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过程工程学报 ›› 2021, Vol. 21 ›› Issue (1): 92-99.DOI: 10.12034/j.issn.1009-606X.220073

• 生化工程 • 上一篇    下一篇

一步离子交换层析从Cohn组分V上清液中分离人血清白蛋白

向 杰1, 张松平2, 张贵锋2, 罗 坚2*, 余 蓉1*   

  1. 1. 四川大学华西药学院生物技术药物学系,靶向药物与释药系统教育部重点实验室,四川省植物来源工程实验室和四川省小分子药物精准化工程技术研究中心,四川 成都 610041 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
  • 收稿日期:2020-03-05 修回日期:2020-04-02 出版日期:2021-01-22 发布日期:2021-01-21
  • 通讯作者: 余蓉
  • 基金资助:
    国家自然科学基金资助项目;国家重点研发计划资助项目

Direct separation of human serum albumin from Cohn fraction V supernatant by one-step ion exchange chromatography

Jie XIANG1, Songping ZHANG2, Guifeng ZHANG2, Jian LUO2*, Rong YU1*   

  1. 1. Department of Biopharmaceutics, West China School of Pharmacy, Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy Sichuan University, Chengdu, Sichuan 610041, China 2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
  • Received:2020-03-05 Revised:2020-04-02 Online:2021-01-22 Published:2021-01-21
  • Contact: YU Rong

摘要: 传统的血浆低温乙醇沉淀工艺中Cohn组分V上清液由于其乙醇浓度高(体积浓度40%),进一步回收残余蛋白困难而被作为废弃组分。本研究探索了采用一步层析从Cohn组分V上清中回收人血清白蛋白的方法。首先以牛血清白蛋白(BSA)为模型蛋白,比较了三种不同类型介质在不同乙醇水溶液中的吸附容量。疏水介质在乙醇水溶液中对BSA吸附能力很弱,增加乙醇浓度导致吸附量趋近于零;阳离子交换介质在低乙醇浓度下有较高的吸附量,但随乙醇体积浓度增高下降较大。相比之下阴离子交换介质展示了最好的吸附性能,在乙醇体积浓度40%的乙醇水溶液中吸附容量仍达34.66 mg/mL。进一步实验表明,在乙醇存在下BSA在阴离子交换介质上的吸附可用Langmuir等温吸附方程描述。将阴离子交换介质DEAE Sepharose Fast Flow装填层析柱,用于从Cohn组分V上清液中分离人血清白蛋白,将40%乙醇的Cohn组分V上清液直接进料阴离子交换层析柱,采用两步洗脱策略,第一步改变pH值由7.0到4.5以洗脱目标产物人血清白蛋白,第二步将氯化钠浓度从0增加到1 mol/L以洗脱紧密结合的部分酸性糖蛋白。SDS-PAGE电泳检测人血清白蛋白纯度为96.35%,得率为43 mg/L Cohn组分V上清液。圆二色谱和荧光光谱检测表明经过离子交换回收的白蛋白二级结构和三级结构与对照的医疗制品无明显差异。活性分析表明回收的白蛋白与配体华法令钠的结合常数与对照的医疗制品的结合常数相似。本研究表明,离子交换层析可用于从含乙醇的溶液中分离蛋白质,一步离子交换层析就可从血浆工业废弃液中回收高纯度的人血清白蛋白。

关键词: 人血清白蛋白, 组分V上清, 离子交换层析, 低温乙醇沉淀, 蛋白结构

Abstract: Fraction V supernatant is an effluent of Cohn fractionation in plasma protein industry. Due to its high ethanol concentration, further recovery of the residual protein has been regarded as non-economical. In this work, a recovery of human serum albumin (HSA) from fraction V supernatant by ion exchange chromatography was reported, which had not been reported in the literature to our knowledge. Firstly, bovine serum albumin (BSA) was used as model protein to compare the adsorption capacity of three different types of chromatographic media in different ethanol-aqueous solutions. The adsorption capacity of the hydrophobic medium to BSA in ethanol-aqueous solution was very weak, and the increase of ethanol concentration led to the adsorption capacity approaching to 0. The cation exchange medium had a high adsorption capacity at low ethanol concentration, but decreased quickly with the increase of the ethanol concentration. In contrast, the anion exchange medium showed the best adsorption performance, and the adsorption capacity in 40% ethanol-aqueous solution still reached 34.66 mg/mL. Further experiments showed that the adsorption of BSA on the anion exchange medium in the presence of ethanol could be described by Langmuir isothermal adsorption equation. The anion exchange medium DEAE Sepharose Fast Flow was packed into a chromatographic column. Real purification of Cohn fraction V supernatant was performed. The Cohn fraction V supernatant, containing about 40% ethanol, was directly loaded to the anion exchange column. A two-step elution strategy was used. The first elution was pH change from 7.0 to 4.5 to obtain the target product human serum albumin, and the second elution was to increase the concentration of sodium chloride from 0 to 1 mol/L to elute glycoproteins. The purity of HSA was 96.35% by electrophoresis, and the activity of binding to the ligand warfarin was comparable to that of commercial HSA product by Cohn fractionation. The total recovery was 43 mg/L Cohn fraction V supernatant.

Key words: human serum albumin, fraction V supernatant, ion exchange chromatography, low-temperature ethanol precipitation, protein structure