Condensation of an additive-free cell extract to mimic the conditions of live cells.
Condensation of an additive-free cell extract to mimic the conditions of live cells.
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DOI：
10.1371/journal.pone.0054155
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Nomura SM
中科院分区：
文献类型：
--
作者：
Fujiwara K;Nomura SM
细胞环境与重构材料的细胞环境不同，主要是因为存在高度浓缩的生物大分子。为了模拟活细胞的环境和条件，我们开发了一种制备无添加剂、高浓缩细胞提取物的方法。首先，我们验证了功能性无细胞翻译提取物对特定盐和缓冲液的需求。不含添加剂的大肠杆菌细胞提取物的 S30 级分表现出足够的无细胞蛋白质产量。接下来，我们建立了一种使用真空干燥器逐渐蒸发来积累生物成分的方法。使用该方法成功地将牛血清白蛋白、绿色荧光蛋白、碱性磷酸酶和稀释的重组蛋白表达系统浓缩成活性形式。制备的细胞提取物的蛋白质浓度升高至180 mg/mL，预计含有约260 mg/mL的大分子，且不损失无细胞蛋白质表达活性。这种浓缩的细胞提取物可能有助于研究细胞和重组材料之间的差异，并可能有助于开发未来从细胞提取物合成细胞的方法。
The cellular environment differs from that of reconstituted materials mainly because of the presence of highly condensed biomacromolecules. To mimic the environment and conditions in living cells, we developed a method to prepare additive-free, highly concentrated cell extracts. First, we verified the requirement for specific salts and buffers for functional cell-free translation extracts. The S30 fraction of Escherichia coli cell extracts without additives exhibited sufficient cell-free protein production. Next, we established a method to accumulate biological components by gradual evaporation by using a vacuum desiccator. Bovine serum albumin, green fluorescent protein, alkaline phosphatase, and a diluted reconstituted protein expression system were successfully condensed in their active forms using this method. The protein concentration of the prepared cell extract was elevated to 180 mg/mL, which was expected to contain approximately 260 mg/mL macromolecules, without the loss of cell-free protein expression activity. Such a condensed cell extract may be useful for investigating the differences between cells and reconstituted materials and may contribute to the development of methods to synthesize cells from cell extracts in the future.
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