High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli.
The collagen prolyl 4-hydroxylases (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, play a central role in the synthesis of all collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers in which the two catalytic sites are located in the alpha subunits, and protein disulfide isomerase serves as the beta subunit. All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful, but assembly of a recombinant enzyme has been reported in several cell types by coexpression of the two types of subunit. An active type I C-P4H tetramer was obtained here by periplasmic expression in Escherichia coli strains BL21 and RB791. Further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human type I C-P4H tetramer. The specific activity of the C-P4H tetramer purified from the cytoplasmic expression was within the range of values reported for human type I C-P4H isolated as a nonrecombinant enzyme or produced in the endoplasmic reticulum of insect cells, but the expression level, about 25 mg/l in a fermenter, is about 5-10 times that obtained in insect cells. The enzyme expressed in E. coli differed from those present in vivo and those produced in other hosts in that it lacked the N glycosylation of its alpha subunits, which may be advantageous in crystallization experiments.
Neubauer
Antje
A
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, P. O. Box 5000, University of Oulu, FIN-90014 Oulu, Finland.
Neubauer
Peter
P
Myllyharju
Johanna
J
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D020134:Catalytic Domain; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007313:Insecta; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; D010455:Peptides; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase; D019704:Protein Disulfide-Isomerases