Preliminary study on preparation of E.coli cell-free system for protein expression |
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Authors: | Haiqin Chen Zhinan Xu Cheng Wang Yuhua Liao and Peilin Cen |
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Affiliation: | (1) Bioengineering Research Institute, Zhejiang University, Hangzhou, 310027, China;(2) State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China;(3) Zhejiang Yangshengtang Natural Medicines Institute, Hangzhou, 310007, China |
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Abstract: | In the new era of “Omics”, the traditional techniques of protein expression in vivo can not come up with the exponential increase
of genetic information. The cellfree protein synthesis system provides a new strategy of protein expression with advantages
of rapid, convenient and high-throughput expression. The preparation of cell extracts, the optimization of substrate concentrations
and the energy regeneration system are the key factors for the successful construction of cell-free protein expression system.
In this work, the cell extract was prepared from RNase I- defective strain E. coli A19. The cell growth phase, the pressure for cell disruption and the storage condition of cell extracts were optimized. Meanwhile,
the optimal substrate concentrations and the energy regeneration system were selected. Under the optimized conditions, the
green fluorescent protein (GFP) reporter gene was expressed in the E. coli cell-free system with high expression level (Ca. 154 μg/mL) which was 29 times higher than the expression level before optimization.
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Translated from Journal of Zhejiang University (Engineering Science), 2007, 41(5): 843–847 译自: 浙江大学学报(工学版)] |
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Keywords: | protein biosynthesis cell-free protein expression system green fluorescent protein (GFP) |
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