Fusion order controls expression level and activity of elastin‐like polypeptide fusion proteins

Abstract

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin-like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C-terminus of the target protein (protein-ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP-protein constructs have a lower specific activity than protein-ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein-ELP and ELPprotein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N-terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C-terminal end of the protein.

Elastin-based protein polymer nanoparticles carrying drug at both corona and core suppress tumor growth in vivo

“…1a), which are similar in composition to I48S48 (Table 1). The DNA sequence of FKBP was inserted to the N-terminus of the ELP, where superior protein activity has been reported [24]. DLS demonstrated that FSI fusion proteins assembled particles with a 23.8 nm hydrodynamic radius (Fig.…”

Injectable protease-operated depots of glucagon-like peptide-1 provide extended and tunable glucose control

“…The fused ELP component of PODs enabled their facile, chromatographyfree purification by inverse transition cycling (ITC) (Fig. S2) (24), with yields of ∼150 mg/L of purified PODs in a shaker flask culture. By tuning the composition of the ELP, we were able to create GLP-1 PODs with a T t below body temperature (POD forming, ELP Low ) or above body temperature (soluble control, ELP High ) (Fig.…”

Expression and purification of the antimicrobial peptide cecropin AD by fusion with cationic elastin-like polypeptides

“…In this study, 1.2 mg CAD was obtained from 100 ml culture, less than P. pastoris and B. subtilis. It was reported that placing the ELP at the N-terminus of the target protein decreased the expression level of the ELP fusion proteins, probably due to the fact that this placement increased the fraction of misfolded protein that are more likely to be degraded by E. coli proteases during intracellular translation [19]. In this study, CELP was fused at the N-terminus of CAD; otherwise there would be one or two extra amino acid residues left on CAD which might decrease the antimicrobial activity of the peptide [20].…”