High level expression and facile purification of recombinant silk-elastin-like polymers in auto induction shake flask cultures

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.

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

“…The gene for SELP-59-A had already been inserted in a modified pET25b(+)vector (Novagen) for expression in E. coli BL21(DE3)12, this vector contains the ampicillin resistance selective marker gene encoding β-lactamase.…”

“…SELP production levels for the various constructs were determined by weight measurements of purified product as previously described1222. A previously developed non-chromatographic approach was used for purification12.…”

“…We have synthesised a number of novel artificial block copolymers combining the repetitive amino acid sequence motifs of silk and elastin12. These silk-elastin-like proteins (SELPs) combine the crystallinity and mechanical strength of silk with the high resilience and water solubility of elastin in a single structure.…”

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag

“…Around 154 mg of the fusion protein was purified from a 12-L fermentation culture and the yield of ELP was$1.8 mg/L after cleavage by factor Xa and purification. Finally, higher levels of ELP20 production could be foreseen after extensive optimization of the process, as was recently described for the production of silk-elastin-like polymers [33][34][35].…”

Adding Functions to Biomaterial Surfaces through Protein Incorporation

“…Recombinant expression of spider silks is essential due to the territorial behavior of the natural hosts [120] and has been carried out in E. coli, [121] yeast, [122] silk worms, [123] and, in a scaledup production system, transgenic goats. [124] Silk-elastin-like PROGRESS REPORT polymers (SELPs), which contain repetitive amino acid sequence motifs derived from both silk fibroin and mammalian elastin, [125] have been produced recombinantly and at up to gram per liter yields in E. coli, [126] enabling their increasingly widespread investigation in structural [127] and controlled release/drug delivery applications. [127a,128] …”