Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cycling

Abstract

A crucial and challenging problem in proteomics is purification, identification, and characterization of proteins, some of which are expressed at very low levels. The preferred method for purification of low abundance proteins exploits multiple affinity purification tags on a single recombinant protein e.g. tandem affinity purification (TAP) [1]. However TAP is both experimentally lengthy, involving many sequential binding, washing and elution steps and costly, requiring two different and expensive resins to recover the purified recombinant protein. Hence processing large amounts of cell lysate makes it prohibitively expensive especially for scale-up. Here we present a new and simple strategy to purify soluble recombinant proteins from E. coli at a protein concentration that approaches the limit of a single protein molecule per cell. This method utilizes the unique aggregation properties of elastin-like polypeptides (ELPs) to capture recombinant fusion proteins composed of a target protein and an ELP tag from cell lysate. ELPs are artificial, genetically encodable polypeptides composed the repeating pentapeptides sequence VPGXG, where the guest residue (X) can be any naturally occurring amino acid except Pro [2,3]. ELPs exhibit a unique reversible inverse phase transition behavior; below a critical transition temperature (Tt) ELPs are highly soluble in aqueous solution, however at temperatures even a few degrees Celsius above Tt, ELP will undergo a solubilityinsolubility phase transition, leading to aggregation of the polypeptide [4]. The Tt of an ELP is a function of a number of variables including identity and stoichiometry of the guest residue, molecular weight, ELP and salt concentration in aqueous solution [5–9]. The environmental sensitivity and reversible solubility of ELPs are retained when expressed as recombinant fusions with proteins. This feature can be exploited for non-chromatographic purification of recombinant proteins by inverse transition cycling (ITC) [10]. In ITC, an ELP fusion protein is selectively separated from other contaminating biomolecules in cell lysate by the sequential and repeated steps of aggregation, centrifugation, and resolubilization of the fusion protein [10,11]. A number of different proteins have been purified by this method using either centrifugation [11–18] or micro-filtration [19]. The direct purification of ELPs has also been extended to the capture of native proteins by ELP-tagged capture reagents [14–17]. Proteins expressed at the level of micrograms per liter of culture are difficult to purify by chromatography because of the losses associated with non-specific and irreversible adsorption of the target protein to chromatography resins as well as the relative increase in contamination from host proteins that are non-specifically adsorbed to the chromatography resin are subsequently eluted with the target protein. We demonstrate a new variant of ITC that solves