei...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/16C12N1/19C12R1/84
CPCC12N1/16
Inventor 牛罡郑伟陶真民何林李阳任小飞张晗郭天罡张宁苑德禄徐明波
Owner BEIJING SL PHARMA
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