Expression and purification of the cytoplasmic tail of an endocytic receptor by fusion to a carbohydrate-recognition domain.
Gene fusion has been used to produce the cytoplasmic domain of an endocytic receptor. DNA sequences coding for the 52 COOH-terminal amino acids of the mannose receptor from human macrophages, including the 41-amino acid cytoplasmic tail, were fused to the codons specifying the carbohydrate-recognition domain (CRD) of rat mannose-binding protein. The fusion protein was expressed in Escherichia coli and purified in one step on mannose-Sepharose, making use of the carbohydrate-binding activity of the CRD. The tail peptide was released from the fusion protein using endoproteinase Arg-C. This method provides an alternative to chemical synthesis for the production of midlength peptides.
Taylor
M E
ME
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
Drickamer
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods; D004705:Endocytosis / Q000502:physiology; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D037181:Lectins, C-Type; D008264:Macrophages / Q000737:chemistry; D008358:Mannose / Q000378:metabolism; D000090323:Mannose Receptor; D037601:Mannose-Binding Lectin; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037601:Mannose-Binding Lectin; D037241:Mannose-Binding Lectins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; C069858:mannose binding protein A; D010450:Endopeptidases; D008358:Mannose