Yeast strain expressing C. reniformis prolyl hydroxylase for recombinant marine collagen production

Description

There is an increasing demand for alternative sources of collagens, which are needed for a wide variety of applications (food, cosmetics, healthcare). Sponge collagens have unique physico-chemical properties and as a consequence are a promising resource, but sponge collagens are not available in large quantities. A biotechnology-based approach for a sustainable production of this family of proteins is here shown. The expression system consists of a yeast strain co-transformed with three different expression vectors containing the following coding sequences identified and cloned from Chondrosia reniformis marine sponge: i) the alpha subunit of C. reniformis enzyme prolyl-4-hydroxylase (P4H), equipped with its signal peptide for endoplasmic reticulum localization, ii) the coding sequence of the beta subunit of the same enzyme (Protein disulphite Isomerase, PDI) whose signal peptide has been replaced with that of S. cerevisiae AlfaMating factor (AM) ensuring a better solubility and activity of the quaternary structure of the reconstituted enzyme in the yeast, and iii) the coding sequence of a non-fibrillar collagen from C. reniformis resembling, from homology sequence analysis, mammalian collagen type IV. The yeast strain used is defective for a gene related to the synthesis of adenine (ade2), while the first vector, carrying the P4H sequence, contains the ADE2 gene allowing the transformed yeast to grow on a medium lacking the purine . In turn, the other two expression vectors are equipped with markers for antibiotic resistance such as blasticidin and zeocin for the PDI expression vector and the collagen expression vector, respectively. Co-transformed yeast strains were finally isolated and cloned in plates containing a semi-solid medium lacking adenine and in the presence of both antibiotics. The enzymatic activity of P4H was then assessed by a specific assay on the microsomal fraction of the co-transformed strain. Presence of the recombinant collagen was subsequently confirmed both in the yeast extract and in the cell culture medium. In particular protein expression was assessed by mass spectrometry analysis of triptic peptides derived from digestion of a protein extracted from SDS-PAGE gel with a molecular weight corresponding to that of C. reniformis collagen single chain and found specifically only in the tripletransformed yeast strain.

Additional details