CN-121181687-B - Recombinant human collagen and purification method thereof

Abstract

The invention provides recombinant human collagen and a purification method thereof. After the recombinant human collagen is expressed by microorganisms, the supernatant of the lysate or the extracted product is heated to more than 15 min ℃ at the temperature higher than 60 ℃, solid-liquid separation is carried out, and finally, column chromatography is carried out by utilizing a cation column. Compared with the current common purification method using two chromatographic columns, the method can not only remarkably reduce time and economic cost, but also further improve the purity and activity of the recombinant human collagen. In addition, the whole process of the method can be carried out under neutral conditions, and protein denaturation or new impurity introduction caused by acidic solvents and the like are avoided.

Inventors

• YANG MUQI
• YANG JIAWEI
• YU SHAN
• LIN YINTING
• LIANG GUOLONG
• WEN YALEI
• FENG CHENG

Assignees

• 广东华生元基因工程发展有限公司
• 联康生物制药有限公司

Dates

Publication Date

20260505

Application Date

20250929

Claims (9)

1. 1. A purification method of recombinant human collagen is characterized in that after the recombinant human collagen is expressed by microorganisms, a lysate or supernatant of the extracted product is heated at 60-100 ℃ for 15-120 min, solid-liquid separation is carried out, and finally column chromatography is carried out by utilizing a cation column, wherein the amino acid sequence of the recombinant human collagen is shown as SEQ ID NO. 2.
2. 2. The purification method according to claim 1, wherein the heating temperature is 75-100 ℃.
3. 3. The method of claim 1, wherein the system is further diluted to a conductivity of 0.8 to 1.2 ms/cm prior to the column chromatography.
4. 4. The purification method of claim 1, wherein the cation column is SP Sepharose FF.
5. 5. The method according to claim 1, wherein the eluent used for the column chromatography is sodium phosphate buffer.
6. 6. The method of claim 1, wherein the microorganism is escherichia coli or pichia pastoris.
7. 7. A recombinant human collagen is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
8. 8. Use of the recombinant human collagen according to claim 7 for the preparation of medical or cosmetic products.
9. 9. A medical product or cosmetic comprising the recombinant human collagen according to claim 7.

Description

Recombinant human collagen and purification method thereof Technical Field The invention belongs to the technical field of biological medicine manufacturing, separation and purification. More particularly, to a recombinant human collagen and a purification method thereof. Background Collagen forms a right-handed supercoiled structure (triple helix) from 3 alpha chains intertwined, each chain comprising about 1000 amino acid residues, with 30% glycine (Gly), 25% proline (Pro) and hydroxyproline (Hyp). The high proportion of glycine and hydroxyproline stabilizes the triple helix structure through hydrogen bond and Van der Waals force, and endows collagen with high mechanical strength, thermal stability and controllable biodegradability. Collagen is widely used in the fields of biological medicine manufacturing, cosmetic manufacturing and the like, for example, in the field of biological medicine manufacturing, collagen is often used as a slow-release carrier to wrap medicines, so as to realize sustained release of the medicines and the like, and in the field of cosmetic manufacturing, collagen is often used for manufacturing cosmetics for repairing skin barriers, whitening, resisting oxidation and the like. The existing purification method of the collagen comprises (1) extraction of animal sources, namely extraction from animal tissues by an acid or enzymolysis method, wherein the method has low cost, but impurities (such as lipid and nucleic acid) and pathogens (such as mad cow disease virus) are easy to remain, the purity is low (usually less than 80%), the efficiency is low (multiple times of centrifugation, filtration, salting-out and the like are needed), (2) chemical synthesis method has high cost, can precisely control the structure, is easy to introduce chemical residues, and limits the application of the collagen in the fields of biological medicine manufacturing, cosmetic manufacturing and the like, and (3) chromatography method has the advantages of separating by utilizing the charge difference of the collagen and the impurities, not introducing other impurities, but using two chromatographic columns, and has higher time and economic cost and low efficiency. Therefore, there is a need to find a method for efficiently extracting collagen, which is low in cost, high in purity and free from other impurities, and is very important in fields such as biopharmaceutical manufacturing and cosmetic manufacturing. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to provide a purification method of recombinant human collagen, which can further improve the extraction purity of the recombinant human collagen without introducing other impurities on the premise of using only one chromatographic column and obviously reducing time and economic cost by carrying out heat treatment before column chromatography. The first object of the present invention is to provide a method for purifying recombinant human collagen. A second object of the present invention is to provide a recombinant human collagen. A third object of the present invention is to provide the use of the recombinant human collagen in the preparation of medical products, cosmetics or pharmaceutical products. A fourth object of the invention is to provide a product. The above object of the present invention is achieved by the following technical scheme: The invention provides a purification method of recombinant human collagen, which comprises the steps of expressing recombinant human collagen by microorganisms, cracking a product or extracting supernatant of the product, heating the supernatant to more than 15 min at a temperature higher than 60 ℃, carrying out solid-liquid separation, and finally carrying out column chromatography by utilizing a cation column. Compared with the current common purification method using two chromatographic columns, the method can not only remarkably reduce time and economic cost, but also further improve the purity and activity of the recombinant human collagen. In addition, the whole process of the method can be carried out under neutral conditions, and protein denaturation or new impurity introduction caused by acidic solvents and the like are avoided. Preferably, the amino acid sequence of the recombinant human collagen is shown as SEQ ID NO. 1. When the recombinant human collagen is adopted, the pH value and the salt concentration in the cracking process can not cause protein denaturation, and the purification effect is not obviously influenced, so that the pH value or the salt concentration in the cracking process does not need to be particularly adjusted. Preferably, the microorganism is E.coli or Pichia pastoris. When the microorganism is escherichia coli, the purification method of the recombinant human collagen comprises the steps of expressing the recombinant human collagen by escherichia coli, then cracking the product, heating the product at a temperature higher than 60 ℃ for mor