Rapid evaluation of nickel binding properties of His-tagged lactate dehydrogenases using surface plasmon resonance.
The use of surface plasmon resonance (SPR), for the comparison of metal binding properties of polyhistidine tags, was evaluated. Six different tags containing various number of histidines, either none (tags n and t), three (tags H3A3 and HA2HA2H) or six (tags H6 and His6), were genetically fused to the N-terminal of lactate dehydrogenase (LDH). The binding ability of these constructs to nickel ions, immobilised with nitrilotriacetic acid (NTA), was tested both by conventional immobilised metal ion affinity chromatography (IMAC) and SPR. The relative binding strengths of the tags to nickel were identical using both methods (n approximately t < HA2HA2H < H3A3 < His6 < H6), confirming the value of the SPR technique for investigating metal-protein interactions. Protein modelling has also proved to be useful in supporting the experimental results.
Bernaudat
Florent
F
Department of Pure and Applied Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, Getingevagen 60, 221 00 Lund, Sweden.
Büllow
Leif
L
D000345:Affinity Labels; D001665:Binding Sites; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism*; D009571:Nitrilotriacetic Acid; D011487:Protein Conformation; D020349:Surface Plasmon Resonance
D000345:Affinity Labels; D006639:Histidine; D009532:Nickel; D007770:L-Lactate Dehydrogenase; D009571:Nitrilotriacetic Acid