ASSEMBLY OF HUMAN PROLYL 4-HYDROXYLASE AND TYPE-III COLLAGEN IN THE YEAST PICHIA-PASTORIS - FORMATION OF A STABLE ENZYME TETRAMER REQUIRES COEXPRESSION WITH COLLAGEN AND ASSEMBLY OF A STABLE COLLAGEN REQUIRES COEXPRESSION WITH PROLYL 4-HYDROXYLASE

Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alph a(2) beta(2) tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human a subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI, Coexpression with human type III procollagen unexpect edly increased the assembly level 10-fold, with no increase in the tot al amounts of the subunits, The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pi chia must have a system for transporting all the cosubstrates needed b y the enzyme into the lumen of the endoplasmic reticulum, The 4-hydrox yproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia pr obably has no Hsp47-like protein, The data indicate that collagen synt hesis in Pichia, and probably also in other cells, involves a highly u nusual control mechanism, in that production of a stable prolyl 4-hydr oxylase requires collagen expression while assembly of a stable collag en requires enzyme expression, This Pichia system seems idea! for the high-level production of various recombinant collagens for numerous sc ientific and medical purposes.