PMID: 8592059Jan 1, 1996Paper

The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system

The Journal of Investigative Dermatology
B J KrolH N Yeowell

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, ...Continue Reading

Citations

Sep 2, 1998·Proceedings of the National Academy of Sciences of the United States of America·K PassojaK I Kivirikko
Aug 10, 2000·The Journal of Biological Chemistry·J HeikkinenR Myllylä
Jan 20, 1999·Proceedings of the Association of American Physicians·H N Yeowell, L C Walker
Jan 15, 2021·International Journal of Molecular Sciences·Zdeněk KejíkMilan Jakubek
Jun 18, 1999·Matrix Biology : Journal of the International Society for Matrix Biology·H N Yeowell, L C Walker
Apr 3, 1998·Matrix Biology : Journal of the International Society for Matrix Biology·K I Kivirikko
Jul 25, 2003·Biochemical and Biophysical Research Communications·Derry K MercerSimon P Robins

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