摘要
目的纯化赤子爱胜蚓核酸酶样蛋白,并鉴定其活性。方法将制备的赤子爱胜蚓核酸酶样蛋白粗提液透析后,经Bio-CAD CM柱层析系统非连续梯度洗脱,收集洗脱峰,对有活性的梯度洗脱峰样品进行单向聚丙烯酰胺凝胶电泳(1D-PAGE)及切胶纯化,将切胶回收纯化的蛋白样品进行双向聚丙烯酰胺凝胶电泳(2D-PAGE),回收蛋白,进行活性和浓度检测。结果透析后的蛋白粗提液经Bio-CAD CM柱层析分离,得到3个不同洗脱梯度的可降解DNA的活性峰,3个梯度蛋白的PAGE带型基本一致,经PAGE分离获得3个核酸酶样蛋白纯品EWD1、EWD2和EWD3,得率和纯化倍数分别为:27.9%,11.6;16.7%,11.2;16.7%,18.6。结论成功纯化了赤子爱胜蚓核酸酶样蛋白,并保持了其良好的生物活性。该纯化方法简单、快速、经济、实用,为该蛋白结构及功能的进一步研究奠定了基础。
Objective T o purify nuclease-like proteins from earthworm Eisenia foetida and determine their activities. Methods Crude extracts of nuclease-like proteins of E.foetida were dialyzed,purified by Bio-CAD CM column chromatography and eluted by non-continuous elution.The samples from active elution peaks were identified by 1DPAEG and purified by gel cutting,then further identified by 2D-PAGE and determined for activity and concentration. Results Three peaks with DNA degradation activity were observed on Bio-CAD CM column chromatographic profile of crude protein extracts after dialysis,of which the PAGE patterns were basically in agreement.Four main protein bands were obtained after separation by PAGE and further purified by imidazole-zinc chloride reverse staining and gel cutting, and determined for activity and concentration.Three pure nuclease-like proteins EWD 1,EWD 2 and EWD 3,were obtained,of which the protein recovery rates were 27.9%,16.7% and 16.7%,while the purification folds were 11.6, 11.2 and 18.6,respectively.Conclusion Nuclease-like proteins of earthworm E.foetida were successfully purified and showed high biological activity.The purification method was simple,quick,economical and practical,which laid a foundation of further study on structures and functions of the proteins.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第5期705-709,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(30472251)
山西省自然科学基金青年科技研究基金(2010021035-2)
山西省归国留学人员基金(2010-52)
关键词
赤子爱胜蚓
核酸酶样蛋白
纯化
Eisenia foetida
Nuclease-like protein
Purification
