Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin.
Saxiphilin is a plasma protein from the bullfrog (Rana catesbiana) that binds saxitoxin (STX), a causative agent of paralytic shellfish poisoning. Saxiphilin is homologous to transferrin and consists of two internally homologous domains called the N-lobe and the C-lobe. STX binds to a single site in the C-lobe of saxiphilin. In this study, cloned genes coding for recombinant saxiphilin and C-lobe saxiphilin were modified to contain two tandemly located affinity tags, Flag epitope (DYKDDDDK) and His(6) (HHHHHH), at the protein C-terminus and were expressed in cultured insect cells using baculovirus vectors. Both tagged proteins are readily detected on immunoblots by anti-Flag monoclonal antibody. Flag-His(6)-tagged saxiphilin was purified to homogeneity using Ni(2+)-chelate affinity chromatography and Heparin Sepharose chromatography. Equilibrium analysis of [3H]STX binding to tagged saxiphilin and tagged C-lobe saxiphilin gave K(D) values of 0.75 and 2.7 nM, respectively. Flag-His(6)-tagged saxiphilin was also utilized in a microtiter well solid-phase assay with Reacti-bind metal chelate plates to measure [3H]STX binding and binding competition by unlabeled STX. Such Flag-His(6)-tagged derivatives of saxiphilin have many possible applications in the assay of STX and related toxinological research.
Krishnan
G
G
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066, USA.
Morabito
M A
MA
Moczydlowski
E
E
D029845:Amphibian Proteins; D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000378:metabolism*; D006639:Histidine; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D012530:Saxitoxin / Q000032:analysis* / Q000378:metabolism
D029845:Amphibian Proteins; D002352:Carrier Proteins; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin; D006639:Histidine; C085706:FLAG peptide