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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
Author(s) -
Vuorela Annamari,
Myllyharju Johanna,
Nissi Ritva,
Pihlajaniemi Taina,
Kivirikko Kari I.
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.22.6702
Subject(s) - tetramer , yeast , biology , university hospital , enzyme , biochemistry , medicine , surgery
Prolyl 4‐hydroxylase, the key enzyme of collagen synthesis, is an α 2 β 2 tetramer, the β subunit of which is protein disulfide isomerase (PDI). Coexpression of the human α 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 unexpectedly increased the assembly level 10‐fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4‐hydroxyproline‐containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47‐like protein. The data indicate that collagen synthesis in Pichia , and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4‐hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high‐level production of various recombinant collagens for numerous scientific and medical purposes.