人组织蛋白酶K在毕赤酵母中的表达、纯化及活性测定

Purification and Activity Determination of Human Cathepsin K Expressed in Pichia pastoris

在动物和人的骨代谢疾病中,组织蛋白酶K是木瓜蛋白酶家族中的一种半胱氨酸蛋白酶,是负责骨吸收过程中溶骨的主要关键酶,是新药物的分子作用靶点。通过毕赤酵母表达系统表达出重组组织蛋白酶K,进一步用离子交换和分子筛系统进行纯化得到高纯度有活性的重组组织蛋白酶K。运用FluStar荧光酶标仪测定390 nm(激发光)和460 nm(发射光)波长下荧光强度的变化并计算出组织蛋白酶K的酶活力为119.6 U,为进一步筛选骨质疏松等骨代谢疾病的新药提供参考。

In the animal and human bone metabolic diseases,cathepsin K is a cysteine protease of the papain family,which is responsible for the resorption of the bone matrix and regarded as a key protease in bone resorption,and become the new drug molecule target.Active recombinant Cathepsin K was expressed in Pichia pastoris,and further purified by Ion Exchange chromatography and Size-Exclusion chromatography.Besides,the its changes of fluorescence intensity were measured at 390 nm excitation and 460 nm emission wavelengths by FluStar fluorescence microplate reader to calculate the enzyme activity of cathepsin K.The result showed that the enzyme activity of cathepsin K was 119.6 U.The research could provide a reference for the further screening of new drugs for osteoporosis and bone metabolic disease.