title: Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen.
abstract: Prolyl 4-hydroxylase (P4H) is a α2β2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate 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 recombinant 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 pharmacological interest needing this particular post-translational modification.
authors.affiliation: Department of Earth, Environment and Life Sciences, University of Genova, Via Pastore 3, 16132, Genova, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Pozzolini
authors.affiliation: Department of Earth, Environment and Life Sciences, University of Genova, Via Pastore 3, 16132, Genova, Italy. Electronic address: soniascarfi@unige.it.
authors.forename: Sonia
authors.initials: S
authors.lastname: Scarfì
authors.affiliation: Department of Experimental Medicine, Section of Biochemistry, Viale Benedetto XV, 1, 16132, Genova, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Mussino
authors.affiliation: Department of Earth, Environment and Life Sciences, University of Genova, Via Pastore 3, 16132, Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 5, 16132, Genova, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Salis
authors.affiliation: Department of Experimental Medicine, Section of Biochemistry, Viale Benedetto XV, 1, 16132, Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 5, 16132, Genova, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Damonte
authors.affiliation: Department of Experimental Medicine, Section of Biochemistry, Viale Benedetto XV, 1, 16132, Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 5, 16132, Genova, Italy.
authors.forename: Umberto
authors.initials: U
authors.lastname: Benatti
authors.affiliation: Department of Earth, Environment and Life Sciences, University of Genova, Via Pastore 3, 16132, Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 5, 16132, Genova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Giovine
mesh_terms: D000818:Animals; D059001:Aquatic Organisms / Q000201:enzymology / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D002417:Cattle; D003094:Collagen / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011161:Porifera / Q000201:enzymology / Q000235:genetics; D064828:Prolyl Hydroxylases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D003094:Collagen; D064828:Prolyl Hydroxylases
keywords: Chondrosia reniformis; Collagen; P4H; PDI; Pichia pastoris