Collagen from marine sources is a very attractive alternative to bovine collagen. In biomedicine, several applications of collagens derived from sponges have been described, many of which from the demospongia Chondrosia reniformis. The production of these molecules in recombinant form would allow to obtain well-defined molecular types and to limit the expensive procedures of sample recovery and purification. Here we report the realization of a yeast strain able to produce hydroxylated collagen polypeptides derived from C. reniformis. First the cDNAs coding for the and subunits of the sponge enzyme prolyl-4-hydroxylase (P were identified by PCR approach, then they were cloned in pPink and in pPIC6 vectors respectively and finally they were stably integrated in the genome of the methanotrophic yeast Pichia pastoris. The strain with the highest P4H activity was then transformed with a third expression vector (pPICZ) containing the coding region of a sponge collagen polypeptide in frame with an Histidine-TAG at the 3’-end. Recombinant protein expression was then induced and collagen polypeptides were purified by affinity chromatography and analysed by SDS-PAGE followed by Mass Spectrometry, in order to confirm the sequence and the presence of hydroxy-proline residues. The biocompatibility of the recombinant proteins was evaluated by MTT test in human neonatal fibroblasts and furthermore, tissue culture plates were treated with 10 g/ml recombinant protein solution to test its cell adhesion properties. In conclusion, a first recombinant hydroxylated marine collagen polypeptide was produced in the Pichia system, resulting biocompatible and suggesting its use as porcine gelatin substitute for cell culture. Research supported by EU (FP7 grant agreement n: 266033 SPonge Enzyme End Cell for Innovative AppLication-SPECIAL)
Recombinant production of hydroxylated collagen polypeptides derived from the Demospongia C. reniformis: a new biotechnological source of marine collagen
Marina Pozzolini;Sonia Scarfì;Annalisa Salis;Gianluca Damonte;Marco Giovine
2017-01-01
Abstract
Collagen from marine sources is a very attractive alternative to bovine collagen. In biomedicine, several applications of collagens derived from sponges have been described, many of which from the demospongia Chondrosia reniformis. The production of these molecules in recombinant form would allow to obtain well-defined molecular types and to limit the expensive procedures of sample recovery and purification. Here we report the realization of a yeast strain able to produce hydroxylated collagen polypeptides derived from C. reniformis. First the cDNAs coding for the and subunits of the sponge enzyme prolyl-4-hydroxylase (P were identified by PCR approach, then they were cloned in pPink and in pPIC6 vectors respectively and finally they were stably integrated in the genome of the methanotrophic yeast Pichia pastoris. The strain with the highest P4H activity was then transformed with a third expression vector (pPICZ) containing the coding region of a sponge collagen polypeptide in frame with an Histidine-TAG at the 3’-end. Recombinant protein expression was then induced and collagen polypeptides were purified by affinity chromatography and analysed by SDS-PAGE followed by Mass Spectrometry, in order to confirm the sequence and the presence of hydroxy-proline residues. The biocompatibility of the recombinant proteins was evaluated by MTT test in human neonatal fibroblasts and furthermore, tissue culture plates were treated with 10 g/ml recombinant protein solution to test its cell adhesion properties. In conclusion, a first recombinant hydroxylated marine collagen polypeptide was produced in the Pichia system, resulting biocompatible and suggesting its use as porcine gelatin substitute for cell culture. Research supported by EU (FP7 grant agreement n: 266033 SPonge Enzyme End Cell for Innovative AppLication-SPECIAL)I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.