Low-temperature culture medium for pichia pastoris

A culture medium and low-temperature technology, applied in the direction of microorganism-based methods, fungi, microorganisms, etc., to achieve the effect of increasing the overall yield, reducing the cost of industrial production, and reducing the difficulty of downstream purification treatment

Active Publication Date: 2020-11-10
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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  • Low-temperature culture medium for pichia pastoris
  • Low-temperature culture medium for pichia pastoris
  • Low-temperature culture medium for pichia pastoris

Examples

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Embodiment 1

[0056] The preparation of embodiment 1 culture medium

[0057] In the following examples of the present invention, the culture medium used, by the total volume of the culture medium, the culture medium is composed of the following components:

[0058] Low temperature type modified medium:

[0059]

[0060] Low temperature type improved 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 respectively named as improved I, improved II and improved 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 to the final volume; after the prepared medium is sterilized at high temperature, add PTM1 solution to detect pH, adjusted to the requ...

Embodiment 228～32

[0066] Example 2 BSM+PTM at 28-32°C 1 Medium Fermentation Test

[0067] Adopt BSM medium as fermentation medium, carry out the cultivation and expression of recombinant insulin analogue precursor Pichia pastoris engineered bacterium in 1.5T fermentation scale, comprise the following steps:

[0068] Step 1: Take recombinant insulin analog precursor recombinant Pichia pastoris engineered bacteria, add 1000 μl of working strains to a 500ml Erlenmeyer flask containing 100ml YPG medium, and culture at 28-32°C and 220rpm / min for 52h; Add 750 μl of cultured bacterial solution for 52 hours to a 500-ml Erlenmeyer flask containing 150 ml of YPG medium, and culture at 28-32° C. with shaking at 220 rpm / min for 16 hours to obtain 6 L of secondary seed solution.

[0069] Step 2, prepare 54L fermentation medium (containing 60L medium component amount), add 6L secondary seed liquid in the above-mentioned fermentation medium by 10% inoculum size, carry out tertiary seed culture, control tempe...

Embodiment 2

[0075] In embodiment 2, the thalline wet weight growth situation in the BSM medium sees figure 1 , 108h recombinant protein detection chart see figure 2 , Induced to 108h bacterial wet weight and recombinant protein expression and heterogeneous impurity proportion results are shown in Table 2.

[0076] Table 2

[0077] Fermentation medium Cell wet weight recombinant protein expression Uneven ratio of N-terminal or C-terminal segments BSM 525g / L 5.06g / L 17.677％

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Abstract

The invention discloses a low-temperature culture medium for pichia pastoris. The culture medium is favorable for realizing high-density fermentation of the pichia pastoris under the condition of lowtemperature (18-22 DEG C) while increasing the expression quantity of unit thalli. Various proteases in fermentation broth can be inhibited, so that the stability of the product structure can be maintained, and the generation of related impurities of the product can be reduced; When the method is applied to industrial preparation, the expression yield of exogenous proteins can be remarkably increased, and the downstream purification treatment difficulty is reduced. The preparation cost and the processing difficulty of products such as genetically engineered drugs, enzyme preparations and the like taking the pichia pastoris as the host are reduced.

Description

technical field [0001] The invention belongs to the field of biological fermentation and relates to a culture medium suitable for cultivating Pichia pastoris and expressing exogenous proteins under low temperature (18-22°C) conditions. The composition formula, pH value and culture conditions of the culture medium are included. [0002] technical background [0003] In recent years, Pichia pastoris has been widely used in biopharmaceuticals, enzyme preparations 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" released by Invitrogen to carry out the fermentation and induced expression of such engineering bacteria. BSM+PTM is given in this manual 1 It is recommended to control the culture temperature at 30°C. Under such high-temperature culture conditions, Pichia pastoris can often reach a high density of 500g / L wet cells, which is conducive to the i...

Claims

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Application Information

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Patent Type & Authority Applications(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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