TA-Cloning for Diabetes Treatment: Expressing Corynebacterium Malic Enzyme Gene in E. coli.

Abstract

Diabetes mellitus represents a persistent metabolic condition marked by heightened levels of blood glucose, presenting a considerable worldwide health concern, and finding targeted treatment for it is a crucial priority for global health. Gram-positive aerobic bacteria, predominantly inhabiting water and soil, are known carriers of various enzyme-encoding genetic material, which includes the malic enzyme gene that plays a role in insulin secretion. Corynebacterium glutamicum bacteria (ATCC 21799) were acquired from the Pasteur Institute and confirmed using microbiological and molecular tests, including DNA extraction. After identification, gene purification and cloning of the maeB gene were performed using the TA Cloning method. Additionally, the enhancement of enzyme expression was assessed using the expression vector pET-28a, and validation of simulation results was monitored through a real-time PCR analysis. Based on previous studies, the malic enzyme plays a pivotal role in maintaining glucose homeostasis, and increased expression of this enzyme has been associated with enhanced insulin sensitivity. However, the production of malic enzyme has encountered numerous challenges and difficulties. This study successfully isolated the malic enzyme genes via Corynebacterium glutamicum and introduced them into Escherichia coli for high-yield production. According to the results, the optimum temperature for the activity of enzymes has been identified as 39°C.