Sequence saturation mutagenesis

Wong, T.S., Tee, K.L., Hauer, B. and Schwaneberg, U. (2004) Sequence saturation mutagenesis (SeSaM) a novel method for directed evolution. Nucleic Acids Research, 32, e26. 

Seng Wong, T., Roccatano, D., Loakes, D. et al. (2007) Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+) a random mutagenesis method with consecutive nucleotide exchanges that complements the bias... 

Of the nine thermostabilized S41 variants identified in the first generation, three had substitutions at residue 211, and one contained a substitution at 212. Saturation mutagenesis was performed at both sites in order to explore a greater sequence diversity than that accessible through error-prone PCR (Miyazaki and Arnold, 1999). One hundred and five of the clones from the library that showed reasonable activity were assayed... 

It is thus dear that the combination of epPCR and saturation mutagenesis constitutes an efficient way to explore protein sequence space with respect to enantioselectivity. Indeed, this strategy led to the creation of several other highly (S)-selective variants (ee = 88 - 91 % E= 20 - 25) [9,42], all of them being the descendents of the parent wild-type lipase, which is only slightly (S)-selective. 

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