一种新型尿酸氧化酶四聚体提取纯化方法的建立

Establishment of a new method for urate oxidase tetramer extraction and purification

目的建立一种简便、快速的尿酸氧化酶四聚体提取纯化方法,以适应大规模生产需求。方法首先通过基因工程的方法获得可以稳定发酵生产尿酸氧化酶的大肠埃希菌菌株,再通过一步萃取法从破碎菌体沉淀中获得尿酸氧化酶蛋白混合物。通过硫酸铵沉淀、离子交换和分子筛层析,最终获得纯化的活性尿酸氧化酶四聚体。结果通过优化发酵及提取条件,每升发酵液可得到尿酸氧化酶四聚体蛋白约400 mg,其纯度>95%,比活>10 U/mg。结论建立了一种新的尿酸氧化酶四聚体高效提取纯化方法。

Objective To establish a simple and fast method for extraction and purification of urate oxidase tetramer, to meet large scale production requirement. Methods Genetic engineering was used to obtain an E. coli strain producing urate oxidase effectively. The mixture of urate oxidase was obtained by one-step extraction from precipitant of bacteria lysis. The purified tetramer of urate oxidase was extracted by ammonium sulfate precipitation, ion exchange chromatography and molecular sieve chromatography. Results Approximately 400 mg tetramer protein was obtained from each liter of fermentation with purity >95% and specific activity >10 U/mg. Conclusion An efficient method for extraction and purification of urate oxidase tetramer is established.