Chapter 11 - High-Throughput Purification of Proteins from Escherichia Coli

Abstract

This chapter describes a procedure for the expression and purification of proteins from Escherichia coli in an automatable 96-well format. The first step in high-throughput expression and purification of proteins is obtaining the corresponding protein expression constructs. The expression of proteins often requires that the gene sequences of interest be placed under the control of a specific transcriptional promoter near specific translational signals in association with a particular selectable marker. The addition of peptide tags requires that the un-translated regions flanking the coding sequences be removed. Finally, the importance of ensuring that the expression clone sequences accurately reflect the natural gene sequence cannot be overemphasized. Even subtle changes in the amino acid sequences of proteins can have profound effects on protein function. One needs to resuspend pellets by shaking the plate on a Beckman Thermoshaker for 30–60 min at 750 rpm at room temperature. This Swizzler improves lyses under both denaturing and non-denaturing conditions. If the goal is to recover functional protein by renaturation, the shaking speed should be reduced to 300 rpm after 5 min to minimize harmful oxidation of proteins.