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

Abstract Four human genes, two of them encoding the proα1 and proα2 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 proα1 and proα2 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 proα 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. Copyright © 2001 John Wiley & Sons, Ltd.