From the notebook to recombinant protein production in Escherichia coli: Design of expression vectors and gene cloning

Abstract

Research in recombinant protein expression in microorganism hosts spans half a century. The field has evolved from mostly trial-and-error approaches to more rational strategies, including careful design of the expression vectors and the coding sequence for the protein of interest. It is important to reflect on many aspects about vector construction, such as codon usage, integration site, coding sequence mutagenesis and many others. In this chapter, we overview methods and considerations to generate a suitable construct and anticipate possible experimental roadblocks.

Keywords: Coding sequence; E. coli; Expression vector; Recombinant protein; Restriction free cloning; T7 system.