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Introduction <p>Severe insulin deficiency in animals causes diabetes mellitus, which is characterized by chronic hyperglycemia. Insulin has been used therapeutically for diabetes mellitus since 1921. Improvements in the recombinant insulin produced in bacteria have been pursued using diverse strategies during this time.</p> Methods <p>In the present study, we describe the development of insulin-CBT2, a single-chain insulin (SCI) variant that consists of a single polypeptide chain, like proinsulin. However, whereas proteolytic cleavage is required to transform proinsulin into active insulin, we found that insulin-CBT2 exhibits biological activities without the necessity for proteolytic cleavage.</p> Results <p>Insulin-CBT2 has a similar secondary structure to proinsulin, but the C-peptide is replaced by a flexible linker peptide, which causes a structural reverse turn and permits insulin-CBT2 to activate insulin receptors. Therefore, we investigated the influence of this structural change on the physical and structural characteristics of insulin-CBT2. Although the polarity was increased, and the structure of the insulin was altered, disulfide bond pairing was present at the same positions as in commercial insulin (c-insulin). We also compared the biological activities of insulin-CBT2 and c-insulin, and found that insulin-CBT2 exhibited slightly slower adipocyte differentiation and glucose uptake, in pre-adipocytes.</p> Conclusion <p>Thus, there are biophysical advantages associated with insulin-CBT2, but its bacterial production can also be scaled up without the necessity for proteolytic enzyme digestion and re-purification. This may provide a low-cost means of improving the quality of life of diabetic patients requiring insulin replacement therapy.</p>

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Production of Active Single-Chain Insulin in E. coli: No PTM for Insulin Activation

  • 期刊论文

  • 发表日期：

    2025年6月17日

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  • Byung Chull An,
  • Jaewon Ha,
  • Seungwoo Kim,
  • Yongku Ryu,
  • Myung Jun Chung

摘要

Introduction

Severe insulin deficiency in animals causes diabetes mellitus, which is characterized by chronic hyperglycemia. Insulin has been used therapeutically for diabetes mellitus since 1921. Improvements in the recombinant insulin produced in bacteria have been pursued using diverse strategies during this time.

Methods

In the present study, we describe the development of insulin-CBT2, a single-chain insulin (SCI) variant that consists of a single polypeptide chain, like proinsulin. However, whereas proteolytic cleavage is required to transform proinsulin into active insulin, we found that insulin-CBT2 exhibits biological activities without the necessity for proteolytic cleavage.

Results

Insulin-CBT2 has a similar secondary structure to proinsulin, but the C-peptide is replaced by a flexible linker peptide, which causes a structural reverse turn and permits insulin-CBT2 to activate insulin receptors. Therefore, we investigated the influence of this structural change on the physical and structural characteristics of insulin-CBT2. Although the polarity was increased, and the structure of the insulin was altered, disulfide bond pairing was present at the same positions as in commercial insulin (c-insulin). We also compared the biological activities of insulin-CBT2 and c-insulin, and found that insulin-CBT2 exhibited slightly slower adipocyte differentiation and glucose uptake, in pre-adipocytes.

Conclusion

Thus, there are biophysical advantages associated with insulin-CBT2, but its bacterial production can also be scaled up without the necessity for proteolytic enzyme digestion and re-purification. This may provide a low-cost means of improving the quality of life of diabetic patients requiring insulin replacement therapy.