Abstract
This chapter describes the use of incremental truncation to create libraries of hybrid enzymes. The creation of genetic diversity through mutagenesis and selection is essential for organisms to adapt to the changing environments. In the laboratory, these processes can be enhanced and accelerated to evolve proteins for specific functions. The most commonly used method of protein engineering is DNA shuffling and the numerous variations of this technique. Incremental truncation is successfully used to convert monomeric enzymes into heterodimers and to create libraries of hybrid enzymes. These methods are generally referred to as incremental truncation for the creation of hybrid enzymes (ITCHY). To start the ITCHY method, the plasmid is linearized with restriction enzyme R1and truncated incrementally using Exo III. It is found that for each ITCHY library, checking crossover distribution is also important. Usually, 20 isolates are sequenced from each library and the crossover positions are plotted to check for adequate distribution. It is suggested that libraries can become biased from insufficient or excessive Exo III digestion and need to be reconstructed.
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