Figure

Overview of rational design process. Step 1: The protein of interest is characterized, and a strategy for rationally introducing specific amino acid (aa) changes is planned on the basis of the known protein structure (A). The desired aa changes are typically introduced using site-directed mutagenesis (SDM) (B). Step 2: A plasmid expressing the mutated protein is created and transformed into an appropriate host (e.g., E. coli), and the mutated protein is purified and analyzed for desired properties (C). Step 3: If the mutant protein displays the desired properties, it is isolated. Step 4: The wild-type or variant protein may be subjected to further rounds of SDM and analysis to improve further desired characteristics.

([52-55] and reviewed in [39]), is making rational design an increasingly efficient tool for engineering enzymatic properties. Rational design remains an important component of the protein engineer's toolbox, and its usefulness will no doubt become more apparent as more in-depth knowledge of various industrial and medicinal enzymes accrues. Concomitantly, rational design is complementing our basic understanding and appreciation of fundamental enzymology. Rational design has tremendous potential for the much sought-after area of de novo protein enzyme design [39]. Furthermore, as discussed in Section 2.4, rational design has expanded and is predicted to continue to expand the usefulness of enzymes as medicinal products, an area of tremendous potential economic and health benefits. Despite the fact that rational design has had a tremendous impact on protein and enzyme engineering, the strategy usually requires detailed understanding of the structures and mechanisms. However, this information is not available for the vast majority of enzymes, and even if it was, the actual molecular basis of the desired function may not be [56]. These limitations on rational design have prompted the development of some innovative new technologies, such as directed evolution.

2.2.2 Directed Evolution

Despite the ever-growing body of knowledge surrounding enzyme structure and function, it is quite clear that various aspects of enzyme function cannot be predicted [57]. Rational

1. Denature and anneal oligo with desired change encoded

2. Extend using DNA polymerase, dNTPs and DNA ligase

1. Denature and anneal oligo with desired change encoded

2. Extend using DNA polymerase, dNTPs and DNA ligase

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