br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion Prolyl 4-hydroxylases are oxygenases with key roles in a variety of biological processes including oxygen sensing, siRNA regulation and collagen folding (Gorres and Raines, 2010). Hydroxyproline is particularly abundant in collagenous proteins which present a characteristic helical region composed by the repetition of Xaa-Yaa-Gly sequences, where X and Y frequently are proline and hydroxyproline (Vanderrest and Garrone, 1991). Collagen is widely used in a plethora of industrial and medical applications in the form of animal tissue extracts (Lee et al., 2001). Since these products are not well characterized at the molecular level and may be contaminated with potential immunogenic and infective agents (Lee et al., 2001, Moon et al., 2014), the recombinant technology has been suggested and developed at various degrees of success, to obtain high quality and animal derived contaminant-free collagens (Olsen et al., 2003, Ritala et al., 2008, Vuorela et al., 1997, Xu et al., 2011). In the last years, an increasing interest in sponge collagen production for biomedical and cosmetic purposes has been developed (Nicklas et al., 2009a, Nicklas et al., 2009b, Swatschek et al., 2002), but until now, its recombinant production was hampered by lack of biomolecular information on the collagenous genes and the Fmoc-D-Lys(Boc)-OH involved in their biosynthesis. However, the recent identification and cloning of the first C. reniformis collagen gene (Pozzolini et al., 2012), as well as of an enzyme involved in its post-translational modifications (Pozzolini et al., 2015), has definitely laid the foundations for the setup of sponge collagen large scale production by recombinant technology. The present paper demonstrates that with the optimal combination of expression vectors it\'s possible to integrate the three C. reniformis genes responsible for sponge collagen synthesis (ColCH) and post-translational modifications (α and β P4H) into the P. pastoris genome using a commercial strain without resorting to particular genetic manipulations. Usually the available commercial yeast strains are designed to produce a single recombinant polypeptide at once, typically such yeast strains are defective in a gene coding for enzymes involved in fundamental metabolic pathways and are transformed with expression vectors that can restore this defect. In order to obtain a P. pastoris strain able to produce a stable sponge P4H tetramer, the cloning strategy used was a co-transformation of the commercial Pichia Pink strain with ade+ genotype with the pPink expression vector containing the sponge αP4H gene together with an αpPIC6 vector containing the sponge PDI gene (Scheme 1). The co-transformed strains were then easily selected on blasticidin agar plates lacking adenine. In the αP4H cDNA we maintained the original ER retention signal peptide sequence while in the PDI cDNA the same was replaced with the S. cerevisiae alpha mating factor pre-pro sequence as it had been already reported that this signal peptide gave the highest amount of P4H active tetramer in a previous work contemplating recombinant expression of human α and βP4H in P. pastoris (Vuorela et al., 1997).