Preparation of Very-High-Yield Recombinant Proteins Using Novel High-Cell-Density Bacterial Expression Methods
- Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201, USA
- ↵1Corresponding authors (qil{at}med.wayne.edu; jjwang{at}med.wayne.edu).
INTRODUCTION
Using bacterial hosts such as Escherichia coli to produce recombinant proteins is a common practice in many laboratories. Here, we describe a protocol for preparing very-high-yield recombinant proteins using novel high-cell-density bacterial expression methods. By combining strictly regulated traditional isopropyl-β-D-thiogalactopyranoside (IPTG) induction with high-cell-density auto-induction, we have developed an efficient bacterial expression method that can be used widely and does not require changing expression vectors or fermentation. Beginning with a newly created bacterial expression vector, we outline the specific steps to determine whether the vector expresses the target protein, select high-expression colonies of the target protein, and optimize expression conditions for very-high-yield protein production. This method routinely produces 15-35 mg of pure protein from 50-mL bacterial cell cultures.
