在线单柱二维液相色谱法对蛋清中三种活性蛋白的快速分离纯化

doi:10.6023/A15120761

化学学报 ›› 2016, Vol. 74 ›› Issue (3): 265-270.DOI: 10.6023/A15120761 上一篇下一篇

研究论文

在线单柱二维液相色谱法对蛋清中三种活性蛋白的快速分离纯化

王建山, 夏红军, 万广平, 刘家玮, 金丽花, 白泉

西北大学合成与天然功能分子化学教育部重点实验室西北大学现代分离科学研究所现代分离科学陕西省重点实验室西安 710069

投稿日期:2015-12-04 发布日期:2016-01-29
通讯作者: 白泉 E-mail:baiquan@nwu.edu.cn
基金资助:
项目受国家自然科学基金(No. 21545007)、陕西省科技统筹重点实验室项目(No. 2013SZS18-K01)和陕西省重点实验室科研项目(Nos. 14JS098, 15JS115)资助.

On-line Fast Purification of Three Kinds of Active Proteins from Egg White with Two-dimensional Liquid Chromatography Using a Single Column

Wang Jianshan, Xia Hongjun, Wan Guangping, Liu Jiawei, Jin Lihua, Bai Quan

Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education of China, Institute of Modern Separation Science, Northwest University, Key Laboratory of Modern Separation Science in Shaanxi Province, Xi'an 710069

Received:2015-12-04 Published:2016-01-29
Supported by:
Project supported by the National Natural Science Foundation of China (No. 21545007), Shaanxi Provincial Science and Technology Co-ordinating innovation projects (No. 2013SZS18-K01) and Shaanxi Province Key Laboratory (Nos. 14JS098, 15JS115).

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摘要/Abstract

基于一种新型的强阳离子交换/疏水色谱(SCX/HIC)双功能色谱柱构建了在线单柱二维液相色谱(2DLC-1C), 分离系统实现了自动控制, 可在线进行样品收集、二维进样和二维色谱分离. 采用SCX-HIC 2DLC-1C, 在120 min内可将8种标准蛋白完全分离, 并成功应用于鸡蛋清中三种活性蛋白质的分离纯化. 结果表明利用在线2DLC-1C技术可在70 min内完成对鸡蛋清中溶菌酶、卵清蛋白和卵转铁蛋白三种活性蛋白的快速分离纯化, 其纯度分别为95%、93%和97%. 该方法减少了样品的处理步骤, 对样品的污染小, 纯化速度快, 操作简单, 易于实现自动化和放大, 对活性蛋白的快速分离制备和工业化生产具有广阔的应用前景.

关键词: 在线单柱二维液相色谱, 蛋白质, 分离纯化, 蛋清

With the development of biotechnology and life science, both recombinant protein drug productions and proteomics research depend largely on fast and efficient protein separation technology. Analysis of complex samples has put forward higher and higher requirements to separation science. Therefore, developing new separation material, separation mode and more sensitive detection method should be one of the effective ways to solve these problems. Based on a novel strong cation exchange/hydrophobic interaction chromatography (SCX/HIC) dual-function column, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed and can be employed to separate proteins with SCX and HIC modes, respectively, a new on-line two-dimensional liquid chromatography technology with a single column (2DLC-1C) was developed. An equipment with automatic control system called as “ChromatoExpert” for processing fast protein separation online 2DLC-1C was constructed, which has multiple functions, such as on-line fraction collection from the first separation mode and quantitatively sample re-injection into the second separation mode, on-line buffer exchange, etc. Eight kinds of protein standards were separated using SCX/HIC 2DLC-1C. The unretained and uncompletely separated proteins in SCX mode were collected online and could be separated completely with each other by online re-injection and second dimensional separation of HIC mode. As a result, these eight kinds of proteins can be separated within 2 h. It also was applied to purify three kinds of active intact proteins from egg white successfully. The results indicated that lysozyme, ovotransferrin and ovalbumin from egg white could be purified just within 70 min by online 2DLC-1C. The purity of these proteins obtained was 95%, 93% and 97%, respectively. The new online 2DLC-1C technology can simplify the purification process and lower the separation cost. It is easy to realize automation and scale-up to preparative scale. Therefore, this new technology has a broad application prospect in the fast separation and preparation of active protein.

Key words: online 2DLC-1C, protein, purification, egg white

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王建山, 夏红军, 万广平, 刘家玮, 金丽花, 白泉. 在线单柱二维液相色谱法对蛋清中三种活性蛋白的快速分离纯化[J]. 化学学报, 2016, 74(3): 265-270.

Wang Jianshan, Xia Hongjun, Wan Guangping, Liu Jiawei, Jin Lihua, Bai Quan. On-line Fast Purification of Three Kinds of Active Proteins from Egg White with Two-dimensional Liquid Chromatography Using a Single Column[J]. Acta Chim. Sinica, 2016, 74(3): 265-270.

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在线单柱二维液相色谱法对蛋清中三种活性蛋白的快速分离纯化

On-line Fast Purification of Three Kinds of Active Proteins from Egg White with Two-dimensional Liquid Chromatography Using a Single Column

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