10.4 Human Insulin Production in Bacteria

This document outlines the process of producing human insulin using recombinant DNA technology, where the human insulin gene is inserted into bacteria (e.g., E. coli) to synthesize the protein.

1. The Human Insulin Gene (INS)

• Function: Codes for the hormone insulin, which regulates blood sugar.
• Structure: Consists of two polypeptide chains, chain A (21 amino acids) and chain B (30 amino acids), linked by disulfide bonds.
• Location: Located on human chromosome 11 at position 11p15.5.
• Genetic Makeup: The gene is approximately 1.5 Kbp long, containing 3 exons and 2 introns.
• Synthesis Pathway in Humans:
  1. Pre-pro-insulin (109 amino acids) is first synthesized.
  2. It becomes inactive pro-insulin (86 amino acids) inside the cell.
  3. Proteolytic enzymes cleave it into active insulin (51 amino acids).

2. Steps in Recombinant Insulin Production

Step 1: Identification and Isolation of the Insulin Gene

The gene responsible for producing human insulin (the INS gene) is identified and isolated from a human DNA sample. Because bacteria cannot process introns, synthetic genes corresponding to the final A and B chains are created. This is often done using reverse transcriptase to create cDNA from mRNA.

Step 2: Selection of a Suitable Plasmid Vector

A plasmid (a small, circular DNA molecule from bacteria) is chosen to act as a vector to carry the insulin gene into the host bacterium.

• Common Plasmids:

  • pBR322: Contains resistance genes for ampicillin and tetracycline.
  • pUC18/pUC19: Derivatives of pBR322, contain an ampicillin resistance gene.

• Key Features of a Good Vector:

  • Small size
  • Origin of replication (allows it to be copied by the host cell)
  • High copy number (many copies per cell)
  • Multiple unique restriction sites (for gene insertion)

Step 3: Creation of Recombinant DNA

• The synthetic genes for insulin's chain A and chain B are inserted into two separate plasmids.
• Each gene is inserted next to the lacZ gene, which codes for the enzyme β-galactosidase.
• This creates a fusion gene. A promoter sequence is also included to ensure the gene is expressed by the bacterium.
• DNA Ligase is used to seal the DNA fragments together.

Step 4: Insertion into Host Cells (Transformation)

The recombinant plasmids (one with the gene for chain A, one with the gene for chain B) are introduced into separate host bacteria, typically Escherichia coli. This process is called transformation.

Step 5: Production of Insulin Chains (Expression)

• The transformed bacteria are grown in large fermentation tanks with optimal culture conditions.
• As the bacteria multiply, they replicate the plasmids, cloning the insulin gene.
• The bacteria then express the fusion gene, producing a fusion protein:
  • β-galactosidase-A chain
  • β-galactosidase-B chain
• The large β-galactosidase protein protects the small insulin chains from being destroyed by the bacteria's own protease enzymes.

Step 6: Extraction and Separation

• The bacterial cells are harvested, and the fusion proteins are isolated.
• The insulin chains are separated from the β-galactosidase protein by treatment with cyanogen bromide. This chemical specifically cleaves at the amino acid methionine, which was intentionally engineered to be at the junction between the two proteins.

Step 7: Assembly of Functional Insulin

• The purified A and B chains are mixed together in-vitro.
• They are chemically treated (e.g., with sodium disulphonate and sodium sulphite) to form the correct disulfide bridges between cysteine residues.
• This assembly process creates biologically active human insulin.

Step 8 and 9: Final Purification, Testing, and Packaging

• The active insulin is further purified to ensure it is free of any bacterial by-products.
• It undergoes rigorous testing for quality, safety, and effectiveness.
• Finally, the pure insulin is packaged into vials or insulin pens for medical use.

Biological Significance

This technology provides a reliable and scalable source of human insulin for treating diabetes mellitus, a global health issue. It avoids the immunological reactions and supply limitations associated with animal-derived insulin (e.g., from pigs or cows).