No abstract

TMDSC data have been employed to observe the effect of NaCl on the inverse temperature transition of the model elastin-like polymer (GVGVP)251. NaCl causes a decrease in Tt and an increase in DeltaH. The increase in enthalpy appears both in the enthalpy related with the folding of the polymer and in the contribution associated with disruption of the structured water of hydrophobic hydration. It has been suggested that the presence of NaCl may cause a better formation of water structures surrounding the apolar polymer chains.

An elastomeric polypeptide was produced, with the sequence G-(VPGVG)19-VPGV, as a fusion to glutathione S-transferase using the vector pGEX-3X. The fusion protein was expressed to high levels in Escherichia coli as indicated by SDS-PAGE analysis of induced cells. The fusion protein was affinity purified and cleaved with protease factor Xa, and the elastomeric polypeptide was recovered to a high degree of purity as indicated by SDS-PAGE followed by staining with CuCl2. The physical characterizations of carbon-13 and proton nuclear magnetic resonance and of the temperature profile for turbidity formation for the inverse temperature transition of hydrophobic folding and assembly attest to the successful microbial synthesis of the polypentapeptide of elastin. The results of these studies provide the initial progress toward achieving a more economical and practical means of producing material for elastic protein-based polymer research and applications.

In the present paper, a temperature dependent dielectric relaxation near 5 GHz (at a frequency just lower than that of bulk water) is observed in aqueous solutions of hydrophobic elastic protein-based polymers, such as (GVGVP) 251 and (GV-GIP) 260 . On dilution at low temperatures of the solution, this relaxation becomes more intense approaching different hydrophobicity dependent limits as the hydrophobicity increases from Val (V) with the side chain -CH(CH 3 ) 2 to Ile (I) with the addition of a CH 2 moiety (i.e., -CH(CH 3 )CH 2 CH 3 ). The relaxation decreases in intensity to near 0 as the temperature of solutions of the elastic protein-based polymers are raised from below to above their respective inverse temperature transitions of hydrophobic folding and assembly. Furthermore, using the polymers (GEGXP GVGVP GVGVP GVGVP GVGVP GVGX-P) n where the two X residues are either two V or two Phe (F) residues with the aromatic phenyl side chain of -CH 2 C 6 H 5 , ionization of glutamic acid (E) side chains (i.e., the formation of COOfrom COOH) destroys the majority of the waters of hydrophobic hydration in a charge density dependent manner down to a limit suggestive of remaining pentagonally arranged waters previously observed in crystal structures 1,2 adjacent to hydrophobic moieties. This paper characterizes, for the first time, waters of hydrophobic hydration (N hh ) in terms of the variables of dilution, temperature and polymer charge density. In the absence of charge, N hh appears to be more extensive than the first shell of pentagonally arranged waters. The significance of this characterization resides in the widely held view that the thermodynamics of waters of hydrophobic hydration is central to the hydrophobic folding and function of proteins and proteinbased polymers. [3][4][5][6][7] Previous dielectric relaxation studies extending into the microwave (supra gigahertz) range have been reported on proteins such as myoglobin, 8,9 lysozyme, 10 and collagen, 11 and the ca. 10 GHz relaxation was, indeed, recognized as arising from protein hydration. For several reasons, however, the previous protein studies were unable to correlate with hydrophobic hydration any part of the relaxations ascribed to protein hydration. First, only a very small part of the hydration could be due to the presence of hydrophobic groups on the surface of these native proteins. Second, transitions to hydrophobically unfolded states (e.g., cold denaturation) would have to occur under conditions where a substantial part of the total water was hydrophobic hydration. Third, the transitions would have to be thermally accessible and well-characterized as dominantly hydrophobic. Finally, the variables that favor or disrupt hydrophobic hydration have not been identified in more complex proteins as they have for the elastic protein-based polymers.Characterization of waters of hydrophobic hydration becomes possible with elastic protein-based polymers, because these model proteins exhibit phase transitional behavior. 7 When the temperature is raised...

Five elastic protein-based polymers, designed as variations of polymer I, (GVGVP)251, elicited different responses when injected as subcutaneous implants in the guinea pig, a preclinical test used to evaluate materials for soft tissue augmentation and specifically for correction of urinary incontinence. All six polymers, prepared using recombinant DNA technology, expressed at good levels using transformed E. coli fermentation. These E. coli-produced polymers were purified for the first time to the exacting levels required for use as biomaterials where a large quantity could disperse into the tissues in a few days. Time periods of 2 and 4 weeks were used. Polymer I functioned as a bulking agent around which a fine fibrous capsule formed. Inclusion of (GVGVAP)8, a chemoattractant toward monocytes and elastin-synthesizing fibroblasts in the sequence of polymer I, resulted in an appropriate tissue response of invasion of macrophages. Inclusion of lysine residues, for lysyl oxidase cross-linking, suggested a possible remodeling of the implant toward fibers. Most promising however, when the cell attachment sequence, GRGDSP, was added to polymer I, the implant elicited tissue generation with a normal complement of collagen and elastic fibers, spindle-shaped histiocytes and angiogenesis. If this response is retained over time, the desired soft tissue augmentation and generation will have been achieved. Our working hypothesis is that on formation of elastin, with a half-life of the order of 70 years, a long lasting soft tissue augmentation would result rather than scar tissue as occurs with Contigen, the currently approved injectable implant for soft tissue augmentation.