SeSaM: Sequence Saturation Method
Directed evolution has become a widely accepted and broadly applied method for biocatalyst engineering. The quality of a mutant library is decisive for the success of a directed evolution experiment and many methods have been developed for generating diversity at the gene level. These methods differ significantly in the mutational spectra, mutation frequency and are differently affected by the redundancy of the genetic code (Wong et al., 2006).
The standard method used in directed protein evolution is error-prone PCR (epPCR) due to its robustness and simplicity in use. Current epPCR and whole cell random mutagenesis methods suffer from three fundamental problems:
• Bias of the polymerase or the mutagenic agent that results in mutagenic "hot spots" and thus limits amino acid substitutions
• Significant fraction of stop codons and destabilizing amino acid substitutions (e.g. proline and glycine in helices)
• Lack of subsequent mutations due to methodological limitations
SeSaM is a four step method that:
• targets each nucleotide “equally” (no mutagenic “hot spots”)
• eliminates polymerase bias
• offers control over mutational bias through universal bases
• offers increased diversity due to more transversions and subsequent
mutations are possible (up to 16.7 % observed)
• can be performed in 2-3 days
SeSaM has been patented in EU, USA, China, Japan, Switzerland and others through BASF AG and was commercialized:
- Wong, T. S., Zhurina, D. and Schwaneberg, U. (2006). The diversity challenge in directed protein evolution, Comb. Chem. High Throughput Screen., 9, 271-289.
- Wong, T. S., Tee, K. L., Hauer, B. and Schwaneberg, U. (2005). Sequence Saturation Mutagenesis with tunable mutation frequencies, Anal. Biochem. 341, 187-189.
- Wong, T. S., Tee, K. L. and Schwaneberg, U. (2004). Sequence Saturation Mutagenesis (SeSaM): A novel method for directed evolution, Nucleic Acids Res. 32, e26.