Abstract
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.
Highlights
To understand biochemical reactions in cells, researchers have reconstituted various biological systems using purified components
Preparing S30 Cell Lysates Using distilled Water (DDW) Buffers and salts are considered essential components required for the preparation of functional cell lysates, especially those for cell-free translation
The harvested E. coli cells were dissolved with 20% sucrose solution and washed with DDW
Summary
To understand biochemical reactions in cells, researchers have reconstituted various biological systems using purified components. Cellular components encapsulated in liposomes have been used to study the behaviors of major macromolecules, i.e., DNA, RNA, and protein [7,8,9,10] Both cytosolic and membrane proteins can be functionally expressed within liposomes [9,11,12,13]. In a previous study, we expressed poreforming membrane proteins in liposomes in order to enable the transport of bioactive peptides and small chemicals from liposomes to living cells [14]. These results suggest that artificial cells can be used to mimic living cells
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