Method for preparing human parathyroid hormone 1-34

Abstract

The invention relates to the technical field of biomedical engineering, and relates to a method for preparing human parathyroid hormone (1-34) through fermenting and purifying human parathyroid hormone 1-34 recombinant engineering bacteria constructed by a gene engineering method. The method provided by the invention comprises the following steps: fusing and expressing the genes for coding human parathyroid hormone and a thioredoxin label, and further constructing and finishing genetic engineering bacteria capable of highly expressing human parathyroid hormone; adding IPTG (isopropyl beta D thiogalactopy ranoside) and triton X-100 in the fermentation process by adopting a chemosmosis fermentation technology so as to improve the release of proteins extracellularly in the fermentation process, which is good for the continuous synthesis of proteins, and avoiding the attack of endoproteinase, thus protein high-yield extracellular accumulation is promoted; and simultaneously carrying out heat treatment on a fermentation liquid when the fermentation reaches the final point aiming at the heat stability of thioredoxin fusion, thus initially purified target proteins are directly recycled and obtained from a fermentation liquor supernatant after centrifugation.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, and relates to a method for constructing human parathyroid hormone (1-34) recombinant engineering bacteria by a genetic engineering method, and further fermenting and purifying a method for preparing human parathyroid hormone (1-34). Background technique [0002] Human parathyroid hormone (hPTH) is an active polypeptide secreted by human parathyroid glands. Its main physiological function is to maintain blood calcium balance by regulating bone cells and kidney cells. It is an important peptide for regulating calcium and phosphorus metabolism. Hormones that regulate the rate of bone resorption and bone synthesis and affect bone metabolism. Parathyroid hormone can efficiently and selectively increase the activity and number of osteoblasts to promote bone growth. It can not only prevent the loss of bone mass caused by the decrease of estrogen level, but also reverse the loss of bone ...

AI Technical Summary

Problems solved by technology

Although the E. coli expression system has the advantages of good stability, easy gene manipulation and high expression, this expression system also has disadvantages

One of the obvious disadvantages is that E. coli lacks a proper protein secretion mechanism, which makes most of the foreign proteins expressed in E. coli cells only accumulate intracellularly.

The retention of foreign protein in the cell will affect its continued synthesis, and it is vulnerable to the attack of intracellular proteases, which also makes subsequent separation and purification difficult

In addition, because of the small size of the polypeptide, it is difficult to directly express it, and fusion expression technology is generally required

The fusion expression used in current research is divided into two types: self-tandem fusion expression and fusion expression with other carrier proteins. Most of them accumulate in the cell and are easy to form inclusion bodies. However, refolding biologically active proteins from inclusion bodies requires a series of procedures. Denaturation and refolding process, which is very expensive

Images