Design of Experiments As a Tool for Optimization in Recombinant Protein Biotechnology: From Constructs to Crystals.

Abstract

In this review, the basic concepts and applications of design of experiments (DoE) in recombinant protein biotechnology will be discussed. The production of recombinant proteins usually begins with the construction of an expression vector that is then introduced into a microbial host. The target protein is overexpressed in the host's cells and subsequently, it is isolated using a suitable purification method, its activity is assessed using a biological assay, while its crystallization is often required. Because each protein is unique and due to the complex interactions among the reagents in experiments, it is impossible that one set of reaction conditions would be optimal for all cases. Optimization of experimental conditions is usually carried out by the inefficient one-factor-at-a-time approach that does not take into account the combined effects of factors on a process. On the other hand, DoE approaches with a carefully selected small set of experiments, and therefore with a reduced cost and in a limited amount of time predict the effect of each factor and the effects of their interactions on a process. Importantly, several software packages are available that facilitate the choice of the DoE approach, design of the experiments, and analysis of the results.