Journal of Biotechnology 208 (2015) 28–36 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen Marina Pozzolini a , Sonia Scarfì a,∗ , Francesca Mussino b , Annalisa Salis a,c , Gianluca Damonte b,c , Umberto Benatti b,c , Marco Giovine a,c a Department of Earth, Environment and Life Sciences, University of Genova, Via Pastore 3, 16132, Genova, Italy b Department of Experimental Medicine, Section of Biochemistry, Viale Benedetto XV, 1, 16132, Genova, Italy c Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 5, 16132, Genova, Italy article info Article history: Received 13 February 2015 Received in revised form 12 May 2015 Accepted 15 May 2015 Available online 27 May 2015 Keywords: Collagen P4H Pichia pastoris Chondrosia reniformis PDI abstract Prolyl 4-hydroxylase (P4H) is a ˛ 2 ˇ 2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to gener- ate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens. Here we report on the production and selection, by enzymatic and biomolecular analyses, of a triple transformed Pichia pastoris strain expressing a stable P4H tetramer derived from C. reniformis sponge and a hydroxylated non fibrillar procollagen polypeptide from the same animal. The percentage of recom- binant procollagen hydroxylated prolines inside the transformed yeast was of 36.3% analyzed by mass spectrometry indicating that the recombinant enzyme is active on its natural substrate inside the yeast cell host. Furthermore, the recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions in the Xaa-Yaa-Gly collagenous triplets. In conclusion this Pichia system seems ideal for high-level production of hydroxylated sponge- or marine-derived collagen polypeptides as well as of conotoxins or other marine proteins of high pharma- cological interest needing this particular post-translational modification. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Prolyl 4-hydroxylase (P4H) is a key enzyme of the collagen biosynthesis catalyzing the hydroxylation of proline residues in - Xaa-Pro-Gly-sequences typical of proteins with collagen domains (for review, see Kivirikko et al., 1989; Kivirikko and Myllyharju, 1998; Myllyharju, 2003). The synthesis of 4-hydroxyproline residues is essential to the folding of the newly synthetized chains into the triple helices of collagens. In vertebrates, this enzyme is a ˛ 2 ˇ 2 tetramer, where the ˛ subunits contain the catalytic active site for the proline hydroxylation, while the ˇ subunits, that are identical to protein disulfide-isomerase (PDI), act to maintain the ˛ subunits in a soluble form and to retain the enzyme in the ER lumen (Kivirikko et al., 1989). The pivotal role of this enzyme in collagen ∗ Corresponding author. Tel.: +39 010 35338227. E-mail address: soniascarfi@unige.it (S. Scarfì). metabolism makes it a potential specific target for pharmacological regulation of fibrotic diseases and, for this reason, several studies have been carried out in order to elucidate the details of the hydrox- ylation mechanism (Myllyharju and Kivirikko, 1997; Lamberg et al., 1995). Attempts to assemble an active P4H tetramer in vitro from its individual ˛ and ˇ subunits have been unsuccessful (Koivu and Myllylä, 1986; Nietfeld et al., 1981). Human P4H was produced in recombinant form in insect cells by baculovirus vectors (Vuori et al., 1992) and more recently in E. coli (Kersteen et al., 2004). The recombinant production of a stable P4H tetramer proves to be of great significance not only to perform accurate structure/function analyses but also to promote further studies on the enzymatic activ- ity mechanism by site-specific mutagenesis. Last but not least, this protein is considered a fundamental biotechnological tool for the production of recombinant proteins needing prolyl-hydroxylation in their post-translational modifications as collagen. Due to its several industrial applications, the recombinant pro- duction of collagen and all its derivatives as gelatins has been http://dx.doi.org/10.1016/j.jbiotec.2015.05.007 0168-1656/© 2015 Elsevier B.V. All rights reserved.