Preparation of gene recombinant MAGE1/HSP70/MAGE3 by high cell density culture of E.coli. with DO fed-back control of nutrient feeding

HU Pei-zhen; WEN Jiang-tian; LU Fan; WANG Xiao-gong; LI Xia; SI Shao-yan; GE Wei; HUANG Yang; ZHANG Xiu-min; SUI Yan-fang

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Preparation of gene recombinant MAGE1/HSP70/MAGE3 by high cell density culture of E.coli. with DO fed-back control of nutrient feeding

更新时间：2025-01-15

- Preparation of gene recombinant MAGE1/HSP70/MAGE3 by high cell density culture of E.coli. with DO fed-back control of nutrient feeding
- Journal of Army Medical University Vol. 29, Issue 16, Pages: 1576-1579(2007)
- 作者机构：
  
  第四军医大学:西京医院病理科，西京医院超声诊断科，基础部生物化学与分子生物学教研室
- 作者简介：
- 基金信息：
- DOI：
  CLC： Q784;Q93-335;Q939.121
- Published：2007，
  
  Published Online：30 August 2007，
- 稿件说明：
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HU PEI-ZHEN, WEN JIANG-TIAN, LU FAN, et al. Preparation of gene recombinant MAGE1/HSP70/MAGE3 by high cell density culture of E.coli. with DO fed-back control of nutrient feeding. [J]. Journal of army medical university, 2007, 29(16): 1576-1579.
DOI：

HU PEI-ZHEN, WEN JIANG-TIAN, LU FAN, et al. Preparation of gene recombinant MAGE1/HSP70/MAGE3 by high cell density culture of E.coli. with DO fed-back control of nutrient feeding. [J]. Journal of army medical university, 2007, 29(16): 1576-1579. DOI：

摘要

目的建立人黑色素瘤抗原MAGE1/HSP70/MAGE3融合蛋白基因重组工程菌E.coli. BL21/pET-MHM的高密度发酵工艺。方法以摇瓶发酵结果为基础，扩大至NBS Bioflo Ⅳ 20 L发酵罐发酵，利用溶氧反馈-补料分批培养技术，对影响工程菌生长及目的蛋白表达的因素如发酵培养基、活化时间、诱导浓度及时间、pH值及分批补加营养物质等进行优化。结果保持培养过程中40%的溶解氧，采用半合成培养基、活化至对数生长期时0.2 mmol/L IPTG诱导5 h以及以甘油为碳源连续流加补料的条件发酵，连续3批重复发酵，最终菌密度D(600)均达45~50时，菌体量可提高至70 g/L以上，目的蛋白的表达量占菌体蛋白总量的38%以上。结论确定了周期短、产率高且稳定可靠的发酵工艺。

Abstract

Objective To investigate the optimal high cell density fermentation conditions of recombinant E.coli. BL21/pET-MHM to produce recombinant MAGE1/HSP70/MAGE3 fusion protein． Methods Based on the experimental results obtained in shake flask cultivation

the cells were transferred to NBS Bioflo 20 L DO feed-back fed-batch culture system. The effects of the composition of the fermentation medium

activation time

induction time

the range of pH and fed-batch carbon sources on the expression level of MAGE1/HSP70/MAGE3 and cell output were analyzed． Results By keeping dissolved oxygen at 40%

culturing in semi-defined medium and inducing for 5 h in the presence of 0.2 mmol/L IPTG at 37 ℃ with glycerol as the carbon sources during fed-batch cultivation

we performed a three-time repeated fermentation. The final cell density was D(600) 45 to 50 and more than 70 g of wet bacteria per liter. The expression level of recombinant protein MAGE1/HSP70/MAGE3 was over 38% of the total protein in E.coli. Conclusion A Short cycle，high expression and stabilized fermentation process of recombinant MAGE1/HSP70/MAGE3 has been established

which founds the basis for further purification and large-scale production of MAGE1/HSP70/MAGE3.

关键词

黑色素瘤抗原重组大肠杆菌发酵

Keywords

melanoma antigen-associated generecombinationEscherichia colifermentation

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