BIACORE Analysis of Histidine-Tagged Proteins Using a Chelating NTA Sensor Chip

Nieba, Lars; Nieba-Axmann, Sabine E.; Persson, Anette; Hämäläinen, M.; Edebratt, Fredrik; Hansson, Annelie; Lidholm, Jonas; Magnusson, Karin; Karlsson, Åsa Frostell; Plückthun, Andreas

doi:10.1006/abio.1997.2326

Cited by 331 publications

(282 citation statements)

References 40 publications

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“…[40][41][42][43][44] The calculation of the protein surface coverage from the SPR response is described elsewhere. 15,16 The home-built SPR liquid biosensing system used in this study has been described and characterized in more detail elsewhere.…”

Section: Spr Measurementsmentioning

confidence: 99%

XPS, TOF-SIMS, NEXAFS, and SPR Characterization of Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers for Controllable Immobilization of Proteins

2008

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For immobilization of proteins onto surfaces in a specific and controlled manner it is important to start with a well-defined surface that contains specific binding sites surrounded by a nonfouling background. For immobilizing histidine-tagged (his-tagged) proteins, surfaces containing nitrilotriacetic acid (NTA) headgroups and oligo(ethylene glycol) (OEG) moieties are a widely used model system. The surface composition, structure and reactivity of mixed NTA/OEG selfassembled monolayers (SAMs) on Au substrates were characterized in detail using x-ray photoelectron spectroscopy (XPS), near-edge x-ray absorption fine structure spectroscopy (NEXAFS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and surface plasmon resonance (SPR) biosensoring. XPS results for sequential adsorption of NTA thiols followed by OEG thiols showed that OEG molecules were incorporated into an incompletely formed NTA monolayer until a complete mixed SAM was formed. The surface concentration of NTA headgroups was estimated to be 0.9-1.3 molecule/nm 2 in the mixed NTA/OEG monolayers, compared to 1.9 molecule/nm 2 in pure NTA monolayers. Angle-dependent XPS indicated NTA headgroups were slightly reoriented toward an upright position after OEG incorporation and polarization-dependent NEXAFS results indicated increased ordering of the alkane chains of the molecules. Nitrogen-containing and OEG-related secondary ion fragments from the ToF-SIMS experiments confirmed the presence of NTA headgroups and OEG moieties in the monolayers. A multivariate peak intensity ratio was developed for estimating the relative NTA concentration in the outermost (10 Ǻ) of the monolayers. SPR measurements of a his-tagged, humanized antilysozyme variable fragment (HuLys Fv) immobilized onto Ni(II)-treated mixed NTA/OEG and pure NTA monolayers demonstrated the reversible, site-specific immobilization of his-tagged HuLys Fv (108 -205 ng/cm 2 ) with dissociation rates (k off ) between 1.0x10 −4 and 2.1x10 −5 s −1 , both depending on the NTA surface concentration and orientation. The monolayers without Ni(II) treatment exhibited low nonspecific adsorption of his-tagged HuLys Fv ( < 2ng/cm 2 ).

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Section: Spr Measurementsmentioning

confidence: 99%

XPS, TOF-SIMS, NEXAFS, and SPR Characterization of Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers for Controllable Immobilization of Proteins

2008

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“…There have been further technical developments, such as a His-tag specific BIACORE TM , enabling analysis of His-tagged proteins using the Sensor chip NTA. [16]. Recently, an electrochemical technique using a Histag specific antibody (anti-His 6 )-modified electrode was developed [17].…”

mentioning

confidence: 99%

Variability in the immunodetection of His-tagged recombinant proteins

Debeljak

Feldman

Davis

et al. 2006

Analytical Biochemistry

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Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection. Poly-histidine tags (His-tags) are among the most commonly used affinity tags. We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo), wild type Epo (Epo wt ) and Epo containing an R103A mutation (Epo R103A ). Both were engineered to contain a C-terminal six residue His-tag. The cDNA constructs were stably transfected into CHO cells and COS-7 cells. Clones from the CHO cell transfections were selected for further characterization and larger-scale protein expression. Three chromatographic steps were utilized to achieve pharmacologically pure Epo. Conditioned media from the Epo-expressing cell lines and protein-containing samples from each step of purification were analyzed by SDS-PAGE and dot blot, using both monoclonal anti-human Epo antibody (AE7A5) and anti-His antibodies. While the successful incorporation of the His-tag into our constructs was confirmed by Epo binding to Ni 2+ -NTA resin and by μLC/MS/MS amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti His-tag antibody used. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods. KeywordsEpo; erythropoietin; His-tag; protein purification; His-tag specific antibodies; immunodetection Recombinant DNA technology enables the production of large quantities of highly purified and well-characterized proteins. Methods that will facilitate subsequent protein analysis and purification are of major interest during the initial design of the recombinant protein. Both detection and purification are greatly simplified by engineering the DNA construct so that the encoded protein is fused to a readily detectable affinant peptide partner, a method designated "affinity tagging". There is often a preference for selection of a short affinity tag that can be fused to the target gene more easily using the polymerase chain reaction (PCR) [1,2]. Examples † Corresponding author: Phone: +1 (617) 632-9980, Fax: +1 (617) 632-0401 Email: asytkows@bidmc.harvard.edu. * Address to which proofs should be mailed: Arthur J. Sytkowski, MD, Beth Israel Deaconess Medical Center, 330, Brookline Ave., W/ BL 548, Boston, MA 02215, USA Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In the present study, we generated two cDNAs encoding C-terminal His-tagged variants of recombinant human erythropoietin (Epo) a...

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“…In the literature, various methods for NTA molecule immobilization on the surface of the SPR discs were described and the binding constants for these systems are very similar to the ones described by Nieba et al (~1.0x10 6 M -1 ) [20,[41][42][43]. Nakamura et al have studied different anchors for immobilization of NTA molecules onto the gold surface which were chelated with nickel(II) [25].…”

Section: Spr Binding Responses Of Gst-his 6 -Jak2 Over Dpta-cu(ii) Anmentioning

confidence: 79%

“…The His-tag typically consists of five or six consecutive His residues added to the C-or N-terminus of the protein [20]. The most popular systems for oriented immobilization of His-tagged proteins are complexes of nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) with transition metal ions (Ni 2+ , Cu 2+ ) [20][21][22][23][24][25][26][27][28]. Nitrilotriacetic acid forms a tetradentatechelate with Ni 2+ and Cu 2+ , which is able to bind His-tagged proteins via coordinated bonds [29].…”

Section: Introductionmentioning

confidence: 99%

“…A coordinative complex with a binding affinity of K D equal to 1 mM is simply formed between one molecule of NTA and two imidazole rings on His-tagged protein [20,27]. Dextran-based SPR chips modified with covalently linked NTA molecules are commercially available and have been used for the detection of the complex between Me 2+ -NTA and His-tagged proteins [20,24,26,30].…”

Section: Introductionmentioning

confidence: 99%

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Immobilization of His-tagged kinase JAK2 onto the surface of a plasmon resonance gold disc modified with different copper (II) complexes

Kurzątkowska¹,

Mielecki²,

Grzelak³

et al. 2014

Talanta

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New surface plasmon resonance (SPR) sensing platform swhich consists of copper (II) complexes of a pentetic acid thiol ligand (DPTA-Cu(II)) and of a thiol derivative of dipyrromethene (DPM-Cu(II) created on the surface of gold SPR disc were applied to oriented immobilization of His-tagged Janus kinase 2 (GST-His 6 -JAK2). This method is based on the covalent bond formation between histidine from a His-tag chain of a protein and Cu(II) centres from the complexes. The kinetic and thermodynamic parameters of the oriented immobilization of GST-His 6 -JAK2 protein to DPTA-Cu(II) and DPM-Cu(II) complexes attached to the Au surface of a SPR disc were discussed.

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BIACORE Analysis of Histidine-Tagged Proteins Using a Chelating NTA Sensor Chip

Cited by 331 publications

References 40 publications

XPS, TOF-SIMS, NEXAFS, and SPR Characterization of Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers for Controllable Immobilization of Proteins

XPS, TOF-SIMS, NEXAFS, and SPR Characterization of Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers for Controllable Immobilization of Proteins

Variability in the immunodetection of His-tagged recombinant proteins

Immobilization of His-tagged kinase JAK2 onto the surface of a plasmon resonance gold disc modified with different copper (II) complexes

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