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

Publication ,  Journal Article
Krol, BJ; Murad, S; Walker, LC; Marshall, MK; Clark, WL; Pinnell, SR; Yeowell, HN
Published in: J Invest Dermatol
January 1996

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.

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Published In

J Invest Dermatol

DOI

ISSN

0022-202X

Publication Date

January 1996

Volume

106

Issue

1

Start / End Page

11 / 16

Location

United States

Related Subject Headings

  • Sequence Analysis
  • Recombinant Proteins
  • Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase
  • Molecular Sequence Data
  • Molecular Probes
  • Kinetics
  • Humans
  • Gene Amplification
  • Dermatology & Venereal Diseases
  • DNA, Complementary
 

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Krol, B. J., Murad, S., Walker, L. C., Marshall, M. K., Clark, W. L., Pinnell, S. R., & Yeowell, H. N. (1996). The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system. J Invest Dermatol, 106(1), 11–16. https://doi.org/10.1111/1523-1747.ep12326956
Krol, B. J., S. Murad, L. C. Walker, M. K. Marshall, W. L. Clark, S. R. Pinnell, and H. N. Yeowell. “The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system.J Invest Dermatol 106, no. 1 (January 1996): 11–16. https://doi.org/10.1111/1523-1747.ep12326956.
Krol BJ, Murad S, Walker LC, Marshall MK, Clark WL, Pinnell SR, et al. The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system. J Invest Dermatol. 1996 Jan;106(1):11–6.
Krol, B. J., et al. “The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system.J Invest Dermatol, vol. 106, no. 1, Jan. 1996, pp. 11–16. Pubmed, doi:10.1111/1523-1747.ep12326956.
Krol BJ, Murad S, Walker LC, Marshall MK, Clark WL, Pinnell SR, Yeowell HN. The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system. J Invest Dermatol. 1996 Jan;106(1):11–16.
Journal cover image

Published In

J Invest Dermatol

DOI

ISSN

0022-202X

Publication Date

January 1996

Volume

106

Issue

1

Start / End Page

11 / 16

Location

United States

Related Subject Headings

  • Sequence Analysis
  • Recombinant Proteins
  • Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase
  • Molecular Sequence Data
  • Molecular Probes
  • Kinetics
  • Humans
  • Gene Amplification
  • Dermatology & Venereal Diseases
  • DNA, Complementary