生产与科研应用

重组人Ⅲ型胶原蛋白的分离纯化

  • 梁鑫 ,
  • 张仁怀 ,
  • 吕自力 ,
  • 艾华伟 ,
  • 刘冬 ,
  • 梁波 ,
  • 单旭东 ,
  • 陈浩然
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  • 1 成都中医药大学 医学与生命科学学院,附属生殖妇幼医院,四川 成都,610041
    2 四川省人口和计划生育科学研究所,四川 成都,610041
    3 成都远睿生物技术有限公司,四川 成都,610000
    4 河南多美康生物药业有限公司,河南 郑州,450000
博士,副教授(本文通讯作者,E-mail:liangxin@cdutcm.edu.cn)

收稿日期: 2020-03-17

  修回日期: 2020-04-20

  网络出版日期: 2020-09-17

基金资助

四川省科技厅项目(2018YSZH0028);四川省卫生健康委课题(19PJ033);四川省中医药管理局中医药科研专项(2018JC010)

Isolation and purification of recombinant human type Ⅲ collagen

  • LIANG Xin ,
  • ZHANG Renhuai ,
  • LYU Zili ,
  • AI Huawei ,
  • LIU Dong ,
  • LIANG Bo ,
  • SHAN Xudong ,
  • CHEN Haoran
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  • 1 School of medical and life sciences,Reproductive & Women-Children Hospital, Chengdu University of Traditional Chinese Medicine, Chengdu 610041, China 2 Sichuan Family Planning Research Institute, Chengdu 610041, China
    3 Chengdu Farwits Biotechnology Co., Ltd., Chengdu 610000, China
    4 Henan duomeikang Biological Pharmaceutical Co., Ltd., Zhengzhou 450000, China

Received date: 2020-03-17

  Revised date: 2020-04-20

  Online published: 2020-09-17

摘要

利用分泌表达重组人Ⅲ型胶原蛋白的毕赤酵母发酵液,通过层析条件的优化,建立了重组人Ⅲ型胶原蛋白一步层析纯化工艺。发酵液以硫酸铵(30%饱和度)沉淀胶原蛋白,沉淀用超纯水复溶后超滤转换介质至20 mmol/L 磷酸缓冲液(phosphate buffer,PB)(pH 6.0),然后用SP Sepharose HP阳离子交换柱进行纯化,以20 mmol/L PB (pH 6.0)-150 mmol/L NaCl洗涤杂蛋白,以20 mmol/L PB (pH 6.0)-250 mmol/L NaCl洗脱重组人Ⅲ型胶原蛋白。采用该工艺对60 L发酵液进行重组人Ⅲ型胶原蛋白的纯化,产品纯度97.7%,总回收率68.8%,纯化的重组人Ⅲ型胶原蛋白纯度高,热原含量低,可用于医药领域。该研究建立了一步层析法从毕赤酵母发酵液中高效制备重组人Ⅲ型胶原蛋白的纯化工艺,工艺简便、稳定可靠,离子交换层析放大容易,为重组人Ⅲ型胶原蛋白的工业化生产奠定了基础。

本文引用格式

梁鑫 , 张仁怀 , 吕自力 , 艾华伟 , 刘冬 , 梁波 , 单旭东 , 陈浩然 . 重组人Ⅲ型胶原蛋白的分离纯化[J]. 食品与发酵工业, 2020 , 46(16) : 159 -163 . DOI: 10.13995/j.cnki.11-1802/ts.023967

Abstract

The objective of this study was to establish a one-step chromatographic process, by optimizing the chromatographic conditions, for purifying recombinant human type Ⅲ collagen from Pichia pastoris fermentation broth. Collagen was precipitated from fermentation broth using 30% (mass fraction) saturated ammonium sulfate. Then, the medium was ultrafiltered to 20 mmol/L phosphate buffer (PB) (pH 6.0) after reconstitution of the precipitate with ultrapure water. Following this, a SP Sepharose HP cation exchange column was used for purification, and heteroproteins were washed with 20 mmol/L PB (pH 6.0)-150 mmol/L NaCl. Finally, recombinant human type Ⅲ collagen was eluted with 20 mmol/L PB (pH 6.0)-250 mmol/L NaCl. Using this process to purify recombinant human type Ⅲ collagen from 60 L fermentation broth, the harvested product had a purity of 97.7% and total recovery rate of 68.8%. The purified recombinant human type Ⅲ collagen exhibited high purity and low pyrogen content and thus can be used in medicine industry. This study established a one-step chromatography purification process for the efficient preparation of recombinant human type Ⅲ collagen from Pichia pastoris fermentation broth. The process is simple, stable, and reliable. Ion exchange chromatography is easy to scale up, laying a foundation for industrial production of recombinant human type Ⅲ collagen.

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