Stage-by-stage mixing-induced fermentation method of collagen

Abstract

The invention discloses a collagen phased mixing, induction and fermentation method, which belongs to the technical field of protein engineering. The method comprises the following steps: purifying and preserving a strain, activating the strain and spreading cultivation; shaking a flask, culturing seeds in a seed tank; culturing the strain in a fermentation cylinder and acclimating the strain by methanol, carrying out an initial induction phase and a mixing increased induction phase; and post-processing a broth to obtain the collagen. The mixing increased induction mode by combining an inducer, low temperature and high power is employed, compared with induction carried out by adding the inducer at a single phase, the strain has better adaptation process to carbon source methanol, biomass accumulation can be further realized, induction intensity can be gradually enhanced, and output of target protein can be greatly increased; compared with the situation that only a temperature induction mode is used, correct folding of the target collagen can be guaranteed, massive requirement for the dissolved oxygen by the strain at an inducible expression phase can be satisfied by enhancing the power condition, expression output of the target collagen is increased, and poisoning phenomenon of the strain is avoided.

Description

technical field [0001] The invention belongs to the technical field of protein engineering, and in particular relates to a stage-by-stage mixing induction fermentation method of collagen. Background technique [0002] Collagen, also known as collagen, is the basic protein component in the extracellular matrix, widely distributed in various organs and tissues of the human body, such as skin, blood vessels, bones and cartilage, etc., especially in the skin and connective tissues of the human body, containing a large amount of collagen , about 70%-80% of the extracellular matrix of the skin is collagen, and its total content in the human body accounts for about 25%-33% of the total protein in the human body. These proteins are very important for maintaining the normal physiological functions of cells, tissues, and organs. important role. Collagen has a very complex special superhelical structure, with Gly-X-Pro as the basic repeat to form a left-handed helix, X is other amino ...

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Problems solved by technology

However, due to many disadvantages of bacterial expression, such as safety issues such as endotoxin and heat source, the protein exists in the cell in the form of inclusion body, which is difficult to extract and purify. However, it has high secretion and expression characteristics in Pichia pastoris. In the process of yeast expression, the mode of induction is to add the inducer methanol, and methanol has a certain toxic effect on the bacteria, and the amount should be strictly and accurately controlled. If the amount is small, the induction intensity is not enough, and the protein production is low. Produces toxic effects, causing some bacterial cells to autolyze and die, which brings a lot of trouble to the later extraction and purification, so the protein expression is greatly limited by the inducer methanol

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