Chemical synthesis protein-based polymer

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... 

Precise control of the sequence of amino acid residues Any protein-based polymer sequence utilizing the 20 naturally occurring monomers can be specified using recombinant DNA technology. This allows for equivalent ease of production of diverse protein-based polymer sequences, many of which would otherwise be quite difficult or essentially impossible to prepare due to problems of chemical synthesis and unfavorable energetics in the final polymer. 

As introduced in Chapter 1, the present chapter constitutes Assertion 4 The Applications Assertion of the book. Production and purification are first addressed, as they obviously make up the initial enabling steps in moving toward applications of any materials. The most surefooted path toward materials applications of protein-based polymers, however, intertwines issues of production and purification through a combination of the two methods of preparation—chemical synthesis and biosynthesis. Chemical synthesis proved the biocompatibility of elastic protein-based polymers and therefore opened the door to medical applications. Demonstration of the biocompatibility of the chemically synthesized product made clear the purification required of elastic protein-based polymers produced by E. coli if unlimited medical applications were to be possible. Chemical synthesis also provided a faster route to diverse polymer compositions, which allowed... 

The initial preparation of protein-based polymers utilized solution and solid phase peptide chemistry. This made possible the preparation of more than 1,000 polymer compositions. As discussed in Chapter 5, these compositions were studied for determination of their basic properties, for the development of the set of phenomenological axioms for protein engineering and function, and for the demonstration of the basic mechanism that underlies function. In short, it is the chemical synthesis that has allowed development of much of the basic science and the demonstration of the potential of protein-based materials in a timely manner. Mostly because of the historical relevance, but also because of the unique contributions of chemical synthesis to arriving at satisfactory purification of microbially prepared protein-based polymers, a brief description of the chemical synthesis of protein-based polymers is given below. 

Chemical Synthesis Achieved Many Different Protein-based Polymers Quickly... 

Our research utilizing chemical synthesis of repeating peptide sequences is represented in over 170 scientific publications. It utilized classic solution synthesis and to a much lesser extent solid phase methods. The primary focus in all cases has been development of an understanding relevant to structure, function, and mechanism rather than development of synthetic methodologies, although some of the latter did indeed occur principally due to the expert capacities of T. Ohnishi, K. Okamoto, R. Rapaka, K.U. Prasad, T.P. Parker, and D.C. Gowda. Here we note a few issues relevant to the production of protein-based polymers, that is, of polymers composed of repeating peptide sequences. 

Fig. 14 Molecular structure of synthetic erythropoietin protein (SEP). A Primary amino acid sequence with three ligation sites circled in red. B Structure of the branched, negatively charged poly(ethylene glycol)-based polymer. C Scheme for the synthesis of SEP by chemical ligation. Branched polymers were first attached to the individual peptide segments by oxime-forming ligation followed by native chemical ligation. Reprinted with permission from [62]. Copyright 2003 AAAS...

Uny et also reported the chemical synthesis of protein polymers based on the (Val-Pro- Ala-Val-Gly) repeat sequence in which glycine is replaced by the D-alanine residue. The hetero-chiral Pro- Ala diad would be erqrected on the basis of stereochemical considerations to adopt a type-II p-tum conformation. Stmctural analyses of small-molecule "Pro- Ala turn models support the formation of the type-II p-mm conformation in solution and the solid state. Polymers based on the (Val-Pro- Ala-Val-Gly) repeat sequence display a thermo-reversible phase transition similar to the corresponding polypeptides derived from the parent (Val-Pro-Gly-Val-Gly) sequence, albeit with a shift of the Tt to approximately 5-10 ° G below the latter due to a slight inaease in hydrophobic character due to the presence of the alanine residue. NMR spectroscopic analyses of the (Val-Pro- Ala-Val-Gly) polymer suggest that the repeat unit retains the p-tum stmcture on the basis of comparison to the corresponding behavior of the (Val-Pro-Gly-Val-Gly) polymer. Stress-strain measurements on cross-linked matrices of the (Val-Pro- Ala-Val-Gly) polymer indicate an elastomeric mechanical response in which the elastic modulus does value in comparison to the (Val-Pro-Gly-Val-Gly) polymer. These smdies of glycine suhstitution support the hypothesis that type-II p-tum formation can he associated with the development of elastomeric behavior with native elastins and elastin-derived polypeptide sequences. Several investigators have proposed that the (Val-Pro-Gly-Val-Gly) pentapeptide represents the minimal viscoelastic unit... 

---