Method for preparing thrombolytic medicament Reteplase without inclusion-body renaturation in escherichia coli

Abstract

The invention relates to a method for preparing a thrombolytic medicament Reteplase (rPA) directly through an escherichia coli expression system without inclusion-body renaturation. A gene segment of the rPA is obtained through PCR amplification, and is cloned into a vector pET40b to construct a pET40b-rPA recombinant plasmid, and the rPA is made to perform fusion expression with the protein disulfide isomerase DsbC in the vector pET40b. The recombinant plasmid is transformed into the escherichia coli BL21 (DE3), and induction expression conditions of IPTG and lactose are respectively established optimally. The obtained target fusion protein is mainly expressed by soluble components, and is purified by Ni-NTA affinity chromatography and processed by a Xa factor (or formic acid or thrombin) to form the high-purity rPA target protein. Fibrin plate method detection results show that the fusion protein DsbC-rPA obtained by the method and the rPA protein obtained by removing a label and purifying have obvious thrombolytic activity.

Description

Technical field [0001] The invention belongs to the recombinant protein drug preparation technology in the field of biomedicine, and specifically relates to a method for directly expressing and producing the active thrombolytic drug Reteplase (rPA) in Escherichia coli without the need for inclusion body renaturation. Background technique [0002] At present, cardiovascular diseases, especially thromboembolic diseases, are a major killer of human health. Thrombolytic therapy is a safe and effective treatment for thrombotic diseases. rPA is a single-chain protein with a molecular weight of 39.6kD, consisting of 355 amino acids. It is a deletion variant of human tissue type plasminogen activator (tPA) lacking the amino acid sequence 4-175. The primary structure of rPA does not contain the Kringle1, Finger, and EGF domains in the tPA molecule, but it retains the Kringle2 and serine protease domains. Since rPA lacks part of the domain of tPA, rPA has a slightly weaker binding a...

AI Technical Summary

Problems solved by technology

But the problem that exists in this method is: (1) need two plasmid co-transformation, and therefore need to add ampicillin and kanamycin two kinds of antibiotics simultaneously in culture process, cause recombinant escherichia coli genetic stability to reduce relatively on the one hand On the other hand, it also increases the difficulty of how to avoid antibiotic residue contamination in industrial production and subsequent clinical application; (2) The obtained rPA recombinant protein is only identified by western blot, and its thrombolytic activity cannot be assessed. Validation, cannot ensure correct clinical application

[0004] At present, domestic and foreign studies on the production of rPA by genetic engineering methods are mainly applied to eukaryotic expression systems such as yeast and mammalian cells, which have high costs, long production cycles, and low yields, and the expression products in eukaryotic cells are prone to sugar production. Kylation affects biological activity

The use of prokaryotic expression systems such as Escherichia coli can greatly reduce production costs and increase yields. However, although many scholars have tried to express rPA genes in Escherichia coli systems, because rPA is rich in 9 pairs of disulfide bonds, the Escherichia coli system is difficult. Directly form the correct spatial conformation, resulting in the expression products mostly in the form of inclusion body proteins, which must be separated after the cells are broken. During the separation process, a protein denaturant is used, and finally the protein must be renatured. The renaturation process There are many molecules that will be wrongly connected to form abnormal structures. At the same time, the residual protein of the bacteria is a substance that is not easy to eliminate in the production process of genetic engineering drugs. Not only the yield is low, but also it is easy to affect the quality of the product, thereby affecting the efficacy of the drug.

Images