Stringent regulation and high-level expression of heterologous genes in Escherichia coli using T7 system controllable by the araBAD promoter

Biotechnology Progress
Yun-Peng ChaoWen-Bin Hung

Abstract

The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter. Upon induction with L-arabinose, recombinant cells produced 100-fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol. This protein yield accounts for 30% of total cell protein content. In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20-30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome. In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using a two-stage feeding strategy to compartmentalize cell growth and protein synthesis. In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase. Subsequently, glycerol was fed to the culture broth and L-arabinose was augmented to ind...Continue Reading

Citations

Jun 25, 2003·Protein Expression and Purification·Cheng-Yang HuangYuh-Shyong Yang
May 20, 2011·Journal of Agricultural and Food Chemistry·Zei Wen WangYun-Peng Chao
May 23, 2012·Journal of Agricultural and Food Chemistry·Po Ting ChenJei-Fu Shaw
Jan 5, 2006·Applied and Environmental Microbiology·Jen-You Wang, Yun-Peng Chao
Oct 11, 2012·Bioscience, Biotechnology, and Biochemistry·Ji-Young RyuHor-Gil Hur
Jul 29, 2008·Protein Expression and Purification·Seonghun Kim, Sun Bok Lee
Jan 5, 2010·Biotechnology and Applied Biochemistry·Yanina R SevastsyanovichJeffrey A Cole
May 26, 2005·Protein Expression and Purification·F William Studier
Mar 18, 2021·Advanced Biology·Yang ZhangShuobo Shi

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