Principle
In all organisms, most amino acids are encoded by more than one codon: 61 codons are available for 20 amino acids. But each organism is characterized by a specific “codon bias” (see table below), i.e. it preferentially uses some codons over others. In practice, when a heterologous gene is expressed in E. coli, this gene might exhibit some codons that are common in the original host but rarely used in E. coli. Whereas, the presence of only a small number of rare codons might not severely depress target protein synthesis, the presence of clusters of and/or numerous rare codons generates a demand for one or more rare tRNAs. In turn, the rarity of some tRNAs leads to very low expression of the target protein due to premature translation termination, translation frameshifting, amino acid misincorporation, growth inhibition and plasmid instability. Six rare codons can cause problems in
E. coli B
(e.g.; BL21(DE3) or SE1): AGG and AGA (both encoding arginine using the argU tRNA), AUA (isoleucine, ileX tRNA), CUA (leucine, leuW), GGA (glycine, glyT), and CCC (proline, proL). An analysis of your gene-of-interest can be performed using Staby™ Soft.
In the Staby™Codon T7 kit, we solve the problem by the use of the pSCodon1.2 expression plasmid encoding the tRNA genes of the six rare codons. Hence, this plasmid contains both the T7 promoter for a strong expression and supplies the rare tRNAs (fig.9). Moreover, this plasmid encodes the antidote protein. Consequently, transformed in bacteria containing the natural poison gene ccdB (such as the CYS21 and SE1 bacteria included in the kit), the plasmid is stabilized. If some bacteria lose the vector, they will not obtain a selective (growth speed) advantage but will die (see the StabyExpress™ T7 kit principle). In practice, this additional stabilization technology solves the problem of plasmid instability and insures that during bacterial growth, 100 % of the bacteria will carry the vector. Thus, the production of the protein of interest is higher and purer (lower amount of non-target proteins).
Results
To evaluate the efficiency of the Staby™ Codon T7 kit, a DNA fragment encoding a human protein containing 24 rare codons (10 prolines, 7 arginines, 3 isoleucines, 3 leucines and 1 glycine) was cloned into pStaby1 and pSCodon1. These plasmids were transformed in SE1. As illustrated in figure below the protein is not or very poorly produced using pStaby1 but clearly visible using pSCodon1. The protein was purified and its integrity was confirmed by Mass Spectrometry.
Legal notices
The Staby™Codon kit is covered by worldwide patents. The kit is sold under a license from the
Université Libre de Bruxelles (Belgium). The kit is sold for research purpose only. A license
from Delphi Genetics SA is required for any commercial use (Please, contact Delphi Genetics SA
at
delphigenetics@delphigenetics.com).