Journal
ACS SYNTHETIC BIOLOGY
Volume 7, Issue 11, Pages 2656-2664Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.8b00332
Keywords
rare tRNA genes; genome editing; efficient translation; recombinant protein production; E. coli
Categories
Funding
- National Research, Development and Innovation Office [OTKA K116455, OTKA-PD115404]
- Ministry for National Economy of Hungary [GINOP-2.3.2-15-2016-00001]
- Hungarian Academy of Sciences [bo_329_15]
- Lendulet Program Grant [LP2017-7/2017]
- Drosophila Genomics Resource Center (NIH) [2P40OD010949]
Ask authors/readers for more resources
Escherichia coli is a well-established and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation. Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In the BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a rRNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in the exponential growth phase. Results from expression assays of a panel of recombinant proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available