Herfurth E, Wittmann-Liebold B
Max-Delbrück-Centrum für Molekulare Medizin, Abteilung Proteinchemie, Berlin, Germany.
Biol Chem Hoppe Seyler. 1995 Feb;376(2):81-90. doi: 10.1515/bchm3.1995.376.2.81.
We synthesized six peptides corresponding to regions that are predicted to be surface-exposed of the following ribosomal proteins: protein L2, positions (D263-K272); protein L5, positions (I136-G150); protein L25, positions (Q75-D90); protein S3, positions (Q222-K232) derived from Escherichia coli; and protein L2, positions (K257-K275), and protein S3, positions (R130-T150) from Bacillus stearothermophilus. These peptides were employed to raise ribosomal protein-cross-reactive antibodies. The anti-peptide antisera reacted specifically with their parent proteins, as demonstrated by immunoblotting experiments. In a competition assay proteins L2 from E. coli and B. stearothermophilus as well as proteins L5 and L25 from E. coli were found to be accessible to the respective anti-peptide antibodies in the 50S subunits, but not in 70S ribosomes, proving their location at the 50S interface which is covered by the 30S subunit in the 70S complex. Two of the anti-peptide antisera directed against sequences deduced from protein S3 of E. coli and B. stearothermophilus reacted with 30S subunits as well as with 70S ribosomes, demonstrating their location at the backside, which is exposed to solvent. Thus, by the strategy applied specific short peptide stretches were located at the surface of the ribosome.
我们合成了六种肽段，它们分别对应于以下核糖体蛋白预测的表面暴露区域：大肠杆菌的L2蛋白，位置为（D263 - K272）；L5蛋白，位置为（I136 - G150）；L25蛋白，位置为（Q75 - D90）；S3蛋白，位置为（Q222 - K232）；嗜热栖热放线菌的L2蛋白，位置为（K257 - K275），以及S3蛋白，位置为（R130 - T150）。这些肽段用于产生核糖体蛋白交叉反应抗体。免疫印迹实验表明，抗肽抗血清与它们的亲本蛋白发生特异性反应。在竞争分析中，发现大肠杆菌和嗜热栖热放线菌的L2蛋白以及大肠杆菌的L5和L25蛋白在50S亚基中可被相应的抗肽抗体识别，但在70S核糖体中则不能，这证明它们位于50S界面，在70S复合物中该界面被30S亚基覆盖。两种针对大肠杆菌和嗜热栖热放线菌S3蛋白推导序列的抗肽抗血清与30S亚基以及70S核糖体都发生反应，表明它们位于暴露于溶剂的背面。因此，通过所应用的策略，特定的短肽段被定位在核糖体表面。
