Assembly and Optimization of Expression of Synthetic Genes Derived From the Human Elastin Repeated Motif

Abstract

Naturally occurring proteins often possess interesting properties that make them attractive for the realization of innovative biomaterials. Repetitive artificial polypeptides have been modeled on the repeated domains of mammalian elastin, retaining and even enhancing their thermally responsive behavior. These protein polymers have been produced by recombinant biotechnology and due to their smart properties show a huge potential for a wide range of biomedical and biotechnological applications. For this reason, production of large quantities of highly purified material is a crucial step. We focused our attention on elastin-derived polypeptides based on the hexapeptidic motif typical of human elastin. Synthetic genes were assembled starting from a monomeric unit, and the different conditions were assayed to optimize the yield of the artificial polypeptides. Optimization of Escherichia coli strain and of the extraction procedure led to significant improvement in expression and recovery of the recombinant products. Electron micrographs of expressing bacteria under optimized conditions showed the accumulation of the recombinant product in the induced cells.

In vitro myogenesis induced by human recombinant elastin-like proteins

“…The stiffness measured in control myotubes (49.69±4.19 kPa) differ significantly from that reported by Collinsworth et al[31] for the same cells(12)(13)(14)(15). A possible origin of such a discrepancy is that we measured cell stiffness on samples dehydrated after paraformaldehyde fixation, while Collinsworth at al.…”

“…The third polypeptide employed in this study, HELP1, is composed by the simple repetition of the hydrophobic domain of human elastin (yellow). Similarly to what previously described [13], the three recombinant polypeptides were expressed and purified at a good yield. Once deposited onto coverslips and allowed to dry, they form a coating film whose thickness was evaluated by Atomic Force Microscopy.…”

“…The final construct was verified by sequencing and the recombinant product was expressed in the C3037 E. coli strain (New England Biolabs, Ipswich, MA). Expression and purification were carried out as already detailed [13]. The recombinant polypeptides obtained were analyzed by SDS-PAGE (not shown) and the purified products were lyophilized for long term storage.…”

“…The synthesis of HELP and HELP1 was carried out as previously described [12], [13]. For the synthesis of HELPc, the synthetic gene of the HELP polypeptide was fused with the 41aa coding sequence corresponding to the 854-895 region of the collagen IV α2 chain (NP_001837.2), exploiting the unique DraIII site in the expression vector that allows the in-frame insertion at the Cterminus of the polypeptide [12].…”

The potential of recombinant human elastin-like polypeptides for drug delivery

“…Thus, it seemed interesting to direct efforts toward the realization of a family of protein biopolymers with 'smart' and tunable properties derived from the human sequences that were named human elastin-like polypeptides (HELPs) [5].…”