What Is the T7 Expression System and How Does It Work?

The T7 expression system is a method for producing large amounts of a specific protein within a host organism, most commonly the bacterium Escherichia coli (E. coli). This technique uses the bacterium’s cellular machinery to manufacture a foreign protein from an introduced gene. Its efficiency and simplicity have made it one of the most popular protein expression systems used in laboratories. The system is a tool for generating proteins for study or for use as products, with applications ranging from academic research to the industrial production of enzymes and pharmaceuticals.

Core Components of the System

The engine of the T7 system is T7 RNA polymerase, an enzyme from the T7 bacteriophage. This polymerase is fast and can synthesize long strands of messenger RNA (mRNA) without detaching from the DNA template. A defining feature is its specificity, as it exclusively recognizes and initiates transcription from a DNA sequence known as the T7 promoter, dedicating its activity to the gene of interest.

The most common host is the E. coli strain BL21(DE3). These cells are engineered to contain the gene for T7 RNA polymerase integrated into their chromosome. The production of the polymerase is controlled by an inducible promoter called lacUV5. This setup prevents the host cell from producing T7 RNA polymerase until it receives a specific chemical signal.

The final component is the expression vector, a small, circular piece of DNA called a plasmid that carries the Gene of Interest (GOI). The vector must contain a T7 promoter sequence positioned directly upstream of the GOI. When T7 RNA polymerase is produced, it binds to this promoter and begins transcribing the GOI.

Expression vectors also include a selectable marker, such as a gene for antibiotic resistance. This feature allows researchers to grow the bacteria in a medium containing an antibiotic. This ensures that only cells that have taken up the plasmid can survive and multiply.

The Induction Process

In its resting state, the system is kept off by a lac repressor protein that binds to the lac operator sequence on the bacterial chromosome. This physically blocks the host’s RNA polymerase from transcribing the T7 polymerase gene. The system is activated by adding an inducer, most commonly isopropyl β-D-1-thiogalactopyranoside (IPTG). IPTG binds to the lac repressor, causing it to detach from the DNA.

With the repressor gone, the host’s RNA polymerase transcribes the T7 RNA polymerase gene. The cell’s ribosomes then translate the resulting mRNA into T7 RNA polymerase protein. This new polymerase finds the T7 promoter on the expression vector and transcribes the Gene of Interest at a high rate. The host’s ribosomes translate this new mRNA, producing large quantities of the desired protein.

Controlling Protein Expression

A challenge with this system is “leaky” expression, the low-level production of the target protein without an inducer. This occurs because repression of the T7 RNA polymerase gene is not absolute. If the target protein is toxic to the E. coli host, even this small amount can inhibit cell growth, preventing large-scale induction.

To counteract this, T7 lysozyme is used to tighten the system’s control. T7 lysozyme is a natural inhibitor of T7 RNA polymerase that functions by binding directly to the enzyme. This action blocks its ability to initiate transcription and neutralizes any stray polymerase molecules from leaky expression.

This solution is implemented using host strains that carry an additional plasmid, such as pLysS or pLysE, which produce a constant, low level of T7 lysozyme. This pool of lysozyme inactivates the few T7 RNA polymerase molecules made before induction. The pLysS plasmid produces moderate levels of lysozyme, while pLysE produces higher levels for more stringent control of toxic proteins.

Applications and Common Uses

In academic research, the T7 system produces large quantities of proteins for purification. These proteins are used for structural biology studies, like X-ray crystallography and cryo-electron microscopy (cryo-EM), to determine a protein’s shape. The system also generates enzymes for kinetic studies or proteins used as antigens to raise antibodies.

The T7 system is also used in industrial biotechnology and pharmaceutical development. It is employed for the commercial production of enzymes used in products like detergents. The system also manufactures diagnostic reagents and serves as a platform for producing certain biopharmaceuticals and mRNA-based vaccines.

The system is valuable for protein engineering and high-throughput screening. Scientists create large libraries containing many variants of a single protein. The T7 system allows for the rapid production of each variant, enabling researchers to test thousands of modified proteins to find ones with improved properties.