应用原子力显微镜观测蛋白酶体的结构与功能研究

[Abstract] Applying the atomic force microscopy (AFM) to explore the nanostructure of biomoleculars is a vital research approach in current biostructural study for proteomics. This project selected the proteasome and immunoglobulin A (IgA) as the study subjects, and combinated the molecularbiological technology and biochemical procedure to produce and purify the protein sample for the AFM observation. Establish the stable liquid system suited for observation of proteins with high molecular weight in natural environment. We realized the in real-time detection of the protein under physiological condition by special O shape ring sealing the liquid in detection cell and perfusing the buffer with regulated components in it. The 20S complex of proteasome purified from the yeast cell showed the distribution ranged from 22kDa to 32kDa in SDS-PAGE gel. The histidine tagged subunits of proteasome were constructed and the expression in prokaryotic cell was identified by Western blotting. Secretory IgA was purified from bovine colostrums by salt out, dialysis and ion-exchange chromatography, and the secretory piece, heavy and light chains of IgA were revealed in SDS-PAGE analysis followed by Coomassie blue staining. Under atomic force microscopy, it was showed that the IgA existed in majority as oligomer form in the physiological condition. The monomer's dimension with 2.6 nm×18.0 nm×20.2 nm coincided with the result discerned by electronmicroscopy. The amount of IgA attached on mica increased by the prolonged incubation time, but no aggregration formed among the oligomers and monomer details inside structure of oligomers can be differentiated clearly. The attachment of IgA on poly-lysine treated mica showed dendritic aggregation form after incubated for 30 minutes and no clear structure of monomers in the aggregation proteins. In the range of 300 nm×300 nm scanning field, the aggregation formed a regular radiated arrangement circled with one point. The fibre stripe width was about 20 nm, coinciding with the width of IgA monomer. This aggregation phenomenon tended to be more obvious with prolonging incubation time. These results showed that it was available to observe the microstructure of immunoglobulin in its natural condition by combination of biochemistry and atomic force microscopy techniques, so that it will provide an effective research pathway for the study of structure basis contributing to functional regulation of micromoleculars.

原子粒显微镜是近年来广泛应用于生物大分子结构研究的有力工具。对蛋白酶体一类重要细胞器的纳米级观测，结合生化功能的研究，有望在单分子水平揭示蛋白酶体功能变化的结构基础。对蛋白酶体的生化提纯结合合脂膜的体外构建，建立适合原子力显微镜观测的生物鞍字噬涎逑担蠊婺？咕哂兄匾锕δ艿鞍字式峁沟难芯康於ɑ