Bacteria can be engineered to produce and secrete proteins native to more complex organisms, like animals and plants. This capability is a technique in modern biotechnology that allows for the large-scale production of proteins. While getting a bacterium to manufacture a foreign protein is a standard laboratory practice, convincing it to export that protein presents several biological challenges. The process begins with getting the genetic instructions into the bacterial cell using genetic engineering.
The Genetic Engineering Process for Protein Expression
Expressing a eukaryotic protein in bacteria involves introducing its specific gene into a bacterial host. Scientists first isolate the desired gene from the eukaryotic organism’s DNA and insert it into a small, circular piece of DNA called a plasmid. Plasmids act as vehicles, or vectors, to carry the foreign gene into the host. The resulting combination of a plasmid containing a foreign gene is known as recombinant DNA.
This recombinant plasmid is then introduced into a host bacterium, like a strain of Escherichia coli. The process of getting the bacteria to take up the plasmid is called transformation, which is encouraged using chemical or electrical pulses to make the cell membrane permeable. Once inside, the plasmid replicates independently of the bacterium’s chromosome, creating many copies of the eukaryotic gene.
The bacterium’s cellular machinery reads the inserted eukaryotic gene as if it were one of its own. The plasmid can be thought of as a recipe card added to the bacterium’s kitchen, and the bacterium follows the new instructions to synthesize the eukaryotic protein. Specific DNA sequences on the plasmid, known as promoters, act as on-switches, allowing scientists to control when protein production begins.
Obstacles in Eukaryotic Protein Production
Getting the genetic instructions into the bacterium does not guarantee a functional protein. Several biological mismatches between prokaryotic bacteria and more complex eukaryotic cells create hurdles. The internal environment of a bacterium is not always suited for the production of complex foreign proteins.
A common issue is improper protein folding. Bacteria can produce the eukaryotic protein at such a high rate that the polypeptide chains do not have time to fold into their correct three-dimensional shapes. Instead, they misfold and clump into dense, insoluble aggregates known as inclusion bodies. These protein masses are biologically inactive and require laboratory procedures to solubilize and refold them.
Many eukaryotic proteins also require chemical additions called post-translational modifications (PTMs) to become functional. These modifications, such as attaching sugar molecules (glycosylation), are carried out by molecular machinery found in eukaryotic cells. Bacteria like E. coli lack this machinery, meaning they produce a version of the protein that may not be stable or active.
Another challenge is codon bias. Organisms use three-letter DNA “words,” or codons, to specify which amino acid to add to a protein chain. Different organisms show a preference for using certain codons over others. If a eukaryotic gene contains many codons that are rare in the host bacterium, protein synthesis can slow or stop, reducing the yield.
Mechanisms for Bacterial Secretion
After addressing internal production challenges, the next step is getting the protein out of the cell. Bacteria possess natural systems for exporting their own proteins across their cell membranes. Scientists can hijack these native pathways to transport engineered eukaryotic proteins, which simplifies purification.
This is achieved using a short chain of amino acids called a signal peptide. This sequence acts as a molecular “shipping label” that is genetically fused to the beginning of the eukaryotic protein. The bacterium’s secretion machinery, such as the Sec or Tat pathways, recognizes this signal peptide. The Sec pathway transports unfolded proteins across the inner membrane, while the Tat pathway transports already folded proteins.
Once the signal peptide is recognized, the secretion apparatus pushes the attached eukaryotic protein through a channel in the cell membrane. Gram-negative bacteria like E. coli have two membranes, an inner and an outer, which adds a layer of complexity. Secretion systems like the Type II pathway move the protein into the space between the membranes (the periplasm) before exporting it across the outer membrane. Selecting the right signal peptide directs the eukaryotic protein into a specific secretion pathway.
Practical Applications in Science and Medicine
The production and secretion of eukaryotic proteins by bacteria has many applications in medicine and industrial processes. This technology allows for the cost-effective, large-scale production of proteins that would be difficult to obtain from their natural sources.
In the pharmaceutical field, this process is used to produce important medicines. Human insulin, used to treat diabetes, was an early success of this technology, replacing less efficient and potentially allergenic insulin from pigs and cattle. Human growth hormone is also now produced in bacteria, avoiding risks associated with its previous extraction from human cadavers.
Beyond medicine, bacterially-secreted proteins are used in various industries. Enzymes, which are proteins that speed up chemical reactions, are manufactured in large quantities for commercial use. For example, proteases and lipases are ingredients in laundry detergents, where they break down protein and fat stains. In the food industry, these enzymes are used for tasks like cheese making and clarifying fruit juices.