Acta Chim. Sinica ›› 2016, Vol. 74 ›› Issue (3): 265-270.DOI: 10.6023/A15120761 Previous Articles     Next Articles



王建山, 夏红军, 万广平, 刘家玮, 金丽花, 白泉   

  1. 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
  • 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   

  1. 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).

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