GenScript's codon optimization technology

The expression of functional proteins in heterologous hosts is the cornerstone of modern biotechnology. Unfortunately, many proteins are difficult to express outside their original contexts. They may contain expression-limiting regulatory elements, come from organisms that use non-canonical nucleotide codes or from a gene rife with codons rarely used in the desired host. Improvements in the speed and efficiency of gene synthesis have rendered feasible complete gene redesign for maximum protein expression. For example, protein expression can improve dramatically when the codon frequency of the gene under study is matched to that of the host expression system. This was found to be the case with green fluorescent protein (GFP), naturally expressed in jellyfish, which showed a strong increase in expression after GenScript optimization. GenScript's redesign strategy includes not only the use of optimum codon biases, but also the alteration of mRNA structural elements and the modification of translation and initiation regions.

Codon bias and protein expression
Codon bias has been identified as the single most important factor in prokaryotic gene expression. The degree to which a given codon appears in the genetic code varies significantly between organisms, between proteins expressed at high and low levels and even between different portions of the same operon. The reason for this is almost certainly because preferred codons correlate with the abundance of cognate tRNAs available within the cell. This relationship serves to optimize the translational system and to balance codon concentration with isoacceptor tRNA concentration. In E.coli, for example, the tRNA molucule that reads the infrequently used AGG and AGA codons for arginine is present only at very low levels. It is likely that codon usage and tRNA isoacceptor concentrations have coevolved, and that the selection pressure for this coevolution is more pronounced for highly expressed genes than genes expressed at low levels.

Replace infrequently used codons with those preferred by the desired host organism
In general, the more rare codons that a gene contains, the less likely it is that the heterologous protein will be expressed at a reasonable level within that specific host system. These levels become even lower if the rare codons appear in clusters or in the N-terminal portion of the protein. Replacing rare codons with others that more closely reflect the host system's codon bias without modifying the amino acid sequence can increase the levels of functional protein expression.

Eliminate problematic codons
Any codon that an organism uses less than 5% to 10% of the time may cause problems, regardless of where it is from. Again, close or adjacent codons can have more affect on protein expression than they could separately. Eliminating rare codons and codons that could be read as termination signals can prevent cases of low or nonexistent expression.

Express viral proteins in mammalian hosts
Even viral genes can be successfully expressed in mammalian cell lines if the gene is properly prepared. Viral genes' dense information loads frequently result in overlapping reading frames. Many viral genes also encode cis-acting negative regulatory sequences within the coding sequence. Viral genes can be resynthesized not only to express only the desired protein but also to disrupt regulatory elements, thereby enhancing protein production by an average of 28%. Viral codon optimization is especially useful in DNA vaccine research because it increases the immunogenicity of the target.

Other constraints
Although codon bias plays a large role in gene expression, the choice of expression vectors and transcriptional promoters is also important. The nucleotide sequences surrounding the N-terminal region of the protein are particularly sensitive, both to the presence of rare codons and to the identities of the codons immediately adjacent to the initiation AUG. There is also some interplay between translation and mRNA stability, which has not been completely explained, although reduced translational efficiency may be accompanied by a lower mRNA level because decreased ribosomal protection of mRNA increases its exposure to endo-RNAses. Overly stable mRNA secondary structures, particularly near the 5' end of the molecule can also have a significant effect. Strategies using short upstream open reading frames for translational coupling of target genes have proved successful in improving the efficiency of expression of some problem genes.

Additional features of our codon optimization service:

• Manipulation of restriction enzyme cutting sites: These sites can be added or removed during the design process to facilitate future manipulation of the construct.
• Design of fusion proteins: Any epitope tag sequences (e.g, HIS tag and GST tag) or protease cutting site can be incorporated into the designed gene for the expession of fusion proteins. The delivered synthetic gene construct is ready for expression and purification without further manipulation.
• Creation of gene variants (mutant forms): Multiple variant forms (or variant libraries) can be designed for functional study and screening.

Note: You may use our secure web server or to submit sequence for optimization and synthesis. In your message, please include:

• Protein or ORF DNA sequence.
• Intended host expression system.
• Restriction enzyme cutting sites at both ends.
• Restriction enzyme cutting sites that you want to avoid in the optimized sequence.
• Restriction enzyme cutting sites that you want to keep in the original sequence.

• Barrett John, et al. Optimization of Codon Usage of Poxvirus Genes allows for Improved Transient Expression in Mammalian Cells. Virus Genes. Aug 2006; 33(1): 15-26. Kim CH, et al. Codon optimization for high-level expression of human erythropoietin (EPO) in mammalian cells. Gene. 1997; 199: 293-301.
• Ravi Vijaya Satya, et al. Codon Optimization for DNA Vaccines and Gene Therapy Using Pattern Matching. CSB. Aug 2003.
• Frank Gronlund Jorgensen, et al. Heterogeneity in Regional GC Content and Differential Usage of Codons and Amino Acids in GC-Poor and GC-Rich Regions of the Genome of Apis mellifera. Molecular Biology and Evolution. Dec 2006.

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