The second design flaw in pET28a is in the TIR. Translation initiation is affected by ad hoc plasmid assembly In the remaining pET plasmids, the lac operator was not fused and the T7p CONS is intact. 88 of the 103 plasmids Supplementary Fig. This design flaw is present in all pET expression plasmids containing T7 lac (i.e. The truncation of the T7 promoter is therefore a design flaw that reduces protein production. The experiment indicates that T7p CONS is more efficient than the truncated variant (−17 to +2) that is currently used in pET28a. The restoration of the T7 promoter to T7p CONS did not change the sequence of the lac operator or its proximity from the coding sequence, and we still observed repression prior to induction (Supplementary Fig. 2a, b) and when T7p CONS was engineered into pET15b, which also includes the same T7 lac promoter as pET28a (Supplementary Fig. Similar results were observed when using alternative strains such as C41 and C43 8 (Supplementary Fig. We noted a three-fold increase in production yields of sfGFP in BL21( DE3) pLysS when T7p CONS was used (Fig. To determine if the +3 to +6 nucleotides are important in the context of pET28a, we compared the expression levels of the superfolder green fluorescent protein (His 6-TPS-sfGFP, hereafter referred to as sfGFP) from the commercially available pET28a and a variant where we had engineered in the T7p CONS. Subsequent work suggested that divergence from the consensus T7 promoter (designated T7p CONS) sequence decreases productive transcription initiation 7. At the time it was stated that the lac operator has little effect on induced protein expression levels. We noted that pET28a only contains the −17 to +2 region, as four nucleotides were removed when the lac operator sequence was originally introduced in the early generation pET plasmids (designated T7 lac) 5. The nucleotide sequence is derived from the consensus φ10 promoter in the T7 phage, which is 23 nucleotides long and sits −17 to +6 relative to the messenger RNA (mRNA) start site (Fig. The first design flaw in pET28a is in the T7 promoter. The T7 lac promoter lacks the complete T7 consensus sequence The salient features of pET28a are presented in Fig. In a typical experiment, the coding sequence to be expressed is cloned downstream of, and in frame with, the coding sequence for a poly-histidine purification tag (His 6) and a thrombin protease recognition site (TPS) so that the recombinant protein produced can be easily purified using standardised protocols. Translation initiation is mediated by a Shine–Dalgarno (SD) sequence originating from the major capsid protein of T7 ( gene 10 protein). It contains the T7 promoter and an adjacent lac operator sequence that is included to suppress uninduced expression 5. PET28a is the most popular expression plasmid on the market (described in >40,000 published articles). To date, they have been described in >220,000 published research studies (>12,000 per year Supplementary Fig. These expression plasmids support high levels of transcription in strains of Escherichia coli that contain a lysogenised DE3 phage fragment encoding the T7 RNA polymerase and they have become a workhorse for the scientific community 3, 4. Novagen and Invitrogen subsequently expanded the series to 103 unique expression plasmids. They integrated the strong φ10 promoter for the T7 RNA polymerase (T7 promoter) and the Tφ transcription terminator (T7 terminator) into the pBR322 backbone and established the pET nomenclature (plasmid for expression by T7 RNA polymerase). Studier and co-workers 1, 2 described the first pET expression plasmid more than thirty years ago.
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