A recombinant polypeptide, composed of the alpha-helical neck region and the carbohydrate recognition domain of conglutinin, self-associates to give a functionally intact homotrimer.
A recombinant polypeptide composed of the alpha-helical neck region and carbohydrate recognition domain (CRD) of bovine conglutinin was expressed in Escherichia coli. The recombinant protein formed inclusion bodies but could be solubilised using a denaturation-renaturation cycle based on urea and then purified by affinity chromatography on a TSK-N-acetylglucosamide column. The purified product behaved as a homotrimer in non-dissociating conditions, with three CRDs held together by the alpha-helical neck regions. The trimer, although lacking the N-terminal and collagen regions of the native conglutinin, showed the same binding carbohydrate specificities as the native molecule, for the complement fragment C3b and for lipopolysaccharides derived from Gram-negative bacteria.
Wang
J Y
JY
MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, UK.
Kishore
U
U
Reid
K B
KB
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002847:Chromatography, Agarose; D037222:Collectins; D003179:Complement C3b / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D037102:Lectins / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012712:Serum Globulins / Q000235:genetics / Q000378:metabolism*
D037222:Collectins; D037102:Lectins; D008070:Lipopolysaccharides; D009005:Monosaccharides; D010455:Peptides; D011994:Recombinant Proteins; D012712:Serum Globulins; C024014:conglutinin; D003179:Complement C3b; D004492:Edetic Acid; D002118:Calcium