The modulation of the lower critical solution temperature (LCST) of two elastin-like polypeptides (ELPs) was investigated in the presence of 11 sodium salts that span the Hofmeister series for anions. It was found that the hydrophobic collapse/aggregation of these ELPs generally followed the series. Specifically, kosmotropic anions decreased the LCST by polarizing interfacial water molecules involved in hydrating amide groups on the ELPs. On the other hand, chaotropic anions lowered the LCST through a surface tension effect. Additionally, chaotropic anions showed salting-in properties at low salt concentrations that were related to the saturation binding of anions with the biopolymers. These overall mechanistic effects were similar to those previously found for the hydrophobic collapse and aggregation of poly(N-isopropylacrylamide), PNIPAM. There is, however, a crucial difference between PNIPAM and ELPs. Namely, PNIPAM undergoes a two-step collapse process as a function of temperature in the presence of sufficient concentrations of kosmotropic salts. By contrast, ELPs undergo collapse in a single step in all cases studied herein. This suggests that the removal of water molecules from around the amide moieties triggers the removal of hydrophobic hydration waters in a highly coupled process. There are also some key differences between the LCST behavior of the two ELPs. Specifically, the more hydrophilic ELP V5A2G3-120 construct displays collapse/aggregation behavior that is consistent with a higher concentration of anions partitioning to polymer/aqueous interface as compared to the more hydrophobic ELP V5-120. It was also found that larger anions could bind with ELP V5A2G3-120 more readily in comparison with ELP V5-120. These latter results were interpreted in terms of relative binding site accessibility of the anion for the ELP.

Elastin-like polypeptides (ELPs) are artificial polypeptides, derived from Val-Pro-Gly-Xaa-Gly (VPGXG) pentapeptide repeats found in human tropoelastin, that reversibly coacervate above a critical temperature. Genetically encodable ELPs are monodisperse, stimuli responsive, and biocompatible, properties that make them attractive for drug delivery and tissue engineering. The potential of ELPs to self-assemble into nanostructures in response to environmental triggers is another interesting feature of these polypeptides that promises to lead to a host of new applications.

This unit presents a recombinant protein purification method that employs an Elastin-like polypeptide (ELP) as a purification tag. ELPs undergo a sharp and reversible phase transition when heated above their lower critical solution temperature (LCST). ELPs retain this behavior when they are fused to a protein, and thereby provide a simple method to isolate a recombinant ELP fusion protein from cell contaminants by cycling the solution through the insoluble and soluble phase of the ELP fusion protein by a method that we have termed Inverse Transition Cycling (ITC). This method does not require the use of chromatography, so that it is costeffective, easy to scale up and to multiplex.

This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (R h ): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (R h : 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles.drug delivery | polymer bioconjugate | protein engineering P olymer modification of therapeutically relevant proteins is important because it can improve their stability, increase their solubility, enhance their systemic circulation, and also potentially reduce their immunogenicity and antigenicity (1-4). Traditionally, synthetic polymers have been conjugated to proteins by reaction of the polymer with the reactive side chains of protein residues such as lysine or cysteine (5-10). Attachment of PEG to proteins, termed PEGylation, is the most widely used polymer conjugation methodology to improve the pharmacological profiles of proteins (11)(12)(13)(14). Recently, in situ polymerization directly from the surface of a protein, in which a polymerization initiator is conjugated to the protein, followed by in situ growth of the polymer has emerged as a promising alternative to postpolymerization conjugation to synthesize protein-polymer conjugates with high yield (15)(16)(17)(18)(19)(20). However the chemically reactive residues in most proteins are typically promiscuously distributed on their surface, which can result in the growth of polymers from many sites from the protein, resulting in heterogeneous protein-polymer conjugates with poorly controlled stoichiometry and decreased biological activity compared to the protein, thereby limiting the utility of these methods. Hence, the major challenge in protein-polymer conjugation is to create: (i) site-specific, (ii) stoichiometric protein-polymer conjugates, with (iii) high yield, (iv) good retention of protein activity, and (v) significantly improved pharmacological properties over the native protein.Recently, we demonstrated a general strategy for the in situ growth of a stoichiometric, PEG-like polymer from the N terminus of a protein by a N-terminal transamination reaction followed by a chemoselective aldehyde-hydroxylamine reaction to ins...