The refolding, purification, and activity analysis of a rice Bowman-Birk inhibitor expressed in Escherichia coli.
The refolding, purification, and activity analysis of a rice Bowman-Birk inhibitor expressed in Escherichia coli.
复制标题
在大肠杆菌中表达的水稻 Bowman-Birk 抑制剂的重折叠、纯化和活性分析。
DOI：
--
复制
发表时间：
1999
影响因子：
1.6
通讯作者：
Zhang
中科院分区：
文献类型：
--
作者：
Na Li;Li;Yueyi Liu;Qin Li;H. Gu;Zhang;Zhang
Bowman-Birk 家族的推定水稻胰蛋白酶/糜蛋白酶抑制剂 RBBI-8（约 20 kDa）在大肠杆菌中表达为带有 N 末端 (His)6 纯化标签的融合蛋白。表达的重组蛋白 rRBBI-8 不溶，并以包涵体形式积累。在还原环境下将不溶性蛋白质溶解在8M尿素中，然后在优化的氧化还原条件下重新折叠成其活性构象。用于优化产量和效率的策略包括选择氧化还原系统、通过逐步添加变性蛋白质来增加重折叠过程中的蛋白质浓度、利用添加剂防止聚集以及选择缓冲液交换条件。然后使用镍螯合物亲和柱纯化复性蛋白质。 rRBBI-8对胰蛋白酶有很强的抑制活性，对胰凝乳蛋白酶有轻微的抑制作用。本研究针对这种原核系统表达的富含半胱氨酸的重组蛋白建立了重折叠和纯化系统。
A putative rice trypsin/chymotrypsin inhibitor of the Bowman-Birk family, RBBI-8 of about 20 kDa, was expressed in Escherichia coli as a fusion protein bearing an N-terminal (His)6 purification tag. The expressed recombinant protein, rRBBI-8, is insoluble and accumulates as inclusion bodies. The insoluble protein was solubilized in 8 M urea under reducing environment and then refolded into its active conformation under optimized redox conditions. Strategies used to optimize yield and efficiency include selecting the redox system, increasing protein concentration during refolding by adding the denatured protein in a stepwise way, utilizing additives to prevent aggregation, and selecting buffer-exchanging conditions. A Ni-chelate affinity column was then employed to purify the renatured protein. rRBBI-8 shows strong inhibitory activity against trypsin and it can slightly inhibit chymotrypsin. In this study, a refolding and purification system was set up for this cysteine-rich recombinant protein expressed in a prokaryotic system.