Expression of recombined bovine pancreas ribonucleic acid hydrolase A and purifying method thereof

Abstract

The invention relates to the expression of reforming bovine insulin ribonucleic acid hydrolytic enzyme A (RNaseA) and a depurant method, the genetic expression and the albumen purification technology in modern biology are adopted to perform an improvement to the preparation method of the reforming RNaseA, and the invention provides the preparation method of the reforming RNaseA which can be widely popular in biological medicine, is safe and feasible, and can greatly reduce the manufacturing cost.

Description

technical field [0001] The invention relates to a method for expressing and purifying recombinant bovine pancreatic ribonucleic acid hydrolase A (RNaseA). Background technique [0002] With the rapid development and widespread application of molecular biology, newly established biomedical companies have sprung up like mushrooms after rain. For biomedical companies involved in the production and preparation of products closely related to human health, such as pharmaceuticals, food and vaccines, relevant government departments have strict requirements and standards for their production and preparation processes and raw materials used. For example, in the process of preparing DNA for the production of third-generation vaccines, it is necessary to hydrolyze and remove a large number of intracellular RNAs (ribonucleic acid), which requires the addition of RNaseA (bovine pancreatic ribonuclease A) to complete. Currently known methods for preparing RNaseA are represented by bioche...

AI Technical Summary

Problems solved by technology

However, a major problem with this method is that most of the RNaseA produced is an inactive protein polymer, which must undergo complex procedures such as denaturation and dissolution, and then gradually dialysis and refolding to produce a small amount of active RNaseA. Such an operation consumes a lot of time. and cost, and greatly reduces production efficiency

The other is to secrete bovine RNaseA enzyme into the periplasm in the form of fusion protein of maltose binding protein, which has the advantages of solubility and better activity, but the disadvantage of this method is that the yield is too low, less than 1mg / L; It is expressed by Pichia pastoris and secretes recombinant RNaseA into the medium. The problem with this method is that the expression level is low, less than 5 mg / L, and about half of it is glycosylated, and its activity is much lower than that of normal RNaseA

Due to concerns that mad cow disease is a zoonotic disease, we cannot safely use ribonuclease products extracted from bovine pancreas in medicine, food, etc.