title: High-level secretory expression and purification of unhydroxylated human collagen α1(III) chain in Pichia pastoris GS115.
abstract: Recombinant collagen and gelatin have been applied in biomedical materials field because of products from genetically engineered microorganisms with improved safety, traceability, reproducibility, and homogeneous quality. To obtain high-level secretory expression of single-chain full-length human collagen α1(III) chain (COL3A1) without the N and C telopeptides, the cDNA coding for the human COL3A1 gene was cloned into the secretory expression vector pPIC9K and integrated into Pichia pastoris GS115. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis of culture supernatant from the recombinant methylotrophic yeast suggested that the unhydroxylated recombinant human COL3A1 (rhCOL3A1) was secreted into the culture medium, and exhibited an apparent molecular mass of approximately 130 kDa, which is 1.4 times higher than the theoretical one. Finally, the unhydroxylated rhCOL3A1 was purified to greater than 90% purity using a four-step approach. In addition, methylthiazolydiphenyl-tetrazolium bromide experiments indicated that low concentration of rhCOL3A1 could promote Baby hamster kidney cell (BHK21) proliferation effectively. The production and purification of rhCOL3A1 described in this study offer a new method for obtaining high level of rhCOL3A1 in relatively pure form, which is suitable for biomedical materials application.
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, Shaanxi, People's Republic of China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors.forename: Linbo
authors.initials: L
authors.lastname: Li
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, Shaanxi, People's Republic of China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors.forename: Daidi
authors.initials: D
authors.lastname: Fan
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, Shaanxi, People's Republic of China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors.forename: Xiaoxuan
authors.initials: X
authors.lastname: Ma
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, Shaanxi, People's Republic of China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Deng
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, Northwest University, Shaanxi, People's Republic of China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, Xi'an, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: He
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D024061:Collagen Type III / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D006224:Cricetinae; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
chemical_list: C439971:COL3A1 protein, human; D024061:Collagen Type III; D011994:Recombinant Proteins
keywords: Pichia pastoris; purification; rhCOL3A1; secretory expression