High-level production of human type I collagen in the yeast Pichia pastoris

Yeast. 2001 Jun 30;18(9):797-806. doi: 10.1002/yea.730.

Abstract

Four human genes, two of them encoding the proalpha1 and proalpha2 chains of type I procollagen and two of them the two types of subunit of prolyl 4-hydroxylase (4-PH), were integrated into the genome of Pichia pastoris. The proalpha1 and proalpha2 chains expressed formed type I procollagen molecules with the correct 2:1 chain ratio, and the 4-PH subunits formed an active enzyme tetramer that fully hydroxylated the proalpha chains. Chains lacking their N but not C propeptides formed pCcollagen molecules with the 2:1 chain ratio and, surprisingly, the expression levels of pCcollagen were 1.5-3-fold relative to those of procollagen. Both types of molecule could be converted by pepsin treatment to collagen molecules that formed native-type fibrils in vitro. The expression levels obtained for the pCcollagen using only single copies of each of the four genes and a 2 l fermenter ranged up to 0.5 g/l, indicating that it should be possible to optimize this system for high-level production of recombinant human type I collagen for numerous medical applications.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acids / analysis
  • Bioreactors
  • Biotechnology / methods
  • Collagen Type I / biosynthesis*
  • Collagen Type I / chemistry
  • Collagen Type I / genetics*
  • Collagen Type I / ultrastructure
  • Gene Expression
  • Genetic Vectors / genetics
  • Humans
  • Pichia / genetics*
  • Procollagen / biosynthesis
  • Procollagen / chemistry
  • Procollagen / genetics
  • Procollagen-Proline Dioxygenase / biosynthesis
  • Procollagen-Proline Dioxygenase / chemistry
  • Procollagen-Proline Dioxygenase / genetics
  • Protein Folding
  • Protein Structure, Quaternary
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / ultrastructure

Substances

  • Amino Acids
  • Collagen Type I
  • Procollagen
  • Recombinant Fusion Proteins
  • Procollagen-Proline Dioxygenase