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Co‐expression of recombinant human prolyl with human collagen α 1 ( III ) chains in two yeast systems
Author(s) -
Xu J.,
Wang L.N.,
Zhu C.H.,
Fan D.D.,
Ma X.X.,
Mi Y.,
Xing J.Y.
Publication year - 2015
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12447
Subject(s) - pichia pastoris , recombinant dna , plasmid , hydroxylation , yeast , proline , electroporation , biology , microbiology and biotechnology , gene , biochemistry , chemistry , enzyme , amino acid
In this study, we co‐expressed the human prolyl 4‐hydroxylases (P4H) with human collagen α 1 ( III ) ( COL 3A1) in an inducible system: Pichia pastoris ( pPICZB ), and one constitutive system: P. pastoris ( pGAPZ α B). 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 pGAPZ α B plasmid and pPICZB plasmid respectively. These two plasmids were transformed in P. pastoris #1 that carrying COL 3A1. Colony PCR analysis and sequencing after electroporation P. pastoris GS 115 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 rh COL 3A1 demonstrated that the P4H was expressed successfully. Besides, it is noted that low copy number, constitutive system was suitable for hydroxylated rhCOL3A1. Significance and Impact of the Study Successful co‐expression of recombinant human collagen α 1 ( III ) (rh COL 3A1) and human prolyl 4‐hydroxylases (P4H) in Picha pastoris GS 115, simultaneously results in the acquisition of rh COL 3A1 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 rh COL 3A1. Selecting more appropriate strains can express high degree Hyp of rh COL 3A1. This work will be helpful to the collagen structure study.