The use of simple, fast-growing bacteria as factories for producing complex human, animal, or plant proteins is a significant advancement in biotechnology. This process involves inserting a eukaryotic gene into a bacterial cell, compelling the prokaryotic organism to manufacture the foreign compound. The goal is often to have the bacteria secrete the protein outside the cell wall into the surrounding growth medium. Secretion drastically simplifies the purification process and lowers the final production cost. This technology has transformed the manufacture of biopharmaceuticals, making previously rare and expensive proteins widely accessible for medical and industrial use.
The Fundamental Biological Challenge of Production
Simply inserting a eukaryotic gene into a bacterial cell does not guarantee the production of a functional protein. The primary difficulty lies in the difference between the cellular machinery of prokaryotes and eukaryotes. Eukaryotic proteins often require specific chemical modifications after translation, known as Post-Translational Modification (PTM). Bacteria lack the specialized cellular compartments, such as the endoplasmic reticulum and Golgi apparatus, that perform complex PTMs like glycosylation (attaching sugar chains).
The absence of this machinery means that many complex eukaryotic proteins produced in bacteria will be structurally incomplete or non-functional. Furthermore, the bacterial cytoplasm is a reductive environment, which prevents the formation of disulfide bonds—chemical cross-links necessary to stabilize the three-dimensional structure of many secreted proteins. When foreign proteins are expressed too quickly, the bacterial cell’s folding mechanisms become overwhelmed. This causes misfolded proteins to aggregate into inactive, insoluble clumps called “inclusion bodies.” These inclusion bodies require a costly, complex process of extraction, solubilization, and refolding to potentially become functional.
Strategies for Secretion and Export
To overcome incorrect folding and inclusion body formation, scientists engineer bacteria to secrete the target protein. Secretion moves the protein out of the cytoplasm into the periplasm or external medium, providing a better environment for proper folding and simplifying downstream purification. This is achieved by hijacking the bacteria’s native protein export systems, primarily the General Secretion (Sec) pathway and the Twin-Arginine Translocation (Tat) pathway.
The key to directing the protein to these pathways is the use of a “signal peptide,” a short sequence of amino acids fused to the beginning of the eukaryotic protein. This signal peptide acts like a postal code, recognized by the bacterial export machinery. The Sec pathway transports proteins in an unfolded state across the inner cell membrane, allowing them to fold correctly in the more oxidative environment of the periplasm, which is conducive to disulfide bond formation.
Conversely, the Tat pathway transports proteins that are already folded and assembled in the cytoplasm. This pathway is often utilized for proteins that require cofactor binding or complex assembly before they can function. Specific bacterial strains, such as modified Escherichia coli and Bacillus subtilis, are optimized for these secretion pathways to increase yield and efficiency. Bacillus subtilis is frequently chosen for its natural ability to secrete proteins directly into the growth medium, minimizing the need to break open the cells.
Real-World Applications in Medicine and Biotechnology
The technology of using bacteria to express and secrete eukaryotic proteins has been foundational to the modern biopharmaceutical industry. The most well-known example is the production of human insulin. Before this technique, insulin for diabetic patients was harvested from the pancreases of pigs and cows, leading to supply issues and sometimes adverse immune reactions.
Today, recombinant human insulin is mass-produced by E. coli genetically engineered to express the human gene. This process ensures a consistent, high-purity, and cost-effective supply, demonstrating scalability. Another significant therapeutic application is the production of human growth hormone (hGH), used to treat growth deficiency.
Many other therapeutic proteins, including specific cytokines like Interleukin-2 (IL-2), are also produced using engineered bacteria. Although IL-2 is naturally glycosylated in human cells, the non-glycosylated version produced in E. coli retains its full biological activity, making bacterial expression an efficient route. Beyond medicine, this technology produces industrial enzymes for detergents, food processing, and textile manufacturing. The benefits of bacterial expression systems—rapid growth, simple nutritional requirements, and high-density fermentation—make them the preferred choice for large-scale, low-cost production of many non-glycosylated or simple eukaryotic proteins.