Question

Give the purification scheme for isolation of Humulin from E. coli.

Hint:

Focus on both the source organism, and the different methods that need to be employed when working with intracellular proteins.

Answer 1

Fermentation Broth: E. coli} containing the recombinant plasmid (carrying the gene for human insulin, “Humulin") is cultured in a fermenter. Cell Harvest and Lysis: After the fermentation is complete, the bacterial cells are harvested and lysed to release all the intracellular contents including the Humulin protein. Cell debris removal and protein solubilization: Cell debris is removed by methods like centrifugation and filtration. The Humulin protein needs to be solubilised by using buffers, before it can be separated from other proteins. Chromatography: The solubilized protein mix is then passed through chromatographic columns to purify the protein using different methods based on size, charge or affinity. Refolding and purification: The protein can be purified and refolded to reach its active form. Final Purification: The resulting product may undergo additional steps of purification to meet the pharmaceutical standards.