从哺乳动物细胞含血清培养基中一步纯化重组高斯荧光素酶。
Single-step purification of recombinant Gaussia luciferase from serum-containing culture medium of mammalian cells.
作者信息
Inouye Satoshi
机构信息
Yokohama Research Center, JNC Co., 5-1 Okawa, Kanazawa-ku, Yokohama, 236-8605, Japan.
出版信息
Protein Expr Purif. 2018 Jan;141:32-38. doi: 10.1016/j.pep.2017.09.001. Epub 2017 Sep 6.
A dihydrofolate reductase-deficient Chinese hamster ovary (CHO-K1/dhfr) cell line stably expressing Gaussia luciferase with a histidine-tag sequence at the carboxyl terminus (GLase-His) was established. Recombinant GLase-His was purified from serum-containing culture medium by single-step Ni-chelate column chromatography in the presence of 2 M NaCl and 0.01% Tween 20. The protein yield of GLase-His with over 95% purity was 0.5 mg from 0.9 L of the cultured medium. The enzymatic properties of purified GLase-His were characterized. Interestingly, non-ionic detergent Tween 20 stabilized and stimulated GLase-His activity and its luminescence activity was stimulated 2-fold by the synergistic effect of 0.01% Tween 20 and 150 mM NaCl.
建立了一种二氢叶酸还原酶缺陷的中国仓鼠卵巢细胞系（CHO-K1/dhfr），该细胞系稳定表达在羧基末端带有组氨酸标签序列的高斯荧光素酶（GLase-His）。重组GLase-His通过在2 M NaCl和0.01%吐温20存在下的单步镍螯合柱色谱法从含血清培养基中纯化得到。从0.9 L培养基中获得的纯度超过95%的GLase-His蛋白产量为0.5 mg。对纯化后的GLase-His的酶学性质进行了表征。有趣地是,非离子去污剂吐温20可稳定并刺激GLase-His的活性，其发光活性在0.01%吐温20和150 mM NaCl的协同作用下提高了2倍。
Zotero 插件