Production of human type I collagen in yeast reveals unexpected new insights into the molecular assembly of collagen trimers

Substantial evidence supports the role of the procollagen C-propeptide in t he initial association of procollagen polypeptides and for triple helix for mation. To evaluate the role of the propeptide domains on triple helix form ation, human recombinant type I procollagen, pN-collagen (procollagen witho ut the C-propeptides), pC-collagen (procollagen without the N-propeptides), and collagen (minus both propeptide domains) heterotrimers were expressed in Saccharomyces cerevisiae. Deletion of the N- or C-propeptide, or both pr opeptide domains, from both pro alpha -chains resulted in correctly aligned triple helical type I collagen. Protease digestion assays demonstrated fol ding of the triple helix in the absence of the N- and C-propeptides from bo th pro alpha -chains. This result suggests that sequences required for fold ing of the triple helix are located in the helical/telopeptide domains of t he collagen molecule. Using a strain that does not contain prolyl hydroxyla se, the same folding mechanism was shown to be operative in the absence of prolyl hydroxylase, Normal collagen fibrils were generated showing the char acteristic banding pattern using this recombinant collagen. This system off ers new opportunities for the study of collagen expression and maturation.