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Crystallization of recombinant human heme oxygenase‐1
Author(s) -
Schuller David J.,
Poulos Thomas L.,
Wilks Angela,
Montellano Paul Ortiz De
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070820
Subject(s) - crystallization , recombinant dna , heme oxygenase , heme , chemistry , oxygenase , biochemistry , enzyme , organic chemistry , gene
Heme oxygenase catalyzes the NADPH, 0 2 , and cytochrome P450 reductase dependent oxidation of heme to biliverdin and carbon monoxide. One of two primary isozymes, HO‐1, is anchored to the endoplasmic reticulum membrane via a stretch of hydrophobic residues at the C‐terminus. While full‐length human HO‐1 consists of 288 residues, a truncated version with residues 1‐265 has been expressed as a soluble active enzyme in Escherichia coli. The recombinant enzyme crystallized from ammonium sulfate solutions but the crystals were not of sufficient quality for diffraction studies. SDS gel analysis indicated that the protein had undergone proteolytic degradation. An increase in the use of protease inhibitors during purification eliminated proteolysis, but the intact protein did not crystallize. N‐terminal sequencing and mass spectral analysis of dissolved crystals indicated that the protein had degraded to two major species consisting of residues 1‐226 and 1‐237. Expression of the 1‐226 and 1‐233 versions of human HO‐1 provided active enzyme that crystallizes in a form suitable for diffraction studies. These crystals belong to space group P2 1 , with unit cell dimensions a = 79.3 Å, b = 56.3 Å, c = 112.8 Å, and β = 101.5°.