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

Four human genes, two of them encoding the pro alpha1 and pro alpha2 chains of type I procollagen and two of them the tno types of subunit of prolyl 4-hydroxylase (4PH), were integrated into the genome of Pichia pastoris. The pro alpha1 and pro alpha2 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 pro alpha 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 21 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 1 collagen for numerous medical applications. Copyright (C) 2001 John Wiley & Sons, Ltd.