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Overexpression, overproduction, purification, and characterization of rhGH in Escherichia coli
Biotechnology and Applied Biochemistry ( IF 3.2 ) Pub Date : 2020-02-24 , DOI: 10.1002/bab.1902 Reyhane Keshavarz 1 , Valiollah Babaeipour 1 , Mehdi Mohammadpour-Aghdam 1 , Ali Asghar Deldar 1
Biotechnology and Applied Biochemistry ( IF 3.2 ) Pub Date : 2020-02-24 , DOI: 10.1002/bab.1902 Reyhane Keshavarz 1 , Valiollah Babaeipour 1 , Mehdi Mohammadpour-Aghdam 1 , Ali Asghar Deldar 1
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Overexpression of insoluble human growth hormone (hGH) in cytoplasm was achieved by E. coli Rosetta‐gami B(DE3) [pET21a (+)‐hGH]). For overexpression of hGH, effects of eight factors including temperature, type and concentration of carbon source, IPTG and MgSO4, buffering capacity, induction time, yeast extract/peptone ratio on rhGH production were studied by Plackett–Burman screening. Maximum production of rhGH was 0.681 g/L, and results of statistical analysis showed that induction temperature and glucose have the greatest effect and the presence of MgSO4 increases rhGH expression and reduces biomass concentration. So, the effect of ethanol and MgSO4 concentrations on the rhGH production was examined according to the central composite experimental design. The ANOVA of the results showed rhGH production increases to 1.128 g/L in 4 g/L MgSO4 and 1% ethanol. Then, the impact of glucose concentration and induction time on the rhGH production was evaluated in two levels in the fermenter by Taguchi statistical method. Under optimum conditions, OD600nm 4 and 10 g/L glucose crude rhGH concentration 4.17 g/L was obtained, which is one of the highest value ever reported. Finally, rhGH was purified using the biophysical and biochemical techniques comprising circular dichroism, fluorescent spectroscopy, and dynamic light scattering, and it was confirmed that the produced protein is comparable to the commercial standard sample.
中文翻译：
大肠杆菌中rhGH的过表达，过量生产，纯化和表征
大肠杆菌Rosetta-gami B（DE3） [pET21a（+）-hGH]实现了细胞质中不溶性人类生长激素（hGH）的过表达。对于hGH的过表达，通过Plackett–Burman筛选研究了温度，碳源类型和浓度，IPTG和MgSO 4，缓冲能力，诱导时间，酵母提取物/蛋白pe比这8个因素对rhGH产生的影响。rhGH的最大产量为0.681 g / L，统计分析结果表明，诱导温度和葡萄糖的影响最大，而MgSO 4的存在会增加rhGH的表达并降低生物量的浓度。所以，乙醇和MgSO 4的作用根据中央复合实验设计检查了rhGH产生的浓度。结果的方差分析显示，在4 g / L MgSO 4和1％乙醇中，rhGH产量增加至1.128 g / L。然后，通过田口统计学方法在发酵罐中以两个水平评估葡萄糖浓度和诱导时间对rhGH产生的影响。在最佳条件下，获得OD 600nm 4和10 g / L葡萄糖粗rhGH浓度4.17 g / L，这是有史以来的最高值之一。最后，使用包括圆二色性，荧光光谱和动态光散射在内的生物物理和生化技术纯化rhGH，并证实所产生的蛋白质与商业标准样品相当。
更新日期：2020-02-24
中文翻译：
大肠杆菌中rhGH的过表达，过量生产，纯化和表征
大肠杆菌Rosetta-gami B（DE3） [pET21a（+）-hGH]实现了细胞质中不溶性人类生长激素（hGH）的过表达。对于hGH的过表达，通过Plackett–Burman筛选研究了温度，碳源类型和浓度，IPTG和MgSO 4，缓冲能力，诱导时间，酵母提取物/蛋白pe比这8个因素对rhGH产生的影响。rhGH的最大产量为0.681 g / L，统计分析结果表明，诱导温度和葡萄糖的影响最大，而MgSO 4的存在会增加rhGH的表达并降低生物量的浓度。所以，乙醇和MgSO 4的作用根据中央复合实验设计检查了rhGH产生的浓度。结果的方差分析显示，在4 g / L MgSO 4和1％乙醇中，rhGH产量增加至1.128 g / L。然后，通过田口统计学方法在发酵罐中以两个水平评估葡萄糖浓度和诱导时间对rhGH产生的影响。在最佳条件下，获得OD 600nm 4和10 g / L葡萄糖粗rhGH浓度4.17 g / L，这是有史以来的最高值之一。最后，使用包括圆二色性，荧光光谱和动态光散射在内的生物物理和生化技术纯化rhGH，并证实所产生的蛋白质与商业标准样品相当。
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