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Cloning,expression of the nattokinase gene and the activity assay
Author(s): TAN Huan-bo1, 2, ZHANG Guo-jun1, 2, XU Ming-kai1, ZHANG Hui-wen1
Pages: 195-
198
Year: 2012
Issue:
18
Journal: Science and Technology of Food Industry
Keyword: 纳豆激酶; 克隆表达; 活性分析; 冷冻干燥;
Abstract: 应用PCR的方法从分泌纳豆激酶的枯草杆菌基因组DNA中扩增得到全长为1056bp的前导肽+成熟肽纳豆激酶原基因(pro-NK),并构建了纳豆激酶的重组表达载体pET-28a-pro-NK;转化E.coliBL21(DE3)后,在IPTG的诱导下实现了纳豆激酶的高效表达,经SDS-PAGE显示在28ku处有一特异带;表达产物经Ni2+亲和层析纯化后,纤维平板法测得的纳豆激酶的比活力为34245.1IU/mg;纯蛋白经透析和冷冻干燥处理后,纳豆激酶的比活力为16271.5IU/mg;在此基础上,对冻干保护剂的添加进行优化,最佳保护效果为甘露醇与纳豆激酶质量比为1:2,可比不加保护剂时比活力提高了30.9%。这可为基因工程方法生产纳豆激酶纯品,稳定其活性便于贮运,并将其开发成为临床药物提供技术参考。
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