title: The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system.
abstract: This study reports the expression of functional human lysyl hydroxylase (LH), a post-translational modifying enzyme that catalyzes the hydroxylation of the lysine residues essential for cross-linking in collagen biosynthesis. We have developed a novel baculovirus system for the expression of LH, a protein that exists normally within the lumen of the endoplasmic reticulum, using a powerful baculovirus signal sequence for secretion. The supernatant from Sf9 cells infected with the viral recombinant showed significant LH activity that increased linearly with supernatant concentration, whereas there was no detectable LH activity in the cell pellet. Silver staining of the fractions purified from the active supernatant by concanavalin A Sepharose chromatography and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated an 85-kDa protein (the expected size of the LH subunit) that was most prominent in those fractions with the highest LH activity. N-terminal amino acid sequencing verified that the N-terminal primary structure of this 85-kDa protein was identical to human LH. Moreover, the activity of the expressed protein was shown to be dependent on the presence of Fe++, ascorbate, and alpha-ketoglutarate, three essential cofactors for LH activity. We have therefore successfully developed a novel expression system that produces functional human LH and enables this normally nonsecretory enzyme to be secreted, facilitating its separation from the intracellular proteins of insect cells. Future applications should allow characterization of the LH active site by crystallographic studies and site-directed mutagenesis for structure-function comparison.
authors.affiliation: Division of Dermatology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: B J
authors.initials: BJ
authors.lastname: Krol
authors.forename: S
authors.initials: S
authors.lastname: Murad
authors.forename: L C
authors.initials: LC
authors.lastname: Walker
authors.forename: M K
authors.initials: MK
authors.lastname: Marshall
authors.forename: W L
authors.initials: WL
authors.lastname: Clark
authors.forename: S R
authors.initials: SR
authors.lastname: Pinnell
authors.forename: H N
authors.initials: HN
authors.lastname: Yeowell
mesh_terms: D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000201:enzymology*; D003470:Culture Media / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D005784:Gene Amplification; D006801:Humans; D007700:Kinetics; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis
chemical_list: D003470:Culture Media; D018076:DNA, Complementary; D015335:Molecular Probes; D011994:Recombinant Proteins; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase