Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins

Abstract

For structural and functional genomics programs, new high-throughput methods to characterize well-expressing and highly soluble proteins are essential. A faster and more convenient approach to screen expression conditions of recombinant proteins compared to classical in vivo systems is the Escherichia coli cell-free expression system. Here, we describe a rapid procedure to screen for expression and solubility of recombinant proteins using an E. coli cell-free extract. The results presented cover 24 open reading frames of unknown function from different micro-organisms. In order to screen different variables that may interfere with solubility, we expressed the recombinant proteins with a histidine₆ tag, either N-terminal or C-terminal at two temperatures (25° and 30°). The identification of recombinant proteins is performed by the dot blot procedure using an anti-histidine tag antibody. We designed a rapid method that allows the characterization of soluble candidates from a large number of genes or from a large number of variants that is highly compatible with structural genomics expectations.

Abbreviations

IPTG — isopropyl β-d-1 thiogalactopyranoside; Mr — molecular mass; ORF — open reading frame; PCR — polymerase chain reaction; TBST — Tris-buffered saline Tween; Tris — tris(hydroxymethyl)aminomethane.