Expression of recombinant human type I-III collagens in the yeast Pichia pastoris

An efficient expression system for recombinant human collagens will have nu merous scientific and medical applications. However, most recombinant syste ms are unsuitable for this purpose, as they do not have sufficient prolyl 4 -hydroxylase activity. We have developed methods for producing the three ma jor fibril-forming human collagens, types I, II and III, in the methylotrop hic yeast Pichia pastoris. These methods are based on co-expression of proc ollagen polypeptide chains with the alpha- and beta-subunits of prolyl 4-hy droxylase. The triple-helical type-I, -II and -III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast c ells and could be purified from the cell lysates by a procedure that includ ed a pepsin treatment to convert the procollagens into collagens and to dig est most of the non-collagenous proteins. All the purified recombinant coll agens were identical in 4-hydroxyproline content with the corresponding non -recombinant human proteins, and all the recombinant collagens formed nativ e-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.