Chip-based protein-protein interaction studied by atomic force microscopy

• Published in: Biotechnology and bioengineering 2012-10, Vol.109 (10), p.2460-2467
• Main Authors: Kao, Feng-Sheng, Ger, Waylon, Pan, Yun-Ru, Yu, Hui-Chen, Hsu, Ray-Quen, Chen, Hueih-Min
• Format: Article
• Language: English
• Subjects: atomic force microscopy; B7-1 Antigen - metabolism; Biotechnology; CD28 Antigens - metabolism; chip-based; Cinnamates - metabolism; Echinacea purpurea; Flowers & plants; Microscopy; Microscopy, Atomic Force - methods; Pharmacology; Polyphenols; Protein Binding; Protein Interaction Mapping - methods; protein-protein interaction; Proteins; Proteins - metabolism
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.24521

In this article, a technique for accurate direct measurement of protein‐to‐protein interactions before and after the introduction of a drug candidate is developed using atomic force microscopy (AFM). The method is applied to known immunosuppressant drug candidate Echinacea purpurea derived cynarin. T‐cell/CD28 is on‐chip immobilized and B‐cell/CD80 is immobilized on an AFM tip. The difference in unbinding force between these two proteins before and after the introduction of cynarin is measured. The method is described in detail including determination of the loading rates, maximum probability of bindings, and average unbinding forces. At an AFM loading rate of 1.44 × 104 pN/s, binding events were largely reduced from 61 ± 5% to 47 ± 6% after cynarin introduction. Similarly, maximum probability of bindings reduced from 70% to 35% with a blocking effect of about 35% for a fixed contact time of 0.5 s or greater. Furthermore, average unbinding forces were reduced from 61.4 to 38.9 pN with a blocking effect of ∼37% as compared with ∼9% by SPR. AFM, which can provide accurate quantitative measures, is shown to be a good method for drug screening. The method could be applied to a wider variety of drug candidates with advances in bio‐chip technology and a more comprehensive AFM database of protein‐to‐protein interactions. Biotechnol. Bioeng. 2012; 109: 2460–2467. © 2012 Wiley Periodicals, Inc. Efficient and targetable drug screening is established by protein‐immobilization on chip via AFM detection. For example, unbinding force can directly be quantified (q1‐q2) to indicate when candidate component (a blocker between CD28 and CD80) is captured by imm‐CD28 and measured by afm‐CD80. This set‐up can be widely applied to other drugs' discovery.