Question

Outline the important steps for isolation of recombinant insulin (Humulin) from Escherichia coli.

Hint:

Recombinant insulin production involves gene cloning, expression in E. coli, protein extraction, purification, and formulation.

Answer 1

The isolation of recombinant insulin (Humulin) from Escherichia coli involves the following key steps:

1. Gene Cloning: The human insulin gene is inserted into a suitable plasmid vector.
2. Transformation: The recombinant plasmid is introduced into E. coli cells.
3. Expression: Transformed E. coli are cultured to express the insulin protein.
4. Harvesting: Bacterial cells are collected after sufficient growth.
5. Cell Lysis: Cells are broken open to release the insulin protein.
6. Purification: Insulin protein is purified using techniques such as chromatography to remove bacterial contaminants.
7. Folding and Processing: The purified insulin is refolded to its active form if expressed as inactive precursors.
8. Formulation: The final insulin product is formulated for medical use.

These steps ensure safe, large-scale production of human insulin for diabetic patients.