Influence of the Molecular Weight on the Inverse Temperature Transition of a Model Genetically Engineered Elastin-like pH-Responsive Polymer

Abstract

The pH-responsive elastin-like polymers, [(PGVGV) 2-(PGEGV)-(PGVGV)2]n with n ) 5, 9, 15, 30, 45, were obtained using genetic engineering and microbial protein expression. These were intended to study the effects of the molecular weight (MW) on the properties of their inverse temperature transition (ITT) and its dependence on pH. As a result, the transition temperature decreased and the transition enthalpy increased as the molecular weight increased, especially for the lowest MWs. More strikingly, the apparent pK a for the γ-carboxyl residue of the glutamic acid also depends on MW. The apparent pKa is lower for lower MWs. In summary, the modification in the ITT caused by changes in MW is similar to the one caused by changes in the mean polarity of the polymer as described in the literature. A reduction in the molecular weight is equivalent to a decrease in the mean hydrophobicity of the polymer.

De Novo Design of Supercharged, Unfolded Protein Polymers, and Their Assembly into Supramolecular Aggregates

“…ELPs with charged amino acids as guest residues have already been produced, but with much lower charge densities than reported herein. [16] So far only a single-folded protein, green fluorescent protein (GFP), was equipped with similar amounts and densities of charges. [29] However, within GFP the charges were by far not as equally distributed as in the ELP backbone.…”

“…[16] Here, we took advantage of the flexibility of amino acid composition at the fourth position within the repeat to transform ELPs into unprecedented highly charged anionic and cationic polyelectrolytes. These structures of biosynthetic origin are much better defined than their chemically synthesized counterparts.…”

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

“…ELPs exhibit a unique reversible inverse phase transition behavior; below a critical transition temperature (T t ) ELPs are highly soluble in aqueous solution, however at temperatures even a few degrees Celsius above T t , ELP will undergo a solubilityinsolubility phase transition, leading to aggregation of the polypeptide [4]. The T t 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][6][7][8][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].…”

Elastin‐Like Peptides (ELPs) – Building Blocks for Stimuli‐Responsive Self‐Assembled Materials

“…[14][15][16][17][18] Several ELPs have been reported to be light- [19] and pH-sensitive. [20][21][22] However, ELPs have generated the most interest due to their thermoresponsiveness, particularly for targeted drug delivery applications, [7] since local mild hyperthermia can be carried out in a clinical setting using microwaves, [19] radio frequency, [23] or ultrasound. [24] Similarly to tropoelastin, ELPs can exhibit thermoresponsive LCST phase-transition behaviour.…”

“…[27,28] Studies of genetically engineered ELPs have shown that the T t of short polypeptides has an inverse dependence on the length, and subsequently, the molecular weight of the polypeptide: an increase in the ELP M w leads to a decrease of the T t . [20] The characteristics of the guest residue (X in VPGXG) have also been shown to have a marked effect on the T t : while hydrophobic guest residues (Trp and Tyr being the most effective) decrease the T t , hydrophilic guest residues elevate it. [13] The T t of ELPs can also be controlled by varying the salt concentration of the solution, as reducing the salt concentration has been shown to increase the ELPs T t .…”