An efficient expression system for recombinant human collagens will have numerous scientific and medical applications. However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity. We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methyl-otrophic yeast Pichia pastoris. These methods are based on co-expression of procollagen polypeptide chains with the α- and β-subunits of prolyl 4-hydroxylase. The triple-helical type-I, -II and -III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins. All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils. The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g/l depending on the collagen type.

This content is only available as a PDF.
You do not currently have access to this content.