Reichelt Wieland N, Kaineder Andreas, Brillmann Markus, Neutsch Lukas, Taschauer Alexander, Lohninger Hans, Herwig Christoph
Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
Research Division Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
Biotechnol J. 2017 Jun;12(6). doi: 10.1002/biot.201600471. Epub 2017 Apr 28.
The expression of pharmaceutical relevant proteins in Escherichia coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In the scientific literature, substantial effort has been devoted to the quantification of IB size. However, particle-based methods used up to this point to analyze the physical properties of representative numbers of IBs lack sensitivity and/or orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells. TEM imaging in combination with manual grey scale image segmentation allowed the quantification of relative areas covered by the inclusion body within the cytosol. As a high throughput method nano particle tracking analysis (NTA) enables one to derive the diameter of inclusion bodies in cell homogenate based on a measurement of the Brownian motion. The NTA analysis of fixated (glutaraldehyde) and non-fixated IBs suggests that high pressure homogenization annihilates the native physiological shape of IBs. Nevertheless, the ratio of particle counts of non-fixated and fixated samples could potentially serve as factor for particle stickiness. In this contribution, we establish image segmentation of TEM pictures as an orthogonal method to size biologic particles in the cytosol of cells. More importantly, NTA has been established as a particle-based, fast and high throughput method (1000-3000 particles), thus constituting a much more accurate and representative analysis than currently available methods.
药物相关蛋白在大肠杆菌中的表达常常会引发因蛋白质聚集导致的包涵体（IB）形成。在科学文献中，已有大量工作致力于包涵体大小的量化。然而，截至目前用于分析代表性数量包涵体物理性质的基于颗粒的方法缺乏灵敏度和/或正交验证。通过使用高压冷冻和自动冷冻置换技术进行透射电子显微镜（TEM）观察，细胞内的胞质包涵体结构得以保存。TEM成像结合手动灰度图像分割，能够对胞质内包涵体覆盖的相对面积进行量化。作为一种高通量方法，纳米颗粒跟踪分析（NTA）能够基于对布朗运动的测量得出细胞匀浆中包涵体的直径。对固定化（戊二醛）和未固定化包涵体的NTA分析表明，高压匀浆破坏了包涵体的天然生理形状。尽管如此，未固定化和固定化样品的颗粒计数比率可能可作为颗粒粘性的一个因素。在本研究中，我们将TEM图片的图像分割确立为一种用于确定细胞胞质中生物颗粒大小的正交方法。更重要的是，NTA已被确立为一种基于颗粒的快速高通量方法（1000 - 3000个颗粒）, 因此构成了一种比现有方法更准确且更具代表性的分析方法。
