Enzymatic Protein Immobilization for Nanobody Array

Abstract

Antibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to corresponding chemical groups on the substrate surface. However, this method does not control the antibody's anchoring point and orientation, potentially leading to reduced binding efficiency and overall performance. Another method using anti-antibodies as intermediate molecules to control the orientation can be used but it demonstrates lower efficiency. Here, we demonstrate a site-specific protein immobilization strategy utilizing OaAEP1 (asparaginyl endopeptidase) for building a nanobody array. Moreover, we used a nanobody-targeting enhanced green fluorescent protein (eGFP) as the model system to validate the protein immobilization method for building a nanobody array. Finally, by rapidly enriching eGFP, this method further highlights its potential for rapid diagnostic applications. This approach, characterized by its simplicity, high efficiency, and specificity, offers an advancement in the development of surface-modified protein arrays. It promises to enhance the sensitivity and accuracy of biomolecule detection, paving the way for broader applications in various research and diagnostic fields.

Keywords: OaAEP1; enzymatic ligation; nanobody; protein immobilization.

Figure 1

(a–c) The structure of the nanobody (PDB 7SAI) targeting eGFP is shown. (d) Native PAGE gel confirmed the interaction between the nanobody and eGFP. The migration of visible eGFP bands indicated the complex formation, which is highlighted by the red box. (e) Schematic representation shows the addition of a GL-peptide on amino-functionalized glass. First, the surface was coated with N₃ from NH₂; then, the Maleimide group was added. Finally, the target peptide GL was added [45]. (f) Nanobody with an NGL tag was immobilized on the GL-functionalized surface catalyzed by OaAEP1 [27].

Figure 2

Verification of immobilized eGFP by fluorescent imaging. (a) A step-by step procedure for constructing an antibody array to recruit eGFP. (b) Hand-pipetted immobilized eGFP on glass exhibits a distinct fluorescent signal, with its boundary outlined by a dashed line in the bright-field image. (c) eGFP was added in a 2 × 2 nanobody microarray, resulting in a clear fluorescence pattern. (d) Quantification of fluorescent intensity per unit area for these methods, which includes an experiment and a control, was used to confirm the feasibility of this approach. ImageJ was used for quantification, and the values were normalized. The histograms depict the relative intensity of eGFP in the presence (green) and absence (black) of OaAEP1.

Figure 3

(a) Schematic diagram of trace detection of enriched eGFP protein. (b) Diagram of rapid recruitment of eGFP protein from lysate. (c) Cell lysates derived from E. coli cells transformed with an eGFP-expressing plasmid were subjected to SDS-PAGE analysis, with “(+)” representing the original cell lysate and “(−)” representing the lysate from nanobody array treatment. The green and gray dashed boxes represent the bands of the eGFP protein before and after the reaction of the lysate with the nanobody array. (d) eGFP was spotted onto the immobilized nanobody. Recruitment boundary was clearly observed between eGFP and blank (magnified section indicated by dashed lines).