Secreted production of an elastin-like polypeptide by Pichia pastoris

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium

Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection

UVB-induced cutaneous photodamage/photoaging is characterized by qualitative and quantitative deterioration in dermal extracellular matrix (ECM) components such as collagen and elastic fibers. Disappearance of microfibrillar-associated protein 4 (MFAP-4), a possible limiting factor for cutaneous elasticity, was documented in photoaged dermis, but its function is poorly understood. To characterize its possible contribution to photoprotection, MFAP-4 expression was either augmented or inhibited in a human skin xenograft photodamage murine model and human fibroblasts. Xenografted skin with enhanced MFAP-4 expression was protected from UVB-induced photodamage/photoaging accompanied by the prevention of ECM degradation and aggravated elasticity. Additionally, remarkably increased or decreased fibrillin-1-based microfibril development was observed when fibroblasts were treated with recombinant MFAP-4 or with MFAP-4-specific siRNA, respectively. Immunoprecipitation analysis confirmed direct interaction between MFAP-4 and fibrillin-1. Taken together, our findings reveal the essential role of MFAP-4 in photoprotection and offer new therapeutic opportunities to prevent skin-associated pathologies

Molecular basis of elastic fiber formation: Critical interactions and a tropoelastin-fibrillin-1 cross-link

We have investigated the molecular basis of elastic fiber formation on fibrillin microfibrils. Binding assays revealed high affinity calcium-independent binding of two overlapping fibrillin-1 fragments (encoded by central exons 18-25 and 24-30) to tropoelastin, which, in microfibrils, map to an exposed "arms" feature adjacent to the beads. A further binding site within an adjacent fragment (encoded by exons 9-17) was within an eight-cysteine motif designated TB2 (encoded by exons 16 and 17). Binding to TB2 was ablated by the presence of N-terminal domains (encoded by exons 1-8) and reduced after deleting the proline-rich region. A novel transglutaminase cross-link between tropoelastin and fibrillin-1 fragment (encoded by exons 9-17) was localized by mass spectrometry to a sequence encoded by exon 17. The high affinity binding and cross-linking of tropoelastin to a central fibrillin-1 sequence confirm that this association is fundamental to elastic fiber formation. Microfibril-associated glycoprotein-1 showed calcium-dependent binding of moderate affinity to fibrillin-1 N-terminal fragment (encoded by exons 1-8), which localize to the beads. Microfibril-associated glycoprotein-1 thus contributes to microfibril organization but may also form secondary interactions with adjacent microfibril-bound tropoelastin