Prokaryotic expression vectors: Vectors capable of carrying an exogenous target gene into prokaryotic cells for expression. Bacillus subtilis expression vectors, E. coli expression vectors, and Streptomyces expression vectors are the three major types of prokaryotic expression vectors. Among these, E. coli expression vectors are the most widely used and the most mature. The main reasons for this are that E. coli has a well-characterized genetic background, high levels of target gene expression, a short culture cycle, and strong resistance to contamination.

     The E. coli expression system primarily consists of the host strain (i.e., E. coli) and the expression vector. An expression vector is based on a cloning vector with additional expression elements. In addition to possessing replication capability, suitable restriction sites, and a selection marker, it is also capable of introducing an exogenous target gene into E. coli and expressing it.

    Expression vectors have the following characteristics: ① Stable genetic replication capability, allowing them to persist stably within E. coli in the absence of selection pressure; ② A dominant selection marker; ③ The transcription level of the promoter is regulatable, with a low background transcription level when not activated; ④ The mRNA transcribed by the promoter terminates at an appropriate position, and the transcription process does not interfere with the replication of the expression vector; ⑤ It possesses restriction sites suitable for the insertion of exogenous genes.

    In summary, prokaryotic protein expression vector plasmids include a replicon, a promoter, a selection marker, a terminator, a ribosome binding site, a multicloning site (MCS), and a fusion tag removal strategy. Common prokaryotic expression vectors include pET-28a(+), pGEX-4T-1, and pET SUMO

Expression Elements of Prokaryotic Expression Vectors

①. Promoters: Promoters play a crucial role in influencing protein expression levels. The lac, trp, tac, T7 phage, and IPL promoters are the primary promoters used in prokaryotic expression vectors. The lac promoter is regulated by IPTG. The lac hybrid promoter is a combination of lac and trp and is also induced by IPTG; its promotional activity far exceeds that of lac or trp alone. The tryptophan (trp) promoter is induced by tryptophan. The T7 phage promoter is highly specific and is controlled by T7 RNA polymerase, whose activity far exceeds that of E. coli polymerase, thereby enabling highly efficient expression of the target gene. The IPL promoter is temperature-regulated and exhibits greater activity than the trp promoter.

②. Transcription terminator: To maintain the stability of the vector system and prevent interference with the expression of the cloned exogenous gene, a transcription terminator is inserted downstream of the multicloning site.

③. Ribosome Binding Site: After mRNA transcription is complete, the host cell’s protein synthesis system is required to fully translate the target protein. Therefore, the mRNA of the inserted exogenous gene must contain a binding site for the host cell’s ribosomes, allowing translation to begin at the start codon and the protein to be synthesized.

Expression Modes of Prokaryotic Expression Vectors

①. Constitutive Expression: Constitutive promoters include the T7 phage promoter, among others. When the target gene is controlled by such promoters, the exogenous target gene is expressed under all conditions. While this method yields high yields, it is not recommended for proteins that are harmful to certain host cells—a characteristic of constitutive expression.

② Inducible Expression: lac and the hybrid promoter tac are IPTG-inducible promoters. trp is a tryptophan-inducible promoter. IPL is a temperature-regulated promoter. Regardless of the regulatory factors involved, inducible expression avoids adverse effects on the host’s early growth and reduces the degradation of the expressed product by proteases, making it suitable for the expression of toxic proteins.

③. Fusion expression: The multiclonal site of the expression vector contains various fusion tags. The target protein is co-expressed with the tag as a fusion protein, which facilitates subsequent protein separation, purification, and detection. Common tags include the GST tag, SUMO tag, and His tag.

④ Secreted Expression: A signal peptide is inserted between the start codon and the target gene, enabling the target protein to traverse the cell membrane and be secreted into the extracellular space. Additionally, the secreted expression product is generally soluble, eliminating the need for a renaturation step.

    Selecting an appropriate expression vector is crucial for the successful expression of the target protein. We should comprehensively evaluate which vector to use for recombinant protein expression and design the experimental protocol based on the characteristics of the target gene and the protein’s downstream applications.