Characterization of a functional hyaluronan-binding domain from the human CD44 molecule expressed in Escherichia coli.
The CD44 molecule is a widely distributed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan. The ligand-binding site which is located in the membrane distal portion of the molecule encompasses a region of approximately 100 amino acids termed the Link domain, a structural unit that is conserved among members of the Hyaladherin superfamily which includes cartilage link protein, aggrecan, and tumor necrosis factor-stimulated gene-6 (TSG-6). In contrast to these other Hyaladherins, however, the ligand-binding domain of CD44 appears to extend beyond the Link domain to involve additional basic residues located toward the membrane proximal region. Furthermore, recent molecular modeling studies indicate that within the CD44 Link domain itself, the spatial arrangement of critical residues involved in HA binding is likely to differ significantly from the prototypic TSG-6 Link module. In order to obtain material to solve the CD44 solution structure we have developed an optimized method for the expression and purification of functionally active CD44 ectodomains encompassing both the Link module and the additional downstream HA-binding residues in Escherichia coli. Here we describe the details of the method which involves solubilization of recombinant CD44 from inclusion bodies in 8 M urea, followed by refolding and purification of intact monomers using size-exclusion and reverse-phase chromatography. We show the method yields CD44 molecules that (1) retain reactivity with a panel of conformation-sensitive antibodies, (2) possess similar hyaluronan-binding characteristics to authentically folded CD44 molecules expressed in eukaryotic cells, and (3) display one-dimensional NMR spectra that indicate the presence of a single conformational species. This method should enable sufficient amounts of functional CD44 Link module to be produced for comprehensive structural analyses by multidimensional NMR spectroscopy.
Banerji
S
S
Molecular Immunology Group, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 9DU, United Kingdom. sbanerji@worf.molbiol.ox.ac.uk
Day
A J
AJ
Kahmann
J D
JD
Jackson
D G
DG
D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D002479:Inclusion Bodies / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D018960:Hyaluronan Receptors; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D006820:Hyaluronic Acid