Method for producing recombinant human-like collagen by expression by Pichia pastoris

Abstract

The invention discloses a method for producing recombinant human-like collagen by expression by Pichia pastoris, which adopts Pichia pastoris C13 with a collection number CGMCC No.4437 as a strain and comprises: culturing primary seeds, culturing secondary seeds, culturing in a fermentation tank and inducing expression; and centrifuging fermentation liquor obtained after induced expression, collecting supernate after centrifugation, and performing concentration, gel column chromatography, precipitate decolorization, ion-exchange column chromatography and ultrafiltration for desalination in turn to obtain the recombinant human-like collagen. Compared with other purification methods, the method is simpler in process and higher in target protein yield, realizes a recovery rate of over 60 percent and a purity of over 95 percent, can realize complete decolorization and is suitable for industrial large-scale production of the recombinant human-like collagen.

Description

technical field [0001] The invention belongs to the technical field of protein engineering, and in particular relates to a production method for expressing recombinant human-like collagen in Pichia pastoris. Background technique [0002] Collagen is the most abundant protein in the body, accounting for about 25% to 33% of human protein. It is widely distributed in various tissues and organs of the body, such as skin, bones, cartilage, cornea, blood vessels, etc., especially in the skin and connective tissue of the human body. Tissues contain a lot of collagen. As a kind of adhesive substance of connective tissue, collagen plays an important role in maintaining the normal physiological functions of cells, tissues and organs. [0003] As a natural biological resource, collagen has been widely used in biomedicine, cosmetics, food and other industries. At present, the demand for collagen in the market is increasing, and its main source is extracted from animal bodies by acid-b...

AI Technical Summary

Problems solved by technology

At present, the demand for collagen in the market is increasing, and its main source is extracted from animal bodies by acid-base method. However, although the yield of this collagen is high, it has poor water solubility and low purity. It is prone to rejection and has the disadvantages of virus hidden dangers, which inhibits the application of collagen in various industries.

With the development of biotechnology, people have also achieved success in expressing collagen by using transgenic and gene recombination technologies, such as using mammalian cells, insect cell expression systems and E. coli expression systems, etc. Although mammalian cells and insect cell expression systems can express Eukaryotic cell protein components with complex structure, but complex operation, low expression level, high cost of large-scale production, and difficult to be widely used. Although the Escherichia coli expression system has a high expression level, it is easy to form inclusion bodies, resulting in relatively low Problems such as multi-heteroproteins, which lead to difficulties in later purification and low recovery

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