The molecular basis for the inverse temperature transition of elastin11Edited by A. R. Fersht

Improved Non-chromatographic Purification of a Recombinant Protein by Cationic Elastin-like Polypeptides

“…Because this minor contamination product likely consists of the cleaved ELP fragment of the fusion, it is co-purified with the Trx-ELP fusion through multiple rounds of ITC and hence, is present in the final purified product. Furthermore, we hypothesize that ELP[KV 2 F] fusions are more susceptible to proteolysis than ELP[KV 7 F] fusions because of conformational differences caused by different interactions between Trx and ELP domains in the fusion proteins. Clues to these conformational differences can be elucidated from analysis of the thermal properties of these ELPs and their Trx fusions (vide infra).…”

“…13 Figure 3 shows that the T t s of these cationic ELPs and Trx-ELP fusions also exhibit a linear relationship with 1/length of ELP pentapeptides. ELP[KV 2 F] polypeptides and Trx fusions have steeper slopes than their corresponding ELP[KV 7 F] counterparts, which indicates that the fraction of Lys residues in these cationic ELPs dominates their change in T t with ELP length. Although we were only able to measure the T t at 25 μM in PBS for a small subset of the ELP and Trx-ELP proteins (T t <90°C…”

“…We note that the latter possibility is unique to the cationic ELPs studied here, as the aliphatic ELP[V 5 A 2 G 3 -90] in the previous study only had Val, Ala and Gly at the guest residue position. The T t s of ELP[KV 7 F] proteins are 16 to 42 °C lower than ELP[KV 2 F] polypeptides indicating that for the same concentration and similar molecular weight they are more hydrophobic; however, when fused to Trx, ELP [KV 2 F] polypeptides exhibit a greater negative fusion ΔT t parameter, indicating that in this case it is not the net hydrophobic character of the ELP that is responsible for the changes in the fusion ΔT t parameter. Instead, the sign and magnitude of their fusion ΔT t parameter are consistent with electrostatic attraction between the positively charged lysine residues on these ELPs and negatively charged Asp and Glu residues present on the surface of Trx.…”

“…Electrostatic interaction between the cationic ELPs and Trx then results in the neutralization of charged moieties in the ELP, which lowers the T t of the fused ELP, resulting in a negative ΔT t parameter. As ELP [KV 2 F] tags have more than twice the fraction of charged lysine residues, their T t s are depressed to greater extent by fusion to Trx than ELP[KV 7 F], so that they have greater negative ΔT t parameters. Figure 4 shows the effect of NaCl concentration on the T t of the ELPs (Figures 4A and 4C) and their Trx fusions (Figures 4B and 4D).…”

“…Trx-ELP [KV 7 F-36] has more than 1.6-fold greater Trx yield than that from Trx-ELP[KV 7 F-72], which is more than 2.5-fold greater than that from Trx-ELP[V 5 A 2 G 3 -90], which utilizes the most commonly used ELP purification tag. Furthermore, the Trx yield from Trx-ELP[KV 7 To deconvolute the coupled effects that ELP T t , salt sensitivity, and Trx-ELP expression yield have on the purification efficiency of Trx-ELP fusion proteins, the T t of Trx-ELP fusion proteins was measured at a fixed concentration in E. coli soluble lysate as well as in PBS. Figure 7 shows the difference in T t between 0.5 M and 1.0 M NaCl (ΔT t (NaCl)) added to 100 μM Trx-ELP solutions in PBS and soluble E. coli lysate for Trx-ELP[KV 7 .…”