New strategy for expression of recombinant hydroxylated human collagen α1(III) chains in Pichia pastorisGS115

Abstract Type III collagen is one of the most abundant proteins in the human body, which forms collagen fibrils and provides the stiff, resilient characteristics of many tissues. In this paper, a new method for secretory expression of recombinant hydroxylated human collagen α1( III ) chain in P ichia pastoris GS 115 was applied. The gene encoding for full‐length human collagen α1( III ) chain ( COL 3 A 1) without N ‐terminal propeptide and C ‐terminal propeptide was cloned in the p PIC 9 K expression vector. The prolyl 4‐hydroxylase ( P 4 H , EC 1.14.11.2) α‐subunit ( P 4 H α ) and β‐subunit ( P 4 H β ) genes were cloned in the same expression vector, p PICZB . Fluorogenic quantitative PCR indicates that COL 3 A 1 and P 4 H genes have been expressed in m RNA level. SDS ‐ PAGE shows that secretory expression of recombinant human collagen α1( III ) chain was successfully achieved in P . pastoris GS 115. In addition, the result of amino acids composition analysis shows that the recombinant human collagen α1( III ) chain contains hydroxyproline by coexpression with the P4H. Furthermore, liquid chromatography coupled with tandem mass spectrometry analysis demonstrates that proline residues of the recombinant human collagen α1( III ) chain were hydroxylated in the X or Y positions of G ly– X – Y triplets.