乳腺生物反应器表达的重组人乳清白蛋白的高效分离纯化

中国生物工程杂志

2011, Vol. 31

Issue (8): 66-72

技术与方法

乳腺生物反应器表达的重组人乳清白蛋白的高效分离纯化

李由^1,2, 张焱², 苏海佳¹, 罗坚², 白倩¹, 马光辉², 苏志国²

1. 北京化工大学生命科学与技术学院北京 100029;
2. 中国科学院过程工程研究所生化工程国家重点实验室北京 100190

Purification of Recombinant Human Lactoferrin Expressed in A Cattle Mammary Bioreactor

LI You^1,2, ZHANG Yan², SU Hai-jia¹, LUO Jian², BAI Qian¹, MA Guang-hui², SU Zhi-guo²

1. College of Life Science and Technology,Beijing University of Chemical Technology,Beijing 100029,China;
2. National Key Laboratory of Biochemistry Engineering,Chinese Academy of Sciences,Beijing 100190,China

全文: PDF(920 KB) HTML

摘要：

利用乳腺生物反应器高效地表达重组人乳清白蛋白,但是目标产物分离纯化的难度较大。通过分子模拟计算比较待分离原料中主要蛋白组分的物化性质,包括表面电势和表面疏水性,在此基础上设计了高分辨率、快速的分离纯化工艺。通过硫酸铵沉淀的正交试验条件优化,有效地去除了干扰层析精制过程的IgG杂质,提高了后续疏水层析的稳定性,从而成功地分离开目标蛋白及与其同源的牛乳清白蛋白杂质,得到纯度>95%的rHLA纯品,工艺回收率48.6%。乳糖合成活性和圆二色谱检测结果表明,纯化rHLA具有调节β-1,4-半乳糖苷转移酶活性和天然的空间结构。

关键词： 乳腺生物反应器; 乳清白蛋白; 疏水层析; 同源蛋白质

Abstract:

Recombinant human α-lactalbumin(rHLA)could be expressed in mammary gland bioreactor effectively. However,it is very difficult to purify the target protein. Primary proteins’ properties,such as surface hydrophobicity and electric potential,were compared through molecular simulation and calculation,based on which a fast purification process with high resolution was designed. Immunoglobulin G,which would disturb chromatographic purification process,was firstly removed using ammonium sulfate precipitation with orthogonal optimization. Then the subsequent hydrophobic interaction chromatography(HIC)became more stable and rHLA with 95% purity was obtained through separating successfully from its homologous courterpart,i.e.,bovine α-lactalbumin in HIC. The overall recovery of rHLA was up to 48.6%. Activity detection and circular dichroism spectroscopy(CD)confirmed the regulatory activity for β-1,4-galactosyltransferase and native structure of purified rHLA.

Key words: Mammary gland bioreactor α-lactalbumin Hydrophobic interaction chromatography Homologous proteins

收稿日期: 2011-04-28 出版日期: 2011-08-25

ZTFLH:

Q819

基金资助:

国家"863"计划(2007AA100506,2010AA100501)、国家自然科学基金(20706052)资助项目

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引用本文:

李由, 张焱, 苏海佳, 罗坚, 白倩, 马光辉, 苏志国. 乳腺生物反应器表达的重组人乳清白蛋白的高效分离纯化[J]. 中国生物工程杂志, 2011, 31(8): 66-72.

LI You, ZHANG Yan, SU Hai-jia, LUO Jian, BAI Qian, MA Guang-hui, SU Zhi-guo. Purification of Recombinant Human Lactoferrin Expressed in A Cattle Mammary Bioreactor. China Biotechnology, 2011, 31(8): 66-72.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I8/66

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