Product ReviewsNovagen pET Vector Expression System Transcription & Translation of Target Genes pET Vector Cloning System The pET DNA vector cloning system is the most commonly used bacterial system for the overexpression of genes, and probably the most renowned bacterial expression system available. With humble beginnings more than 25 years ago, the pET plasmid collection now boasts more than 40 different vectors. Modifications to the vectors include hybrid promoters to reduce target gene basal level expression, incorporation of cleavable leader sequences to modify the target protein destination in the cell, fusion partners to facilitate target protein purification, cleavage sites to remove fusion partners, and modified vector DNA multiple cloning sites with more desirable restriction sites. The pET expression system is based on the bacteriophage T7 gene 1 RNA polymerase, an extremely processive DNA-dependent RNA polymerase with minimal promoter requirements. In fact, the stand-alone compact promoter is a lean 20-nucleotide sequence that is unrecognized by the host E. coli RNA polymerase. Thus, a clear advantage of the pET system is the ability to maintain or propagate DNA in the absence of basal level expression of the target gene. Moreover, the T7 gene 3.5 amidase, more commonly referred to as T7 lysozyme, is a natural inhibitor of T7 RNA polymerase. This repression is achieved by cotransforming the pET construct with an additional plasmid encoding T7 lysozyme. This plasmid comes in two flavors, pLysS and pLysE, depending on the desired level of lysozyme expression. In pLysS the lysozyme gene is silent with respect to a cognate promoter, while in pLysE the lysozyme is in an expressed orientation. It is noteworthy that the presence of lysozyme can severely limit protein yields because it remains present throughout the induction. Most often, expression of the gene of interest is accomplished by transforming the pET construct into BL21 (DE3), a B strain E. coli harboring a bacteriophage lambda lysogen with bacteriophage 21 immunity. (Thus the name BL21.) The B strain E. coli is much healthier than the prevalent K-12 strains routinely used in molecular biology and thus, affords more robust growth and potentially improved levels of expression. Moreover, this strain is deficient in both the lon protease and the membrane bound protease OmpT, which may afford improved yields of some proteins. The cryptic DE3 lysogen harbors a single copy of T7 gene 1 (T7 RNA polymerase) under the placUV5 promoter (¡¥Cryptic¡| means that the lysogen cannot enter a vegetative state and lyse the cells because introduction of the T7 gene 1 has ablated lambda integrase gene expression.) The lacUV5 promoter is a mutated version of the lac promoter whose basal activity is dramatically less sensitive to intracellular levels of cyclic AMP¡Xa molecule known to participate in derepression of the lac promoter. Cyclic AMP levels correlate with increasing bacterial culture densities, therefore, the lacUV5 promoter is less sensitive or less ¡§leaky¡¨ at higher culture densities. Incorporation of 1% glucose into the culture medium can significantly enhance repression at this promoter and improve expression yields of particularly toxic target genes. The combination of the lacUV5 promoter and glucose can combine to afford a substantial decrease in the basal level of expression, which, in turn, may effectively increase target protein yields. Many of the pET plasmids also harbor a hybrid T7/lac promoter providing yet another layer of protection against basal expression. Derepression or ¡§induction¡¨ of the system is accomplished by the simple addition of IPTG, which results in the displacement of Lac repressor protein (LacI) from promoters servicing both the T7 gene 1 on the chromosome and the target gene on the pET construct. The flavor of LacI can also be modified, as there are lac alleles that display stronger affinities for the repressor-binding site on the DNA. For example, NovaBlue (DE3) encodes lacIq. Unfortunately, this is a K-12 strain of E. coli. Nearly all of the pET vectors harbor a copy of the lacI gene. Regrettably, all are wild-type LacI. Novagen offers a variety of improvements and modifications to this extremely functional system, including vectors for Blue-White screening, ligation-independent cloning strategies, and altered antibiotic resistance markers. They have also recently added a lacY- BL21 derivative to the repertoire of expression hosts that allows the end user to titrate the level of expression or perform graded inductions with IPTG. In summary, the pET system has numerous options and performs extremely well. It is highly recommended and deserves five out of five stars. It is the ultimate paradigm of knowledge acquired through basic research placed in the public domain to advance and enhance research on numerous forefronts. Advantages: -Robust performance. -Extremely well-documented track record. -Exceptionally diverse variety of options from which to select. -Especially useful for expression of toxic target genes! Disadvantages: -Just one: Why is 10 micrograms of DNA so pricy!? Gripes: -No lacIq1 available on the plasmid. Review by socalsmithers |
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