title: New strategy for expression of recombinant hydroxylated human collagen α1(III) chains in Pichia pastoris GS115.
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 Pichia pastoris GS115 was applied. The gene encoding for full-length human collagen α1(III) chain (COL3A1) without N-terminal propeptide and C-terminal propeptide was cloned in the pPIC9K expression vector. The prolyl 4-hydroxylase (P4H, EC 1.14.11.2) α-subunit (P4Hα) and β-subunit (P4Hβ) genes were cloned in the same expression vector, pPICZB. Fluorogenic quantitative PCR indicates that COL3A1 and P4H genes have been expressed in mRNA level. SDS-PAGE shows that secretory expression of recombinant human collagen α1(III) chain was successfully achieved in P. pastoris GS115. 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 Gly-X-Y triplets.
authors: Shaanxi R&D Centre of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors: Jing
authors: J
authors: He
authors: Shaanxi R&D Centre of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors: Xiaoxuan
authors: X
authors: Ma
authors: Shaanxi R&D Centre of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors: Fenglong
authors: F
authors: Zhang
authors: Shaanxi R&D Centre of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors: Linbo
authors: L
authors: Li
authors: Shaanxi R&D Centre of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors: Jianjun
authors: J
authors: Deng
authors: Shaanxi R&D Centre of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors: Wenjiao
authors: W
authors: Xue
authors: Shaanxi R&D Centre of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors: Chenhui
authors: C
authors: Zhu
authors: Shaanxi R&D Centre of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors: Daidi
authors: D
authors: Fan
mesh_terms: D003001:Cloning, Molecular; D024061:Collagen Type III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006900:Hydroxylation; D006909:Hydroxyproline / Q000032:analysis / Q000737:chemistry; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C439971:COL3A1 protein, human; D024061:Collagen Type III; D011994:Recombinant Proteins; D006909:Hydroxyproline
keywords: 4-hydroxyproline; Pichia pastoris GS115; coexpression; human collagen α1(III)chains; hydroxylation; prolyl 4-hydroxylase