THE EXPRESSION OF A FUNCTIONAL, SECRETED HUMAN LYSYL HYDROXYLASE IN ABACULOVIRUS SYSTEM

This study reports the expression of functional human lysyl hydroxylas e (LH), a post-translational modifying enzyme that catalyzes the hydro xylation of the lysine residues essential for cross-linking in collage n biosynthesis. We have developed a novel baculovirus system for the e xpression of LH, a protein that exists normally within the lumen of th e 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 wit h supernatant concentration, whereas there was no detectable LH activi ty 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) t hat was most prominent in those fractions with the highest LH activity . N-terminal amino acid sequencing verified that the N-terminal primar y structure of this 85-kDa protein was identical to human LH. Moreover , the activity of the expressed protein was shown to be dependent on t he presence of Fe++, ascorbate, and cu-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 sep aration from the intracellular proteins of insect cells. Future applic ations should allow characterization of the LH active site by crystall ographic studies and site-directed mutagenesis for structure-function comparison.