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 bin ds saxitoxin (STX), a causative agent of paralytic shellfish poisoning. Sax iphilin is homologous to transferrin and consists of two internally homolog ous 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 recombina nt saxiphilin and C-lobe saxiphilin were modified to contain two tandemly l ocated affinity tags, Flag epitope (DYKDDDDK) and His(6) (HHHHHH), at the p rotein C-terminus and were expressed in cultured insect cells using baculov irus vectors. Both tagged proteins are readily detected on immunoblots by a nti-Flag monoclonal antibody. Flag-His(6)-tagged saxiphilin was purified to homogeneity using Ni2+-chelate affinity chromatography and Heparin Sepharo se chromatography. Equilibrium analysis of [H-3]STX binding to tagged saxip hilin and tagged C-lobe saxiphilin gave K-D values of 0.75 and 2.7 nM, resp ectively. Flag-His(6)-tagged saxiphilin was also utilized in a microtiter w ell solid-phase assay with Reacti(TM)-bind metal chelate plates to measure [H-3]STX binding and binding competition by unlabeled STX. Such Flag-Hiss-l agged derivatives of saxiphilin have many possible applications in the assa y of STX and related toxinological research. (C) 2000 Elsevier Science Ltd. All rights reserved.