Before the 1980s, insulin was extracted from cows and pigs, often causing immune reactions in humans. The advent of recombinant DNA technology allowed scientists to produce human insulin using Escherichia coli (E. coli), a fast-growing and easily engineered bacterium. The process involves gene construction, transformation, fermentation, and purification to yield safe, effective insulin. While E. coli is highly efficient for insulin production, its inability to perform complex post-translational modifications limits its use for some advanced therapeutic proteins.

From Animal Pancreas to Engineered Bacteria

Prior to recombinant DNA technology, insulin therapy relied on animal-derived insulin from cows and pigs. Although effective, these sources had several drawbacks:

  • It could trigger immune reactions in some patients.
  • Supply was limited and inconsistent.

The breakthrough came when scientists used E. coli, a common lab bacterium, as a host to produce human insulin, revolutionizing diabetes treatment and biotechnology.

Why Use E. coli for Insulin Production?

E. coli is ideal for producing recombinant proteins because:

  • Grows quickly and efficiently.
  • It is genetically easy to manipulate.
  • It can be cultured at industrial scales.

In 1978, Genentech successfully produced the first human insulin using E. coli. By inserting the human insulin gene into the bacteria, the cells began producing insulin identical to that made by the human body, eliminating the need for animal extraction.

Key Steps in Insulin Production Using E. coli

Producing insulin in E. coli involves several critical steps:

1. Gene Construction

Scientists create cDNA sequences for insulin’s two chains, Chain A and Chain B, and insert them into a plasmid. The plasmid also carries a kanamycin resistance gene to ensure only successfully transformed bacteria survive.

2. Transformation of E. coli

The plasmid is introduced into E. coli cells via transformation. Only bacteria that incorporate the plasmid survive in a medium containing kanamycin, ensuring selection of the correct transformants.

3. Cell Growth and Proinsulin Production

Transformed E. coli cells are cultured in nutrient-rich media like tryptic soy broth, which supports rapid division. The plasmid drives production of proinsulin, the inactive precursor to insulin.

4. Scaling Up in Bioreactors

To increase yield, cells are transferred to bioreactors, large tanks that maintain optimal conditions for bacterial growth and protein synthesis. This step maximizes proinsulin production.

5. Cell Harvesting and Inclusion Body Isolation

Once sufficient proinsulin is produced, cells are harvested via filtration and centrifugation. Proinsulin accumulates as inclusion bodies inside the cells. The bacteria are lysed, and inclusion bodies are isolated for further processing.

6. Purification and Conversion to Active Insulin

Inclusion bodies are purified using chromatography and filtration. Chemical steps remove the connecting peptide between Chains A and B, producing active human insulin ready for therapeutic use.

Limitations of Using E. coli

While E. coli is highly effective for insulin, it has limitations:

Lack of Post-Translational Modifications (PTMs)

As a prokaryote, E. coli cannot perform certain modifications common in human cells:

  • Disulfide bond formation: Important for protein stability.
  • Glycosylation: Sugar attachment affecting protein folding and function.
  • Phosphorylation: Critical for signaling proteins.
  • Proteolytic processing: Precise protein trimming.

Because of these limitations, E. coli is unsuitable for some complex biologics. Alternative systems like yeast, mammalian cells, or plant-based systems are used when PTMs are essential.

A Breakthrough with Boundaries

The use of E. coli for insulin was a milestone in biotechnology:

  • Made insulin more accessible, consistent, and safe.
  • Reduced reliance on animal sources.
  • Laid the foundation for modern recombinant protein production.

However, the simplicity that makes E. coli efficient also limits its ability to produce highly complex proteins. For insulin and similar proteins, it remains one of the most reliable tools in biotech.

Why This Matters

Understanding how E. coli produces insulin highlights:

  • The power of genetic engineering in medicine.
  • How microbial systems can revolutionize drug production.
  • The balance between efficiency and complexity in biotechnology.

For millions of people living with diabetes, this process is not just scientific; it’s life-changing.