Site-Specific and Covalent Attachment of His-Tagged Proteins by Chelation Assisted Photoimmobilization: A Strategy for Microarraying of Protein Ligands

Abstract

A novel strategy for site-specific and covalent attachment of proteins has been developed, intended for robust and controllable immobilization of histidine (His)-tagged ligands in protein microarrays. The method is termed chelation assisted photoimmobilization (CAP) and was demonstrated using human IgG-Fc modified with C-terminal hexahistidines (His-IgGFc) as the ligand and protein A as the analyte. Alkanethiols terminated with either nitrilotriacetic acid (NTA), benzophenone (BP), or oligo(ethylene glycol) were synthesized and mixed self-assembled monolayers (SAMs) were prepared on gold and thoroughly characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry, and contact angle goniometry. In the process of CAP, NTA chelates Ni(2+) and the complex coordinates the His-tagged ligand in an oriented assembly. The ligand is then photoimmobilized via BP, which forms covalent bonds upon UV light activation. In the development of affinity biosensors and protein microarrays, site-specific attachment of ligands in a fashion where analyte binding sites are available is often preferred to random coupling. Analyte binding performance of ligands immobilized either by CAP or by standard amine coupling was characterized by surface plasmon resonance in combination with IRAS. The relative analyte response with randomly coupled ligand was 2.5 times higher than when site-specific attachment was used. This is a reminder that also when immobilizing ligands via residues far from the binding site, there are many other factors influencing availability and activity. Still, CAP provides a valuable expansion of protein immobilization techniques since it offers attractive microarraying possibilities amenable to applications within proteomics.

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“…This is in agreement with other reports confirming that His-tagged fusion antigens can be directly immobilized on different surfaces with protein orientations completely compatible with antibody recognition. [37][38][39][40] To simplify and reduce the cost and time of the assay, we carried out direct immobilization of S1 protein for specific Ig detection.…”

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“…Several strategies have been described to improve the affinity between the NTA and the His 6 -tagged recombinant proteins in the context of biosensing. , Wegner et al (2013) described a stable immobilization of His 6 -tagged proteins to NTA by oxidizing the chelating metal cobalt, Co­(II), to Co­(III). This step reduces the dissociation constant of the NTA-Co­(III)-His 6 -tagged protein, thereby generating a more stable complex. , This approach was validated using a quartz crystal microbalance with dissipation (QCM-D) and biolayer interferometry (BLI) .…”

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“…DLL1 was attached in the subsequent step by taking advantage of the strong binding affinity between the nickel complex and the nitrogen atoms of imidazole rings provided by a six-fold histidine sequence (His 6 -tag) on the C-terminus of DLL1. This binding strategy via the His 6 -Tag to decorate nanostructures with proteins proved also in other studies to be efficient and specific (Aydin et al, 2009;Deeg et al, 2013;Ericsson et al, 2013;Wegner and Spatz, 2013). In this way, the protein could be immobilized in a specific orientation ensuring that the receptor binding side was available for the cells as also shown in previous studies by the Spatz group (Glass et al, 2003;Deeg et al, 2013).…”