초강력 합성 결합쌍, 쿠커비투[7]릴-페로센 유도체의 생물학적 응용: 친화성 단백질 정제 시스템 및 센서

Abstract

Because of the highly specific and strong non-covalent interaction (K ~ 1013 - 1015 M-1), the ligand-receptor pair, biotin-avidin (or streptavidin) has been widely used in biotechnology for various purposes including purification, sensing and immobilization of biomolecules. However, the natural system suffers some shortcomings including denaturation by organic solvents or elevated temperatures, degradation by enzymes, and high production cost. Developing a synthetic ligand−receptor pair that can replace the biotin−avidin system is thus important not only for deeper understanding of noncovalent interactions but also for practical applications.Synthetic receptor-ligand pair, cucurbit[7]uril (CB[7])-ferrocene derivatives with an exceptionally high binding affinity (K ~ 1 × 1012 - 1015 M-1) has recently received much attention as a prominent candidate for a replacement of the streptavidin-biotin pair. Having high chemical and biological stability, CB[7]-ferrocene derivatives can overcome the shortcomings of the natural system. The aim of this thesis is to explore biological applications of the synthetic binding pair system based on CB[7] and ferrocene derivatives, which can ultimately lead to novel biotechnical tools showing higher performances than conventional ones based on the streptavidin-biotin system.Chapter 2 presents a protein labeling and capturing system based on synthetic receptor-ligand pair, CB[7] and ferrocene derivative, which serve as a more efficient protein purification system than the conventional method using the streptavidin-biotin pair. To prepare labeled model proteins, a ferrocene derivative having a reactive functional group, ferroceneaminetetraethyleneglycol-carboxylic acid (AFc) was synthesized and conjugated to model proteins. CB[7]-immobilized sepharose beads (CB[7]-beads), prepared by conjugating (HO)nCB[7] (n ≈ 6) to N-hydroxysuccimidyl sepharose beads selectively isolated AFc-labeled proteins from heterogeneous protein mixtures. The capturing efficiency of CB[7]-beads for the ferrocene labeled proteins was higher than that of streptavidin conjugated beads (Streptavidin-beads) for biotin-labeled protein. This result demonstrated the potential utility of CB[7]-AFc pair for protein purification.Chapter 3 describes a supramolecular fishing method for membrane proteins using the ultrastable synthetic binding pair CB[7] and AFc. The plasma membrane proteins located on cell surface were selectively labeled with AFc and efficiently captured by CB[7]-beads, minimizing contamination from cytosolic proteins, as demonstrated by SDS-PAGE and immunoblotting. The captured proteins can be recovered easily at room temperature by treatment with a strong competitor such as 1,1′-bis(trimethylammoniomethyl)ferrocene. Enzymatic digestion of the captured proteins on the beads without elution step was also possible over a synthetic nature of CB[7]-AFc binding pair. With these unique properties, which were not achievable with streptavidin-biotin pair, this synthetic but biocompatible host-guest system may be a useful alternative to streptavidin-biotin for membrane proteomics as well as other biological and biotechnological applications.Aptamers are artificial single-stranded DNA or RNA sequences that fold into secondary and tertiary structures making them bind to certain targets with extremely high specificity. Though several advantages of aptamers over antibodies including high stability and easily modifiable structure make them promising sensing element for biosensor, only a few methods for immobilizing the aptamers on a sensor chip has been developed including the ones using the streptavidin-biotin pair. Chapter 4 describes our efforts to develop a surface plasmon resonance (SPR) sensor for specific protein detection using aptamer as a sensing unit and CB[7]-AFc pair as a novel non-covalent linker for immobilizing aptamers. The 37 KDa protein thrombin and 15-mer oligonucleotide thrombin binding aptamer (TBA) were chosen as a model target protein and aptamer, respectively, to demonstrate the sensing ability of the system. The monolayer of synthetic receptor CB[7] on a gold surface produced by spontaneous adsorption successfully immobilized AFc-labeled TBA by specific host-guest interaction between CB[7] and AFc. The immobilized TBA selectively sensed the injected thrombin within the concentration range of 10-9 - 10-7 M. The simple and modular method for surface immobilization using the synthetic receptor-ligand pair may broaden the applications of aptamer-based sensor.