Method for preparing aspergillus flavus urate oxidase by utilizing intracellular expression of yeast

Abstract

The invention relates to a method for preparing aspergillus flavus urate oxidase by utilizing intracellular expression of yeast, which comprises the following steps: after intracellular expression of aspergillus flavus urate oxidase in induced recombinant yeast, putting thalli into a cell breaking buffer solution to break cells; wherein the cell disruption buffer solution contains an additive, and the additive is selected from one or a combination of more of dithiothreitol, tris (2-carboxyethyl) phosphine, cysteine and glutathione. According to the method, degradation of intracellular protein of yeast is inhibited by optimizing a homogenate buffer solution, so that the integrity of target protein is ensured; the cell breaking condition can be improved, such as the homogenizing pressure is reduced, so that the production energy consumption is reduced, and the requirement on equipment is reduced.

Description

technical field [0001] The invention belongs to the field of protein expression, separation and purification, and in particular relates to a method for preparing Aspergillus flavus uric acid oxidase by using yeast intracellular expression. Background technique [0002] Urate oxidase (urate oxidase) is an enzyme in the process of purine metabolism in vivo, which can catalyze the reaction of uric acid to produce allantoin. Humans lost urate oxidase during evolution and cannot further degrade uric acid. Due to the poor solubility of uric acid, its excretion is more difficult, which makes humans susceptible to diseases caused by the accumulation of uric acid. [0003] Tumor lysis syndrome (TLS) is a complication that occurs when a large number of tumor cells are lysed by chemotherapy to produce more potassium, phosphorus, nucleic acids and cytokines than the body's homeostatic mechanism can cope with. Since there is no endogenous urate oxidase in the human body, the uric acid ...

AI Technical Summary

Problems solved by technology

However, this expression also faces some limitations

For example, because the thickness of the cell wall of yeast is thicker than that of prokaryotes, chemical methods, enzymatic methods, ultrasonic methods, freeze-thaw methods and other lysis methods are not suitable for industrial production, and mechanical methods are required to break the cells to release intracellular proteins. There are many operating parameters, the optimization is more complicated, and the effective energy exchange rate is only about 1% ("Bioseparation Engineering", third edition, written by Sun Yan); while the pressure of high-pressure homogenization method needs to reach 1800-2000bar, the equipment requirements higher

In addition, intracellular proteins are prone to degradation during the homogenization process; the release of intracellular impurities such as foreign proteins, nucleic acids, and lipids also poses challenges to the separation and purification of target proteins

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