Protein-based polymers Poly

Table 1. Hydrophobicity scale for protein-based polymers and proteins based on the properties of the inverse temperature transition of elastic protein-based polymers, poly[/v(GVGVP), (GXGVP)]. ...

With the protein-based polymer poly [0.8GVGVP),0.2GEGVP)], at low pH when aU 4 of the Glu (E) residues/lOO residues are as COOH, the transition temperature is near 25° C and the heat of the transition, AH, = 0.97kcal/mole-pentamer (see Rgure 5.28). On raising the pH to the point of less than two C00"/100 residues, the heat of the transition has been reduced, and AH, = 0.27kcal/mole pentamers. The preferred interpretation over a decade ago was (1) that the formation of 2 C00 /100 residues structured almost three-fourths of the thermodynamically measured waters of hydrophobic hydration, and (2) that there exists a competition for hydration between apolar and polar residues, referred to as an apolar-polar repulsive free energy of hydration. 

Early in our studies it was expected that the post-translational modification of proline hydroxylation, so important to proper collagen structure and function, would raise the value of the temperature, T, for the onset of the inverse temperature transition for models of elastin. Accordingly, hydroxyproline (Hyp) was incorporated by chemical synthesis into the basic repeating sequence to give the protein-based polymers poly[fvs,i(Val-Pro-Gly-Val-Gly), fHyp( al-Hyp-Gly-Val-Gly)], where f sl -i- fnyp = 1 and values of fnyp were 0, 0.01, and 0.1. The effect of prolyl hydroxylation is shown in Figure 7.49. Replacement of proline by hydroxyproline markedly raises the temperature for hydrophobic association. Prolyl hydroxylation moves the movable cusp of... 

Bezemer JM, Grijpma DW, Dijkstra PJ, Van Bhtterswijk CA, Feijen J. A controlled release system for proteins based on poly(ether-ester) block-copolymers polymer network characterization. J Control Release 1999 62 393—405. 

For our model protein, poly(GVGVP), replacement of either of the Gly residues or the Pro residue markedly alters the structure and properties of the resulting protein-based polymer. For example, substitution of the Gly and Pro residues can destroy the favorable elasticity of the polymers. These positions will not be used for the comparisons of interest here. Substitution of the Val preceding the Pro is possible by most amino acid residues, but not all. Fortunately, any one of the 20 naturally occurring amino acid residues can replace the V of GVG without significant change to the basic structure and function. 

Figure 5.9. Experimental data for development of the T,-based hydrophobicity scale. The general composition for the protein-based polymer is poly [f,(GXGVP),fv(GVGVP)], where X is the guest amino acid residue to be evaluated and fx and E are mole fractions wherein fj -i- E = 1. Part A contains the raw data for a number of guest residues substituted at a mole fraction of 0.2, which means 4 substituted residues per 100 residues of poly(GVGVP). The experimental conditions were 40mg/ml of polymer of a molecular weight of about 100,000 Da in 0.15 N NaCl and 0.01 M phosphate at pH 7.4. Experimental T,-values were obtained as shown in part A for fx = 0.2, and additional polymers were characterized with different fx values such that a plot of fx versus T, could be constructed as in part B. Extrapolation of the linear plots in part B to fx = 1 gave the T,-values that became the basis for the T,-based hydrophobicity scale given in Table 5.1. (Adapted with permission from Urry. )...

Obviously, the effect of raising the temperature of a loaded, cross-linked elastic band composed of elastic protein-based polymers of the poly(GVGVP) family is to drive hydrophobic association with the consequence of lifting of the attached weight. How does this combine with the above understanding of elasticity to perform mechanical work ... 

Consideration of the effects of stretching begins with hydrophobically associated and cross-linked elastomeric matrix composed of the same protein-based polymer as used in the calorimetry studies with poly[0.8(GVGVP),... 

For production by E. coli there is an important issue of the removal of E. coli toxic proteins, particularly, because all animals have abundant antibodies against E. coli antigens. Because poly(GVGVP) has been chemically synthesized and adequately purified, it was established that these elastic protein-based polymers exhibited extraordinary biocompatibility. This awareness provided the impetus for the necessary levels of purification when using E. cofi-produced protein-based polymers, as discussed below. 

Only because the remarkable biocompatibility of chemically synthesized poly(GVGVP) was already known was there adequate impetus to purify microbially prepared (GVGVP)2si. Otherwise, it would have been presumed, as had been widely expected, that the toxicity of inadequately purified (GVGVP)25i was an inherent property of the protein-based polymer. To be left in such a state of misunderstanding would have meant that the dramatic potential of elastic protein-based polymers for use in medical applications would be neither appreciated nor realized. The inflammatory response elicited by an inadequately purifled biosynthetic elastic protein-based polymer would have overwhelmed most considered medical applications. 

Now, it has been shown for materials such as poly(propylene diol) (wherein both the absorption maximum for loss shear modulus and loss permittivity overlap near the frequency of IHz) that their normalized curves perfectly superimpose over their frequency band width. - As shown in Figure 9.15, the lower frequency loss shear modulus curves uniquely overlap with the loss permittivity data at higher frequency. As such the former is melded to calibrate the loss permittivity data to obtain a coarse estimate of the elastic modulus values. This provides an independent demonstration of the mechanic il resonance near 3 kHz and also allows reference to the 5 MHz dielectric relaxation as a mechanical resonance. Thus, as the folding and assembly of the elastic protein-based polymers proceed through the phase (inverse temperature) transition, the pentamers wrap up into a structurally repeating helical arrangement like that represented in Figure 9.17. 

Figure 9.18. TUbes made by y-iiradiation cross-linking of elastic protein-based polymers of different compositions. (A) Poly(GVGVP). (B) Poly[fF(GFGVP), fv(GVGVP)]. (C) Poly(GVGIP). (Adapted with permission from Urry." Now to be credited as www.WorldandIJournal.com)...

Figure 9.21 exemplifies the nexus between cellular mechano-chemical transduction and elastic protein-based polymers containing cell attachment sequences that enables these temporary functional scaffoldings to result in restoration of natural tissue. Figure 9.21 A shows in the absence of cell attachment sequences that the elastic matrix X -poly(GVGVP) does not support attachment of cells. On inclusion of cell...

Convenient purification of microbially produced transductional protein-based polymers, e.g. poly(GVG T), from the cell lysate is based on a methodologv w hich utilizes the fundamental inverse temperature transitional properties (Urry et al., 1995b McPherson et al, 1996). First the bacterial cells are separated from the growth medium either by centrifugation or filtration and resuspened in Tris-HCl buffer, 50 mM, pH 8.0. Then the cells are lysed by ultrasonic disruption or French press to release the cell contents. The cell lysate is cooled to 4 C and centrifuged at high speed (10,000 x g) to remove the cold insoluble materials. The supernatant... 

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