Co-expression of recombinant human collagen α1(III) chain with viral prolyl 4-hydroxylase in Pichia pastoris GS115

Protein Expr Purif. 2023 Jan:201:106184. doi: 10.1016/j.pep.2022.106184. Epub 2022 Oct 1.

Abstract

The Collagen α1(Ш) chain (COL3A1) is an important structural protein on the surface of human skin. The activity of prolyl 4-hydroxylase (P4H) is crucial to maintaining the stable triple-helix structure and function of human COL3A1. To obtain hydroxylated human COL3A1, virus-derived P4H A085R was co-expressed with human COL3A1 in Pichia pastoris GS115. Colony PCR analysis and sequencing after transfection confirmed that the target gene was successfully inserted. Quantitative reverse transcription PCR (RT-qPCR) indicated that human COL3A1 and P4H A085R were expressed at mRNA levels in the clones. SDS-PAGE and Western blot analysis of supernatant from the recombinant methylotrophic yeast culture showed that recombinant human COL3A1 (rhCOL3A1) was secreted into the culture medium with an apparent molecular mass of approximately 130 kDa. It was observed that the amount of secreted rhCOL3A1 was highest at 120 h after induction. Furthermore, mass spectrometry analysis demonstrated that rhCOL3A1 was successfully expressed in P. pastoris. The His-tagged rhCOL3A1 protein was purified by Ni-affinity column chromatography.

Keywords: Hydroxylation; Purification; Recombinant human collagen α1(Ш) chain; Virus prolyl 4-hydroxylase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Collagen / metabolism
  • Collagen Type III / genetics
  • Collagen Type III / metabolism
  • Humans
  • Pichia* / genetics
  • Pichia* / metabolism
  • Prolyl Hydroxylases* / chemistry
  • Prolyl Hydroxylases* / genetics
  • RNA, Messenger / metabolism
  • Recombinant Proteins / chemistry
  • Saccharomycetales

Substances

  • Collagen Type III
  • RNA, Messenger
  • Recombinant Proteins
  • Collagen
  • Prolyl Hydroxylases

Supplementary concepts

  • Komagataella pastoris