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 immobilization of oligohistidine tagged proteins in the flow cell of the 'BIAcore' surface plasmon resonance (SPR) biosensor. The carboxymethylated dextran surface of the flow cell was covalently derivatized with N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA ligand) via its single primary amino group, and the derivatized surface charged with Ni2+. 6His-VP55, an N-terminally tagged derivative of the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase, was immobilized to this surface in a manner that was dependent upon the immobilized NTA ligand, 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 stability of immobilization varied inversely with the amount of 6His-VP55 immobilized and was greatest in buffer of pH 8.0 or greater, containing NaCl at a concentration of 0.1 M. Utilizing these conditions, 6His-VP55 remained stably immobilized during 60 min of buffer flow at moderate flow rates. VP39, the stimulatory subunit of vaccinia poly(A) polymerase, interacted with the immobilized 6His-VP55. Approximately 99% of immobilized 6His-VP55 molecules were available for VP39 binding, in contrast to the approximately 40% availability for 6His-VP55 molecules immobilized covalently, via primary amino groups. Three additional proteins, tagged at either the N- or C-terminus with oligohistidine, were shown to be stably immobilized via the chelating linkage. This simple method permits immobilization of proteins in the BIAcore biosensor via a commonly employed affinity tag, in a stable and reversible manner, and requires only a single biosensor flow cell for the iterative generation of immobilized protein surfaces.
Gershon
P D
PD
Center for Genome Research, Texas A & M University, Houston 77030-3303, USA.
Khilko
S
S
D015374:Biosensing Techniques; D002614:Chelating Agents / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis; D009571:Nitrilotriacetic Acid / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods*; D014616:Vaccinia virus / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
D002614:Chelating Agents; D008565:Membrane Proteins; D014764:Viral Proteins; D006639:Histidine; D009571:Nitrilotriacetic Acid