Department of Biosynthesis and Biotransformation, National Institute of Chemistry, Ljubljana, Slovenia.
Microb Cell Fact. 2008 Dec 1;7:34. doi: 10.1186/1475-2859-7-34.
For a long time IBs were considered to be inactive deposits of accumulated target proteins. In our previous studies, we discovered IBs containing a high percentage of correctly folded protein that can be extracted under non-denaturing conditions in biologically active form without applying any renaturation steps. In order to widen the concept of correctly folded protein inside IBs, G-CSF (granulocyte colony stimulating factor) and three additional proteins were chosen for this study: GFP (Green fluorescent protein), His7dN6TNF-alpha (Truncated form of Tumor necrosis factor alpha with an N-terminal histidine tag) and dN19 LT-alpha (Truncated form of Lymphotoxin alpha).
Four structurally different proteins that accumulate in the bacterial cell in the form of IBs were studied, revealing that distribution of each target protein between the soluble fraction (cytoplasm) and insoluble fraction (IBs) depends on the nature of the target protein.Irrespective of the folding pattern of each protein, spectroscopy studies have shown that proteins in IBs exhibit similar structural characteristics to the biologically active pure protein when produced at low temperature. In the case of the three studied proteins, G-CSF, His7DeltaN6TNF-alpha, and GFP, a significant amount of protein could be extracted from IBs with 0.2% N-lauroyl sarcosine (NLS) and the proteins retained biological activity although no renaturation procedure was applied.
This study shows that the presence of biologically active proteins inside IBs is more general than usually believed. A large amount of properly folded protein is trapped inside IBs prepared at lower temperatures. This protein can be released from IBs with mild detergents under non-denaturing conditions. Therefore, the active protein can be obtained from such IBs without any renaturation procedure. This is of great importance for the biopharmaceutical industry. Furthermore, such IBs composed of active proteins could also be used as pure nanoparticles in diagnostics, as biocatalysts in enzymatic processes, or even as biopharmaceuticals.
长期以来，IBs 被认为是积累的靶蛋白的非活性沉积物。在我们之前的研究中，我们发现了含有高比例正确折叠蛋白的 IBs，这些蛋白可以在非变性条件下以生物活性形式提取，而无需进行任何复性步骤。为了拓宽 IBs 内正确折叠蛋白的概念，我们选择了 G-CSF（粒细胞集落刺激因子）和另外三种蛋白进行了这项研究：GFP（绿色荧光蛋白）、His7dN6TNF-α（带有 N 端组氨酸标签的肿瘤坏死因子α的截断形式）和 dN19 LT-α（淋巴毒素α的截断形式）。
研究了四种以 IBs 形式在细菌细胞中积累的结构不同的蛋白，结果表明，每种靶蛋白在可溶部分（细胞质）和不溶部分（IBs）之间的分布取决于靶蛋白的性质。无论每种蛋白的折叠模式如何，光谱研究表明，在低温下生产时，IBs 中的蛋白表现出与生物活性纯蛋白相似的结构特征。在所研究的三种蛋白（G-CSF、His7DeltaN6TNF-α和 GFP）中，用 0.2% N-月桂酰肌氨酸（NLS）可以从 IBs 中提取大量的蛋白，并且尽管没有进行复性处理，这些蛋白仍保留生物活性。
这项研究表明，IBs 中存在生物活性蛋白比通常认为的更为普遍。大量的正确折叠蛋白被困在较低温度下制备的 IBs 中。这种蛋白可以用温和的去污剂在非变性条件下从 IBs 中释放出来。因此，无需复性处理就可以从这样的 IBs 中获得活性蛋白。这对生物制药行业具有重要意义。此外，这样由活性蛋白组成的 IBs 也可以用作诊断中的纯纳米颗粒、酶促过程中的生物催化剂，甚至可以用作生物制药。
