Carrió Mar, González-Montalbán Nuria, Vera Andrea, Villaverde Antonio, Ventura Salvador
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain.
J Mol Biol. 2005 Apr 15;347(5):1025-37. doi: 10.1016/j.jmb.2005.02.030.
Bacterial inclusion bodies are major bottlenecks in protein production, narrowing the spectrum of relevant polypeptides obtained by recombinant DNA. While regarded as amorphous deposits formed by passive and rather unspecific precipitation of unfolded chains, we prove here that they are instead organized aggregates sharing important structural and biological features with amyloids. By using an Escherichia coli beta-galactosidase variant, we show that aggregation does not necessarily require unfolded polypeptide chains but rather depends on specific interactions between solvent-exposed hydrophobic stretches in partially structured species. In addition, purified inclusion bodies are efficient and highly selective nucleation seeds, promoting deposition of soluble homologous but not heterologous polypeptides in a dose-dependent manner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which, jointly with Fourier transform infra red spectroscopy data, indicates a high level of organized intermolecular beta-sheet structure. The evidences of amyloid-like structure of bacterial inclusion bodies, irrespective of potential applications in bioprocess engineering, prompts the use of bacterial models to explore the molecular determinants of protein aggregation by means of simple biological systems.
细菌包涵体是蛋白质生产中的主要瓶颈，限制了通过重组DNA获得的相关多肽的范围。虽然通常被认为是由未折叠链的被动且相当非特异性沉淀形成的无定形沉积物，但我们在此证明它们实际上是与淀粉样蛋白具有重要结构和生物学特征的有序聚集体。通过使用大肠杆菌β-半乳糖苷酶变体，我们表明聚集不一定需要未折叠的多肽链，而是取决于部分结构化物种中溶剂暴露的疏水片段之间的特定相互作用。此外，纯化的包涵体是高效且高度选择性的成核种子，以剂量依赖的方式促进可溶性同源而非异源多肽的沉积。最后，包涵体与淀粉样蛋白诊断染料结合，这与傅里叶变换红外光谱数据一起表明存在高水平的有序分子间β-折叠结构。细菌包涵体的淀粉样蛋白样结构的证据，无论在生物过程工程中的潜在应用如何，都促使利用细菌模型通过简单的生物系统来探索蛋白质聚集的分子决定因素。
