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), 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 are 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 tRNA), GGA (glycine, glyT tRNA), and CCC (proline, proL tRNA). An analysis of your gene-of-interest can be performed using Staby(TM)Soft.

In the Staby^TMCodon T7 kit, we solve the problem by the use of the pSCodon1 expression plasmid encoding the tRNA genes of the six rare codons. Hence, this plasmid both contains the T7 promoter for a strong expression and supplies the rare tRNAs. Moreover, this plasmid encodes the ccdA antidote gene. Consequently, transformed in bacteria containing the natural selection 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^TM T7 kit description). 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).

The Staby^TMCodon system is compatible with the use of an auto-inducible medium as Staby^TMSwitch.

Results

To evaluate the efficiency of the StabyCodon^TM 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 here, the protein is not or very poorly produced using pSaby1 (lane 3) but clearly visible using pSCodon1 (lane 4). The protein was purified and its integrity was confirmed by mass spectrometry.

Kit Content

The Staby^TMCodon kit combines three technologies: T7 expression, plasmid stabilization and efficient supply of rare tRNAs. The kit contains DNA of the vector (either pSCodon1.3 or pSCodonGST1.2), chemically- or electro- competent bacteria for cloning (CYS21) and expression (SE1), regeneration medium, sequencing primers, positive control, andinstruction manual. Additional CYS21 or SE1 competent bacteria can be purchased separately.

Benefits of the Staby^TMCodon kit

• High yield of heterologous-protein expression even when the protein contains rare codons;
• Not necessary to mutate each rare codon;
• Recombinant plasmid perfectly stabilized even without the use of antibiotics; 
• Reduced background of “parasite proteins;
• No additional plasmid in the bacteria;
• No “satellite” colonies obeserved even after extended incubation time