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 alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human alpha 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.
Vuorela
A
A
Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu, Finland.
Myllyharju
J
J
Nissi
R
R
Pihlajaniemi
T
T
Kivirikko
K I
KI
D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase; D019704:Protein Disulfide-Isomerases