Briefly describe the development of Humulin through rDNA technology.
In the late 1970s, biochemists exploited various tools of rDNA technology for the production of insulin. The first genetically engineered human insulin was marketed as Humulin in 1982. This insulin was more effective compared to pig and cow-derived insulin and was completely free from any allergic response.
To develop Humulin, the insulin gene was isolated from a gene library and inserted into a bacterial plasmid of E. coli. A promoter site derived from the lac operon was inserted next to the insulin gene to facilitate gene expression.
The gene expression produced a fusion protein, where the insulin molecule was fused with the enzyme β-galactosidase (β-gal). Because the processing of preproinsulin was not feasible in bacteria, scientists sidestepped this difficulty by cloning the genes for the A and B insulin chains separately.
The fusion protein, β-gal-insulin, was extracted and treated with cyanogen bromide (CNBr) to separate the β-gal fragment from the A and B chains. Subsequently, the disulfide bonds were re-established between the A and B chains to reconstruct the final product, insulin.
Explanation
The answer is derived from the provided textbook context which outlines the historical timeline (1982), the advantages over animal insulin, and the detailed technical process. It covers the isolation of the gene, the use of E. coli and lac operon, the creation of fusion proteins, the necessity of cloning chains separately due to bacterial limitations, and the final chemical reconstruction using disulfide bonds.
Solution Steps
Step 1: Isolate the insulin gene from a gene library and insert it into an E. coli bacterial plasmid.
Step 2: Insert a lac operon promoter site next to the insulin gene to induce expression.
Step 3: Produce a fusion protein (insulin + β-galactosidase) by cloning genes for A and B chains separately.
Step 4: Treat the extracted fusion protein with cyanogen bromide (CNBr) to cleave the insulin chains.
Step 5: Re-establish disulfide bonds between A and B chains to reconstruct the final insulin product.