STABLE CHELATING LINKAGE FOR REVERSIBLE IMMOBILIZATION OF OLIGOHISTIDINE TAGGED PROTEINS IN THE BIACORE SURFACE-PLASMON RESONANCE DETECTOR

We describe a stable chelating linkage for the reversible immobilizati on of oligohistidine tagged proteins in the flow cell of the 'BIAcore' surface plasmon resonance (SPR) biosensor. The carboxymethylated dext ran surface of the flow cell was covalently derivatized with N-(5-amin o-1-carboxypentyl)iminodiacetic acid (NTA ligand) via its single prima ry amino group, and the derivatized surface charged with Ni2+. 6His-VP 55, an N-terminally tagged derivative of the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase, was immobilized to th is surface in a manner that was dependent upon the immobilized NTA lig and, the prior injection of Ni2+ at a concentration of > 10(-5) M and the 6His tag, and which was reversible upon injection of EDTA. The sta bility of immobilization varied inversely with the amount of 6His-VP55 immobilized and was greatest in buffer of pH 8.0 or greater, containi ng NaCl at a concentration of 0.1 M. Utilizing these conditions, 6His- VP55 remained stably immobilized during 60 min of buffer flow at moder ate flow rates. VP39, the stimulatory subunit of vaccinia poly(A) poly merase, interacted with the immobilized 6His-VP55. similar to 99% of i mmobilized 6His-VP55 molecules were available for VP39 binding, in con trast to the similar to 40% availability for 6His-VP55 molecules immob ilized covalently, via primary amino groups. Three additional proteins , tagged at either the N- or C-terminus with oligohistidine, were show n to be stably immobilized via the chelating linkage. This simple meth od permits immobilization of proteins in the BIAcore biosensor via a c ommonly employed affinity tag, in a stable and reversible manner, and requires only a single biosensor flow cell for the iterative generatio n of immobilized protein surfaces.