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 utilizingOaAEP1 (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:protein immobilization;enzymatic ligation;OaAEP1;nanobody
Xuan Ding,Ziyi Wang,Bin Zheng,Shengchao Shi,Yibing Deng,Hanyang Yu,Peng Zheng
Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy single-molecule force spectroscopy (AFM-SMFS) study of protein mechanics. However, this methodology typically requires the functionalization of the glass surface with a corresponding peptide sequence/tag for enzymatic recognition and multiple steps are needed. Thus, it is time-consuming and a high level of experience is needed for reliable results. To solve this problem, we simplified the procedure using two strategies both based on asparaginyl endopeptidase (AEP). First, we designed a heterobifunctional peptide-based crosslinker, GL-peptide-propargylglycine, which links to an N3-functionalized surface via the click reaction. Then, the target protein with a C-terminal NGL sequence can be immobilized via the AEP-mediated ligation. Furthermore, we took advantage of the direct ligation between primary amino in a small molecule and protein with C-terminal NGL by AEP. Thus, the target protein can be immobilized on an amino-functionalized surface via AEP in one step. Both approaches were successfully applied to the AFM-SMFS study of eGFP, showing consistent single-molecule results.
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