Optimal oligonucleotide design for protein expression

The design of oligonucleotides for PCR amplification of a gene which will be used for protein expression with the StabyExpress^TM T7  kit, Staby^TM Codon T7 kit,  Cherry^TMExpress kit or Cherry^TMCodon kit . This design is useful for optimizing protein production. As 61 codons are available for 20 amino acids, most amino acids are encoded by more than one codon. Thus, it is possible to generate different gene sequences encoding the same protein. The software first generates all the possible sequences encoding a specific protein and then, ranks these sequences according to a set of parameters including the secondary structure of the corresponding mRNA, the secondary structure of the oligonucleotides and the codon usage of E. coli. Ten 5’-primers (beginning of the gene) are presented as a list containing the original sequence and the 9 best modified oligonucleotides as generated by the software. The mutations introduced to optimize the protein expression are coloured in red (see figure below). The result of the codon usage analysis of the beginning of the gene (the first 7 amino acids) is shown after the name of the primer: the worst codon (and its frequency in E. coli) is mentioned between parentheses and the mean frequency across all codons of the primer is mentioned after the comma. Each primer is analyzed for homodimer and hairpin formation. If one of these parameters can cause a problem during the PCR amplification, a flag appears in the corresponding column. In the example below, the original sequence can cause problems regarding homodimer and hairpin formation and has a bad mRNA structure (all the “synonyms” are better). In this case, the second or the third primers are the best choices because they optimize both codon usage and lack of secondary structure.

Codon Usage Analyzer

Only the 7 first codons can be directly mutated through modification of PCR primers (cf. above). Unfortunately, in all organisms, most amino acids are encoded by more than one codon. But each organism is characterized by a specific “codon bias”, 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 frame shifting, 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).

The codon analyzer software is used to check the presence of rare codons according to the genetic code (standard, mitochondrial,…) and the expression-host chosen by the user. The sequence of the gene-of-interest is analyzed and each codon is coloured according to its frequency in the expression host. The exact frequency is given above each codon. Rare codons are coloured in red. The CGA codon is coloured in blue because it is a codon rarely used by Escherichia coli but large amounts of the corresponding tRNA are present, thus, it does not cause a problem during expression. If the analysis reveals the presence of clusters and/or several rare codons, it is recommended to use the Staby^TMCodon T7 kit or Cherry^TMCodon kit instead of the StabyExpress^TM T7 kit or Cherry^TMExpress kit.

Access

The oligonucletide design and the codon usage analyzer are accessible to the Staby-Operating-System customers (StabyExpress^TM T7, Staby^TMCodon T7, StabyCloning^TM, …). Please, send us an email (stabysoft@delphigenetics.com) containing the nucleic acid of your gene-of-interest and you will receive a pdf report. Total confidentiality is guaranteed.