Expression and characterization of a single recombinant proteoglycan tandem repeat domain of link protein that binds zinc and hyaluronate.
The first proteoglycan tandem repeat (PTR) of bovine link protein has been cloned in the pMAL-c vector and overexpressed in fusion with maltose-binding protein (MBP) in Escherichia coli. The fusion protein can be isolated from the soluble phase of the bacterial lysate by amylose affinity chromatography. The PTR domain can be cleaved from the MBP domain with factor Xa protease. Evidence using zinc affinity chromatography is presented which indicates that at least one of the zinc-binding sites of bovine link protein is contained within the first PTR domain. Zinc affinity chromatography was then incorporated as the final purification step of the MBP/PTR protein. Evidence for the binding of MBP/PTR to hyaluronic acid (HA) is demonstrated by coprecipitation with HA using cetylpyridinium chloride. Binding is specific since MBP/PTR does not coprecipitate with chondroitin sulfate. Binding is also demonstrated in an ELISA assay on HA-coated plates. In this assay, binding could be inhibited by the addition of HA or HA oligosaccharides.
Varelas
J B
JB
Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4946, USA.
Kollar
J
J
Huynh
T D
TD
Hering
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D015870:Gene Expression; D006820:Hyaluronic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D015032:Zinc / Q000378:metabolism*; D000099249:Proteoglycan Link Protein
D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid; D011506:Proteins; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015032:Zinc; D000099249:Proteoglycan Link Protein