In Humulin production, the b-galactosidase-insulin fusion proteins are purified by affinity chromatography. True or false In Humulin production, the b-galactosidase-insulin f a. TRUE b. FALSE
The correct answer and explanation is:
Correct Answer: a. TRUE
In Humulin production, β-galactosidase-insulin fusion proteins are indeed purified by affinity chromatography. This method is used during the recombinant DNA technology process where human insulin is produced using genetically engineered bacteria, typically Escherichia coli.
The process begins with the insertion of the human insulin gene into a plasmid, which is then introduced into E. coli. To enhance expression and simplify purification, the insulin gene is fused to a gene coding for β-galactosidase, a bacterial enzyme. This creates a fusion protein that includes both β-galactosidase and insulin. The fusion protein is expressed in large quantities in the host bacteria.
This fusion strategy serves two purposes:
- It increases the stability and solubility of the insulin precursor in bacterial cells.
- It simplifies the process of purification because the β-galactosidase portion of the fusion protein can specifically bind to an affinity matrix.
Affinity chromatography is then used to isolate the fusion protein. This technique relies on the specific interaction between β-galactosidase and a ligand attached to a chromatography resin. When the cell lysate containing the fusion protein is passed through the affinity column, only the β-galactosidase-insulin fusion protein binds to the column, while other proteins wash away.
After purification, chemical or enzymatic methods are used to cleave the insulin segment from the fusion protein. The insulin chains (A and B chains) are then joined correctly through disulfide bonding to form bioactive recombinant human insulin, known commercially as Humulin.
This approach was a breakthrough in biotechnology, allowing for safe, large-scale production of human insulin without relying on animal pancreases.