Novel preparation method for recombinant human epidermal growth factor

Abstract

The invention aims to provide a simple and inexpensive preparation method for an Ssp intein system mediated recombinant human epidermal growth factor. According to the method, the conventional column cutting and chitin affinity chromatography are replaced with liquid phase cutting and ion exchange chromatography. The column cutting adopted in the conventional Ssp intein system mediated protein purification method is replaced with the liquid phase cutting, so that the loss of the target protein caused by the automatic splitting of fusion protein is reduced; and the expensive chitin affinity chromatography is replaced with the ion exchange chromatography, so that the purification cost of the recombinant human epidermal growth factor is greatly reduced, the operation steps are simplified, and the bottleneck of the large-scale industrial production of the recombinant human epidermal growth factor is overcome.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a novel preparation method of recombinant human epidermal growth factor mediated by an Ssp intein system. Background technique [0002] Human epidermal growth factor (hEGF) is a small molecular polypeptide composed of 53 amino acids, with a molecular weight of about 6.2KD and an isoelectric point of 4.2-4.6. It is heat-stable and resistant to trypsin, chymotrypsin and pepsin Digestion. The molecule contains six cysteines, and three disulfide bonds are formed between cysteines 6-20, 14-31, and 33-42 to form three rings respectively. These ring structures make hEGF Proteases have high stability. [0003] Human epidermal growth factor is a powerful cell division promoting factor that can stimulate the growth of epidermis, endothelial cells, epithelial cells, fibroblasts and capillaries, so it can be used in the treatment of skin burns, healing of surgical wounds, and trauma ...

AI Technical Summary

Problems solved by technology

However, the required protease is not only expensive, but also brings other problems: ① The target protein needs to be separated from the affinity tag, fusion protein and protease, which increases the purification steps and reduces the yield of the target protein; ② Sensitive sites for proteases may exist on the target protein, resulting in miscleavage and reduced yield

The chitin bead filler used in this purification method is expensive, 100ml of filler is 2790 yuan, and the filler can only be reused 3-5 times, which is a great limitation to the expansion of production and application

In addition, on-column cleavage will lead to the loss of the target protein. In the experiment, it was found that in the process of expressing protein using NEB's pTWIN1 and pTWIN2 vectors, there was automatic or uncontrollable cleavage between the target protein and the intein. On-column cleavage There was prior loss of the protein of interest, resulting in lower yields of protein obtained

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