Expression, purification, crystallization and crystallographic characterization of dimeric and monomeric human neutrophil gelatinase associated lipocalin (NGAL)

Abstract

Crystals of the monomeric and dimeric forms of human neutrophil gelatinase associated lipocalin have been grown in hanging-drop vapor-diffusion trials using PEG as a precipitating agent with recombinant protein expressed in a baculovirusbased system. Crystals of monomeric NGAL belong to the cubic space group P432 with lattice constants a = b = c = 126.6 A; crystals of dimeric NGAL belong to the tetragonal space group P4~2~2 (or its enantiomorph P432~2) with lattice constants a = b = 54.14 and c = 121.56 A,. Isomorphous crystals of the NGAL dimer can be grown in the presence of ligand: the tripeptide N-formyl-Met-Leu-Phe.

The endocytic receptor megalin binds the iron transporting neutrophil‐gelatinase‐associated lipocalin with high affinity and mediates its cellular uptake

“…Recombinant human NGAL was generated either as apo-NGAL, i.e., without bacterial siderophore, in Sf9 insect cells using the Baculovirus technique as previously described [24], or as NGAL with E. coli siderophore as detailed in [15,25], with the modification that 50 ml overnight culture of E. coli was added to 500 ml LB medium and incubated for 90 min. Then, 0.1 mM IPTG and 0.1 mg/ml desferoxamine (Desferal, Roche) were added to induce bacterial growth (IPTG) and secure iron deprivation and enterobactin production by the bacteria.…”

Neutrophil Gelatinase-associated Lipocalin, a Siderophore-binding Eukaryotic Protein

“…The clue to its direct function came from crystallography carried out by Dr. Roland K. Strong and collaborators who noticed that recombinant NGAL generated by E. coli was deeply red in color in contrast to recombinant NGAL generated by insect cells. It was shown that this red color was due to an iron molecule that was trapped in the siderophore of E.coli, enterochelin, indicating that NGAL is a siderophore binding protein (Strong et al 1998;Goetz et al 2000). Many such are of course known that mediate the uptake of siderophores in bacteria (Neilands 1995), but no eukaryotic, let alone mammalian protein with siderophore binding properties was known.…”

Lipocalin 2 Diminishes Invasiveness and Metastasis of Ras-transformed Cells

“…Crystallography showed that the protein is a carrier of iron bound to a siderophore (15), which is a small organic molecule produced by bacteria (20). Both recombinant and mammalian-expressed lipocalin 2 (16) induce the de novo expression of E-cadherin, the formation of polarized epithelia, and the development of tubules in embryonic mesenchyme in an iron-dependent fashion (17).…”