T7 expression system
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The T7 expression system is used in the field of microbiology to clone recombinant DNA using strains of E. coli.[1] It is the most popular system for expressing recombinant proteins in E. coli.[2] In order to perform this method, an expression vector (most commonly the pET expression vector) is created which integrates two components: a T7 promoter and a gene of interest downstream of the promoter and under its control. The expression vector is transformed into one of several relevant strains of E. coli, most frequently BL21(DE3). The E. coli cell also has its own chromosome, which possesses a gene that is expressed to produce T7 RNA polymerase. (This polymerase originates from the T7 phage which infects bacterial cells and integrates this DNA into the bacterial host DNA.) T7 RNA polymerase is responsible for beginning transcription at the T7 promoter of the transformed vector. The T7 gene is itself under the control of a lac promoter (or more specifically, a variant of the lac promoter known as the lacUV5 promoter). Normally, both the lac promoter and the T7 promoter are repressed in the E. coli cell by the Lac repressor. In order to initiate transcription, an inducer must bind to the lac repressor and prevent it from inhibiting the gene expression of the T7 gene. Once this happens, the gene can be normally transcribed to produce T7 RNA polymerase. T7 RNA polymerase, in turn, can bind to the T7 promoter on the expression vector and begin transcribing its downstream gene of interest. To stimulate this process, the inducer IPTG can be added to the system. IPTG is a reagent which mimics the structure of allolactose, and can therefore bind to the lac repressor and prevent it from inhibiting gene expression. Once enough IPTG is added, the T7 gene is normally transcribed and so transcription of the gene of interest downstream of the T7 promoter also begins.[3]
The BL21 and pET expression system with the use of T7 RNA polymerase was developed in 2008 and is a representative recombinant protein expression system.[3] One of its strongest advantages is the rate at which the recombinant protein is expressed. Expression of a recombinant protein under the control of the T7 RNA poylmerase is eight times faster than it is with a protein whose expression is under the control of E. coli RNA polymerase.[4]
References
- ^ Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89.
- ^ New England BioLabs. "T7 Expression". Accessed Oct 4 2021. https://international.neb.com/applications/protein-expression/protein-expression-in-e-coli/t7-expression
- ^ a b Wagner S, Klepsch MM, Schlegel S, Appel A, de Gier JW. Tuning Escherichia coli for membrane protein overexpression. PNAS. 2008;105:14371–6. doi: 10.1073/pnas.0804090105.
- ^ Iost I, Guillerez J, Dreyfus M (1992) Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo. J Bacteriol 174:619–622.