Abstract
Silk-elastin-like polymers (SELPs) are protein-based polymers composed of repetitive amino acid sequence motifs found in silk fibroin (GAGAGS) and mammalian elastin (VPGVG). These polymers are of much interest, both from a fundamental and applied point of view, finding potential application in biomedicine, nanotechnology and as materials. The successful employment of such polymers in such diverse fields, however, requires the ready availability of a variety of different forms with novel enhanced properties and which can be simply prepared in large quantities on an industrial scale. In an attempt to create new polymer designs with improved properties and applicability, we have developed four novel SELPs wherein the elastomer forming sequence poly(VPGVG) is replaced with a plastic-like forming sequence, poly(VPAVG), and combined in varying proportions with the silk motif. Furthermore, we optimised a simplified production procedure for these, making use of an autoinduction medium to reduce process intervention and with the production level obtained being 6-fold higher than previously reported for other SELPs, with volumetric productivities above 150 mg/L. Finally, we took advantage of the known enhanced stability of these polymers in developing an abridged, non-chromatographic downstream processing and purification protocol. A simple acid treatment allowed for cell disruption and the obtention of relative pure SELP in one-step, with ammonium sulphate precipitation being subsequently used to enable improved purity. These simplified production and purification procedures improve process efficiency and reduce costs in the preparation of these novel polymers and enhances their potential for application.
Highlights
Nature has refined structural proteins to perform defined functions through the association of specific amino acid sequences in various combinations, creating diverse materials with remarkable properties
Initial studies aiming at protein production were performed in Lysogeny Broth (LB) and Terrific Broth (TB) media supplemented with lactose (LB+lac and TB+lac), at 37°C with a liquid to flask volume ratio of 1:5 and analysed through a 24 hour fermentation period
Silk-elastin-like polymers (SELPs)-1020-A expression levels seem to reach a steady state after 18 hours of fermentation in LB+lac (Figure 2A) which was further corroborated by calculating the area of the protein bands (Table 2)
Summary
Nature has refined structural proteins to perform defined functions through the association of specific amino acid sequences in various combinations, creating diverse materials with remarkable properties These have served as a source of inspiration for a new class of bio-engineered materials, the protein-based polymers (PBPs), founded on conservative amino acid motifs found in nature. The silk-like blocks spontaneously form hydrogen-bonded β-sheet crystals and impart thermal and chemical stability (Megeed et al 2002) while the periodic inclusion of the elastomeric sequence reduces the overall crystallinity of the system and increases its flexibility and aqueous solubility (Cappello et al 1990) These copolymers have already demonstrated their potential importance in several biomedical applications (Megeed et al 2002; Gustafson and Ghandehari 2010) and more recently, in the fabrication of micro- and nano-structures (Nagarajan et al 2007; Ner et al 2009; Qiu et al 2009). To fully develop the potential of SELPs, new polymer designs with altered and improved properties are required, allowing for a better understanding of the design of these and leading to an expanded range of novel polymers with potentially enhanced applicability
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