Production process for inhibiting degradation of recombinant human collagen by supplementing competitive protein

A human-derived collagen and production process technology, which is applied in the field of production technology for inhibiting the degradation of recombinant human-derived collagen, can solve the problems that recombinant collagen expression is technically difficult, the stability of recombinant collagen is not as good as that of natural collagen, and the product is easy to degrade, etc. Reduced degradation, improved stability, improved yield and purity

Pending Publication Date: 2021-12-21
汉肽生物医药集团有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

Different from natural collagen, recombinant collagen has a single-chain structure, while natural collagen has a triple-helix structure. In terms of stability, the stability of recombinant collagen is not as good as that of natural collagen, so the expression technology of recombinant collagen is difficult, and the product is easy to degrade

Method used

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  • Production process for inhibiting degradation of recombinant human collagen by supplementing competitive protein
  • Production process for inhibiting degradation of recombinant human collagen by supplementing competitive protein
  • Production process for inhibiting degradation of recombinant human collagen by supplementing competitive protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-6

[0021] After the induction of recombinant Pichia pastoris was completed, DO>20%, and the methanol feeding was stable, the sterilized competitive protein was added at a time of 15g / L, and then normal methanol feeding was induced. After the fermentation, the temperature was raised to 65° C. for 30 minutes. After the end of the temperature rise, the temperature was rapidly lowered to 25° C. Detect the yield of recombinant human collagen (including degraded protein), and detect the yield of the full sequence target collagen. Thus, the stability of the recombinant human collagen was determined.

[0022] In Examples 1-6, several competitive proteins such as yeast peptone, tryptone, bovine peptone, fish peptone, soybean peptone, and acid hydrolyzed casein were selected, and the results are shown in the appendix figure 1 .

Embodiment 7

[0024] In this example, acid hydrolyzed casein was used as a competitive protein to conduct a comparative study of different parameters.

[0025] Select Example 6 to add acid hydrolyzed casein for further research, the target protein yield and protein stability at different heating temperatures and holding times. Group experiments were carried out according to the combination of different heating temperature and holding time.

[0026] After the induction of recombinant Pichia pastoris was completed, DO>20%, and the methanol feeding was stable, sterilized acid hydrolyzed casein was added at one time at an amount of 15g / L, and then normal methanol feeding was induced. After the fermentation is finished, the temperature is raised, and the temperature of the warming is selected to be 60-75° C., and the holding time is selected to be 10-30 minutes. After the heating is completed, the temperature is rapidly dropped to 25° C. Detect the yield of recombinant human collagen (including...

Embodiment 8

[0027] Example 8 Comparative Example

[0028] This example provides a set of comparative experiments, specifically comparing the conventional fermentation production process in the prior art with the method in Example 6, and performing electrophoresis experiments on the proteins obtained by the two methods.

[0029] In the conventional fermentation of the production process in the prior art, after the induction of recombinant Pichia pastoris is completed, DO>20%, normal methanol feed induction is carried out. After the fermentation, the extraction process was carried out normally, and the yield of recombinant human collagen (including degraded protein) reached 17.2g / L, of which the amount of full-sequence target collagen was 2.8g / L. The stability of recombinant human collagen is 16.2%, see the attached electrophoresis image 3 and 4 .

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Abstract

The invention discloses a production process for inhibiting degradation of recombinant human collagen by supplementing competitive protein, and belongs to the technical field of bioengineering fermentation. The production process specifically comprises the following steps of: supplementing the competitive protein into fermentation liquor at one time to inhibit degradation of the human collagen in an induction stage of pichia pastoris fermentation; and meanwhile, after fermentation is ended, heating to inactivate enzyme activity of the fermentation liquor. The production process disclosed by the invention can guarantee stability of the collagen in a purifying process, effectively improves yield and purity, is stable and easy to realize, and is suitable for large-scale fermentation production.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering fermentation, and in particular relates to a production process for inhibiting the degradation of recombinant human collagen by adding competitive proteins. Background technique: [0002] As a kind of fibrous protein in the human body, collagen accounts for 25%-33% of the protein content in the human body. It mainly exists in the skin, muscles, bones, etc., which can improve the elasticity of the skin and muscles, enhance the combination of calcium and bone cells, and protect bone health etc. [0003] Because of its unique biological characteristics, collagen can be widely used in beauty, clinical medicine, food, chemical industry and other fields. With the expansion of demand for skin care, beauty and nutrition and health care in China, the market demand for collagen will usher in a stage of continuous expansion. At the same time, the level of industry regulation It needs to be further i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/78C12R1/84
CPCC07K14/78
Inventor 殷世清朱永真张甲庆徐靖洋张凤龙李艳琪
Owner 汉肽生物医药集团有限公司
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