Expression of recombinant proteins traditionally require a cellular system to transcribe and translate foreign DNA to a desired protein. The process requires special knowledge of the specific cellular metabolism in use and is often time consuming and labour intensive. A cell free expression system provides an opportunity to express recombinant proteins without consideration of the living cell. Instead, a cell free system relies on either a cellular lysate or recombinant proteins to carry out protein synthesis, increasing overall production speed and ease of handling. The one-pot cell free setup is commonly known as an in vitro transcription/translation reaction (IVTT).

Here we focused on a PURE (Protein synthesis Using Recombinant Elements) IVTT system based on recombinant proteins from Escherichia coli. We evaluated the cell free system's ability to express functional insulin analogues compared to Saccharomyces cerevisiae, a well-established system for large scale production of recombinant human insulin and insulin analogues.

Significantly, it was found that correct insulin expression and folding was governed by the inherent properties of the primary amino acids sequence of insulin, whereas the eukaryotic features of the expression system apparently play a minor role. The IVTT system successfully produced insulin analogues identical in structure and with similar insulin receptor affinity to those produced by yeast. In conclusion we demonstrate that the PURE IVTT system is highly suited for expressing soluble molecules with higher order features and multiple disulphide bridges.

重组蛋白的表达传统上需要细胞系统将外源 DNA 转录和翻译成所需的蛋白质。该过程需要对所使用的特定细胞代谢的特殊知识，并且通常是耗时和劳动密集型的。无细胞表达系统提供了在不考虑活细胞的情况下表达重组蛋白的机会。相反，无细胞系统依赖于细胞裂解物或重组蛋白来进行蛋白质合成，从而提高整体生产速度和易于处理。一锅无细胞设置通常称为体外转录/翻译反应 (IVTT)。

在这里，我们专注于基于来自大肠杆菌的重组蛋白的 PURE（使用重组元素的蛋白质合成）IVTT 系统。我们评估了与酿酒酵母相比，无细胞系统表达功能性胰岛素类似物的能力，酿酒酵母是一种用于大规模生产重组人胰岛素和胰岛素类似物的成熟系统。

值得注意的是，发现正确的胰岛素表达和折叠受胰岛素一级氨基酸序列的固有特性支配，而表达系统的真核特征显然起次要作用。IVTT 系统成功地生产了与酵母生产的胰岛素类似物结构相同且具有相似胰岛素受体亲和力的胰岛素类似物。总之，我们证明 PURE IVTT 系统非常适合表达具有更高阶特征和多个二硫键的可溶性分子。