Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain.
Microb Cell Fact. 2010 Sep 17;9:71. doi: 10.1186/1475-2859-9-71.
Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces.
Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10⁻¹ cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features.
The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.
细菌包含体是亚微米级的蛋白质聚集体，通常在重组细菌中发现，这些聚集体一直被认为是蛋白质生产过程中不理想的产物。然而，由于它们具有完全的生物相容性，最近被描述为纳米级惰性材料，可用作组织工程的支架，在生物医学和材料科学中有更广泛的应用。目前从大肠杆菌中分离包含体的方案通常提供 95%至 99%的蛋白质回收率，从实际意义上讲，这可能意味着细菌细胞的严重污染，与包含体在生物界面中的应用不兼容。
我们使用适当的化学和机械细胞破碎方法组合，建立了一种方便的方法来回收细菌包含体，其中活细胞污染水平可检测到低于 10⁻¹ cfu/ml，这些聚集体的颗粒组织在大小和蛋白质折叠特征方面保持不变。
所开发方案的应用允许获得适合用于哺乳动物细胞培养和其他生物界面的无细菌包含体。
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