Mutagenesis combinatorial

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

Finally, the region of accessible protein sequence space was extended by developing a modified version of Stemmer s combinatorial multiple-cassette mutagenesis (CMCM)... 

Figure 2.9 Schematic summary of the directed evolution of enantioselective lipase variants originating from the WT PAL used as catalysts in the hydrolytic kinetic resolution of ester rac-1. CMCM = Combinatorial multiple-cassette mutagenesis [8c,22],...

All these techniques create genetic diversity by recombination and point mutations and are well developed. However, insertions and deletions (indels) are also important types of mutation which are probably underrepresented in many conventional mutagenesis strategies. Methods for incorporation of indels in predefined positions in a combinatorial manner have been developed.Although there are some published studies on their use in the directed evolution of biocatalysts,the full potential of these newer methods of gene mutation for enzyme improvement are yet to be demonstrated. 

Modlich U., Kustikova O.S., Schmidt M., Rudolph C., Meyer J., Li Z., Kamino K., von Neuhoff N., Schlegelberger B., Kuehlcke K., Bunting K.D., Schmidt S., Deichmann A., von Kalle C., Fehse B., Banm C. (2005) Lenkemias following retroviral transfer of multidrug resistance 1 (MDRl) are driven by combinatorial insertional mutagenesis. Blood Mi, 4235-4246. 

Fig. 15. Extended combinatorial multiple-cassette mutagenesis in the evolution of an (S)-selective lipase-variant from P. aeruginosa as a catalyst in the kinetic resolution of 1 (A position 20 A position 161 A position 234 O position 53 position 180 position 272) (49).

Subsequent directed evolution work on Pseudomonas aeruginosa demonstrates that protein sequence space with respect to enantioselectivity is best explored by a three-step procedure (Reetz, 2001) (i) generation of mutants by error-prone PCR at a high-mutation rate (ii) identification of hot regions and spots in the enzyme by error-prone PCR and substantiation by simplified combinatorial multiple-cassette mutagenesis (iii) extension of the process of combinatorial multiple-cassette mutagenesis to cover a defined region of protein sequence space. 

K. -E. Jaeger, Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis, Angew. Chem. Inti Ed. 2001b, 40, 3589-3591. 

Macrocyclic polyketides have so far been the primary focus of combinatorial biosynthesis technologies. The mutagenesis techniques outlined in Sec. VI sug-... 

Reidhaar-Olson JF, Sauer RT, Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences, Science, 241 53-57, 1988. 

Merino E, Osuna F, Bolivar F, Soberon X, A general PCR-based method for single or combinatorial oligonucleotide-directed mutagenesis on pUC-M13 vectors, Bio techniques, 12 508-510, 1992. 

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