Expressing eukaryotic genes in E coli describe important ste

Expressing eukaryotic genes in E. coli: describe important steps needed to express proinsulin in E.coli

Solution

The first step is the production of enough Pro-insulin producing E.coli bacteria.For this an original amount of E.coli cells containing the plasmid for pro-insulin production will be grown in test tubes containing tryptic soy and kanamycin monosulfate.

Inorder to produce insulin from E.coli, the genetic sequence coding the insulin product has to be fused with a bacerial protein genetic sequence with the E.coli cell will normally translate, and the resulting combination protein products are called Fusion Proteins.The genetically engineered insulin coding sequence is usually inserted into the E.coli bacterial cells a sa component in bacterial plasmid. When the bacterial cells undergo division this plasmid/vector is copied in subsequent bacterial cells. under favourable conditions, the E.coli cells will begin to translate the vector sequences, producing the insulin fusion proteins. These proteins are extracted from the bacterial cells and are processsed in the production of human insulin.

In the Pro-insulin method - the A chain and the B chain are translated in a single step linked together by a C peptide bond forming what is known as Pro-insulin. This pro insulin sequence is expressed as a recombinant fusion protein within the microbial cells. The formation of native insulin from Pro-insulin follows two steps-folding and formation of correct disulfide bridges within the insulin molecule and proteolytic cleavage with subsequent release of connecting C-peptide.