Stable immobilisation of His-tagged proteins on BLI biosensor surface using cobalt

SENSORS AND ACTUATORS B-CHEMICAL

卷:243

Article

Auer, Sanna^1,2  Azizi, Latifeh^1,2  Faschinger, Felix³  Blazevic, Vesna⁴  Vesikari, Timo⁴  Gruber, Hermann J.³  Hytonen, Vesa P.^1,2 

[1] Univ Tampere, BioMediTech, Laakarinkatu 1, FI-33520 Tampere, Finland

[2] Fimlab Labs, Biokatu 4, FI-33520 Tampere, Finland

[3] Johannes Kepler Univ Linz, Inst Biophys, Gruberstr 40, A-4020 Linz, Austria

[4] Univ Tampere, Sch Med, Biokatu 8, FI-33520 Tampere, Finland

关键词: His-tagged protein;    Ni-NTA;    Biolayer interferometry;    Avidin-biotin;    Norovirus;    Label-free detection;   

DOI  :  10.1016/j.snb.2016.11.090

来源: Elsevier

【 摘 要 】

In biosensor experiments, ligand molecules need to be immobilized tightly on sensor surfaces for sensitive and robust detection of the analyte of interest. Stretches of histidines (His-tags) are typically inserted into recombinant proteins for affinity purification purposes, but can also be used in the immobilization of biosensors functionalized with Ni-NTA (nitrilotriacetic acid). The His-tag-Ni-NTA bond is, however, easily disrupted by factors including low pH, reducing agents, or chelating agents such as EDTA. In biosensing applications, a stable ligand immobilization that tolerates harsh conditions is preferable, because the sample matrix can vary from bodily fluids to waste waters. We describe here Co(III)-NTA bonding for tight immobilization of His-tagged proteins on Octet BLI-biosensors. The performance of the Ni(II)NTA was compared to that of Co(III)-NTA using His-tagged avidins and norovirus proteins. Comparative studies were performed using biotin-iminodiacetic acid (IDA) for streptavidin sensor functionalization instead of the NTA surfaces provided by the manufacturer. It was noted that Co(III)-NTA offers highly stable immobilization of His-tagged protein, tolerating factors such as 0.7 M imidazole, that is typically used for detaching proteins from Ni(Il)-NTA-agarose used in protein purification columns. The good performance of Co(III)-NTA functionalized BLI sensor was demonstrated by detecting norovirus antibodies from human serum samples with sensors functionalized with His-tagged virus-like particles. The use of Co-NTA sensors for attachment of His-tagged proteins is ideal in conditions, where the immobilized ligands need to be firmly attached and tolerate harsh chemical conditions. (C) 2016 Elsevier B.V. All rights reserved.

【 授权许可】

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