Exploring Vaccinia Virus as a Tool for Large‐Scale Recombinant Protein Expression
Exploring Vaccinia Virus as a Tool for Large‐Scale Recombinant Protein Expression
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DOI：
10.1021/bp0255762
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发表时间：
2003
影响因子：
2.9
通讯作者：
N. Bleckwenn;W. Bentley;J. Shiloach
中科院分区：
文献类型：
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作者：
N. Bleckwenn;W. Bentley;J. Shiloach
使用VOTE表达系统在HeLa细胞中改造重组牛痘病毒，以在T7启动子的控制下表达增强型绿色荧光蛋白（EGFP）。用该病毒感染HeLa细胞并用IPTG诱导，证明了该构建体用于容易地测量蛋白质表达的实用性。该构建体用于评估几个生产参数，特别是感染复数（MOI）、感染期间的体积和感染阶段的血清浓度。在静态培养中，发现感染复数的增加使EGFP的表达增加到MOI为1.0左右的平台。表达也显示在感染阶段随着体积的减小而增加。感染阶段的血清浓度仅在0 - 7.5%范围内具有轻微显著性。发现Cytodex 3微载体具有最佳的HeLa细胞生长特性。这些细胞在微载体转瓶培养中生长和感染，最大表达量为2.2 μg EGFP/（感染时的百万细胞），证明该系统能够成功地在更大规模表达重组蛋白。
A recombinant vaccinia virus was engineered to express enhanced green fluorescent protein (EGFP) under control of the T7 promoter using the VOTE expression system in HeLa cells. Infection of HeLa cells with this virus and induction with IPTG demonstrated the utility of this construct for easily measuring protein expression. This construct was used to evaluate several production parameters, specifically, multiplicity of infection (MOI), volume during infection, and serum concentration during the infection phase. In static culture, increasing multiplicity of infection was found to increase expression of EGFP up to a plateau around MOI of 1.0. Expression was also shown to increase with decreasing volume during the infection phase. Serum concentration during the infection phase was only marginally significant from 0 to 7.5%. Cytodex 3 microcarriers were found to have the best characteristics for HeLa cell growth. These cells were grown and infected in microcarrier spinner flask culture, and the maximum expression was 2.2 μg EGFP/(million cells at the time of infection), demonstrating the ability of this system to successfully express recombinant proteins at larger scale.