Abstract

Insulin has captured researchers’ attention worldwide. There is a rapid global rise in the number of diabetic patients, which increases the demand for insulin. Current methods of insulin production are expensive and time-consuming. A PCR-based strategy was employed for the cloning and verification of human insulin. The human insulin protein was then overexpressed in E. coli on a laboratory scale. Thereafter, optimisation of human insulin expression was conducted. The yield of human insulin produced was approximately 520.92 (mg/L), located in the intracellular fraction. Human insulin was detected using the MALDI-TOF-MS and LC–MS methods. The crude biosynthesised protein sequence was verified using protein sequencing, which had a 100% similarity to the human insulin sequence. The biological activity of human insulin was tested in vitro using a MTT assay, which revealed that the crude biosynthesised human insulin displayed a similar degree of efficacy to the standard human insulin. This study eliminated the use of affinity tags since an untagged pET21b expression vector was employed. Tedious protein renaturation, inclusion body recovery steps, and the expensive enzymatic cleavage of the C-peptide of insulin were eliminated, thereby making this method of biosynthesising human insulin a novel and more efficient method.

中文翻译：

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一种在大肠杆菌（E. coli）中生产人胰岛素的新型高效生物合成方法。

摘要

胰岛素已引起全世界研究人员的关注。全球糖尿病患者数量迅速增加，这增加了对胰岛素的需求。当前的胰岛素生产方法昂贵且费时。基于PCR的策略用于人胰岛素的克隆和验证。然后在大肠杆菌中过表达人胰岛素蛋白在实验室规模上。此后，进行人胰岛素表达的优化。位于细胞内部分的人胰岛素产量约为520.92（mg / L）。使用MALDI-TOF-MS和LC-MS方法检测人胰岛素。使用蛋白质测序验证了粗生物合成的蛋白质序列，该序列与人胰岛素序列具有100％的相似性。使用MTT测定法在体外测试了人胰岛素的生物学活性，这表明粗制的生物合成人胰岛素显示出与标准人胰岛素相似的功效。由于使用了未标记的pET21b表达载体，该研究消除了亲和标签的使用。乏味的蛋白质复性，包涵体恢复步骤，