Methods in protein engineering and screening : from rational design to directed evolution and beyond

Humans have engineered organisms for thousands of years. The domestication of plants and animals are early examples of how humans have changed genotypes of organisms for prized traits that persist today. The genotype to phenotype linkage is often the result of the expression or performance of proteins found within an organism. The modern era of biological engineering has seen the emergence of two fundamentally distinct philosophies on how best to alter the genetic code of an organism or sequence of a particular protein for desired phenotypic outcomes. Rational design and directed evolution are often seen as two competing methodologies working towards the same goal—the engineering of proteins and ultimately organisms. Here, I attempt to bridge the gap between the two engineering strategies and argue that a combination is needed in many cases. Starting with the rationalization of directed evolution experiments using software tools, we begin to understand the predictive power and limitations of rational computational approaches. We then explore the computational resurfacing of an enzyme and discover an unpredicted thermoswitchable phenotype. Next, we rationally design a number of fusion proteins, which allow us to create novel point-of-care diagnostics. We then turn our attention to the directed evolution of a number of DNA polymerases with novel functions. The amalgamation of rational design and directed evolution are then extended to a eukaryotic organism, which enables us to engineer more complex proteins and gives us the ability to create novel drug screening and discovery platforms.