title: Co-expression of recombinant human prolyl with human collagen α1 (III) chains in two yeast systems.
abstract: In this study, we co-expressed the human prolyl 4-hydroxylases (P4H) with human collagen α1 (III) (COL3A1) in an inducible system: Pichia pastoris (pPICZB), and one constitutive system: P. pastoris (pGAPZαB). The P4H catalyses the post-translational hydroxylation of proline residues in collagen strands. Conventional protein expression system such as bacteria and yeasts, which lack endogenous P4H, are not efficient for the production of recombinant collagen. In this study, the P4H gene was constructed in pGAPZαB plasmid and pPICZB plasmid respectively. These two plasmids were transformed in P. pastoris #1 that carrying COL3A1. Colony PCR analysis and sequencing after electroporation P. pastoris GS115 showed that the target gene had inserted successfully. The results of reverse transcript-qPCR, SDS-PAGE, Western blotting and LC-MS/MS analysis of the rhCOL3A1 demonstrated that the P4H was expressed successfully. Besides, it is noted that low copy number, constitutive system was suitable for hydroxylated rhCOL3A1.
  
  
  CONCLUSIONS
  Successful co-expression of recombinant human collagen α1 (III) (rhCOL3A1) and human prolyl 4-hydroxylases (P4H) in Picha pastoris GS115, simultaneously results in the acquisition of rhCOL3A1 with hydroxylation of proline (Hyp). Further, this experiment also discusses that the high or low copy numbers and different promoters affect the Hyp degree of rhCOL3A1. Selecting more appropriate strains can express high degree Hyp of rhCOL3A1. This work will be helpful to the collagen structure study.
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Shaanxi, China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of chemical engineering, Northwest University, Xi'an, China.
authors.forename: J
authors.initials: J
authors.lastname: Xu
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Shaanxi, China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of chemical engineering, Northwest University, Xi'an, China.
authors.forename: L N
authors.initials: LN
authors.lastname: Wang
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Shaanxi, China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of chemical engineering, Northwest University, Xi'an, China.
authors.forename: C H
authors.initials: CH
authors.lastname: Zhu
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Shaanxi, China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of chemical engineering, Northwest University, Xi'an, China.
authors.forename: D D
authors.initials: DD
authors.lastname: Fan
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Shaanxi, China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of chemical engineering, Northwest University, Xi'an, China.
authors.forename: X X
authors.initials: XX
authors.lastname: Ma
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Shaanxi, China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of chemical engineering, Northwest University, Xi'an, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Mi
authors.affiliation: Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Shaanxi, China.|Shaanxi R&D Center of Biomaterials and Fermentation Engineering, School of chemical engineering, Northwest University, Xi'an, China.
authors.forename: J Y
authors.initials: JY
authors.lastname: Xing
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D024061:Collagen Type III / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006900:Hydroxylation; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011392:Proline / Q000378:metabolism; D064828:Prolyl Hydroxylases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D024061:Collagen Type III; D011994:Recombinant Proteins; D011392:Proline; D064828:Prolyl Hydroxylases
keywords: Pichia pastoris GS115; RT-qPCR; co-expression; human collagen α1 (III); pGAPZαB; pPICZB; prolyl 4-hydroxylase