Introduction

Environmental problems require the development of biodegradable plastics of benign production that can be synthesized from renewable resources without the use of toxic and hazardous chemicals and will help in solving the increasing global disposal burden. Protein-based polymers offer a wide range of materials similar to that of petroleum-based polymers such as elastomers and plastics. Protein-based polymers can be prepared of varied design and composition through genetic engineering without the use of hazardous and noxious solvents and can be made biodegradable with chemical clocks to set their half-lives such that they can be environmentally friendly over their complete life cycles of production and disposal. Compositions tested to date have been shown to be extraordinarily biocompatible, allowing for medical applications ranging from the prevention of postsurgical adhesions and tissue reconstruction to programmed drug delivery (I). Among nonmedical applications, there are transducers, molecular machines, super absorbents, biodegradable plastics, and controlled release of agricultural crop enhancement agents like herbicides, pesticides, and growth factors.

Bioelastic materials are based on elastomeric and related polypeptides comprised of repeating peptide sequences (2); they may also be called elastic and plastic protein-based polymers. The parent polymer, (Val1-Pro2-Gly3-Val4-Gly5)„ or poly(VPGVG), derives from sequences that occur in all sequenced mammalian elastin proteins (3). In the most striking example, the sequence (VPGVG)„ occurs in bovine elastin with n = 11 without a single substitution (3). A particularly interesting analog is poly (AVGVP) as it reversibly forms a plastic on raising the temperature.

From Methods m Molecular Biology, vol 63 Recombinant Protein Protocols

Detection and Isolation Edited by R Tuan Humana Press Inc , Totowa, NJ

What is required for the commercial viability of protein-based polymers is a cost of production that would begin to rival that of petroleum-based polymers. The potential to do so resides in low cost bioproduction. We have recently demonstrated a dramatic hyperexpression of an elastin protein-based polymer, (Gly-Val-Gly-Val-Pro)„ or poly(GVGVP), which is a parent polymer for a diverse set of polymers that exhibit inverse temperature transitions of hydrophobic folding, and assembly as the temperature is raised through a transition range and which can exist in hydrogel, elastic, and plastic states. Electron micrographs revealed formation of inclusion bodies in E. coli cells occupying up to 80-90% of the cell volume under optimal growth conditions (3a). The beauty of this approach is the lack of any need for extraneous sequences for the purposes of purification (4) or adequate expression. The usual strategy for expression of a foreign protein or protein-based polymer in an organism such as E coli anticipates that the foreign protein will be injurious to the organism. Accordingly, the transformed cells are grown up to an appropriate stage before expression of the foreign protein is begun and expression is generally considered viable for only a few hours. The situation is quite different for the elastic protein-based polymer considered here. This may result in part due to the extraordinary biocompatibihty exhibited by (GVGVP)„ and its related polymers. The elastic protem-based polymer, (GVGVP)„ and its y-irradiation crosshnked matrix as well as related polymers and matrices appear to be ignored by a range of animal cells and by tissues of the whole animal (5—7). This chapter describes in detail methodologies to accomplish hyperexpression of a protein based polymer m E coli.

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