Generation of an AraC-araBAD promoter-regulated T7 expression system

An alternative and facile delivery system for T7 RNA polymerase has been devised and constructed. T7 gene 1 has been placed under control of the araBAD promoter element regulated by the AraC protein. Cotransformation of the resultant plasmid, pTara, with one containing a target gene under T7 promoter-regulated expression potentially allows repression by glucose and induction by arabinose in the range of 0.5 to 20 mM sugar concentration. To demonstrate the efficacy of this expression system, the p53 gene under T7 promoter control in two different plasmids was expressed in Escherichia coli using pTara as the source of T7 RNA polymerase. Repression and induction of p53 were achieved in both a lower and higher copy number plasmid, although the levels of induction were higher with the lower copy number expression vector. Cotransformation of an expression plasmid with pTara provides a low-cost method of T7 RNA polymerase-regulated expression that can be fine-tuned using glucose and arabinose concentrations to balance protein expression with potential solubility or toxicity problems.     

Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter

The coding sequence for bacteriophage T7 RNA polymerase has been cloned and expressed under control of a cognate T7 promoter, a configuration referred to as an autogene. Cloning a T7 autogene in a derivative of plasmid pBR322 in Escherichia coli was achieved by a combination of blocking initiation at the T7 promoter with bound lac repressor and inhibiting the polymerase itself by T7 lysozyme. Neither type of inhibition by itself was sufficient to control the autogene. Upon unblocking the T7 promoter with added inducer. T7 RNA polymerase produced its own mRNA, leading to autocatalytic production of polymerase protein. T7 autogenes may be useful for developing high-level gene expression systems in a variety of cell types, with little if any need for the host cell RNA polymerase.     

The expression of proUK in Escherichia coli: the vgb promoter replaces IPTG and coexpression of argU compensates for rare codons in a hypoxic induction model

The expression of the proUK gene was improved by the coexpression of the argU gene cloned in a moderate copy number vector. As the proUK gene contains 2% AGG/AGA codons, which is much higher than the normal frequency in E. coli, about 0.14%-0.21%, the argU gene cloned in a multicopy plasmid was coexpressed with the proUK expression vector in our experiments. In E. coli strain BL21(DE3), IPTG is known to induce the expression of T7 RNA polymerase gene and this enzyme can transcribe the proUK gene under the control of the T7 promoter leading to expression of proUK. To replace IPTG by a cheaper alternative on a large scale, we constructed a plasmid in which the vgb promoter--which is known to be activated by the onset of hypoxic conditions--controls the T7RNA polymerase gene expression. Low oxygen conditions were then used to activate the vgb promoter causing T7RNA polymerase gene expression and finally leading to the expression of proUK as inactive inclusion bodies. Our experiments on a large scale in a bioreactor show that the expression of proUK accounts for about 30% of total protein after about 6 h of anaerobic cultivation, so the presented model represents an economical alternative to IPTG induction.     

Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli

Plasmids containing a ColE1 origin of replication are widely used for cloning purposes in Escherichia coli. Among the host factors that affect the copy number of ColE1 plasmids is the E. coli protein poly(A) polymerase I (PAP I), which regulates the intracellular level of RNA I, a ColE1-encoded negative regulator of plasmid replication. In strains that lack PAP I, RNA I levels are elevated, resulting in reduced levels of ColE1 plasmids in the cell. PAP I is encoded by the gene pcnB. We devised a genetic approach, based on the identification of multicopy suppressor clones, to identify trans-acting factors that can help offset the ColE1 plasmid copy number defect in a pcnB (-) genetic background. Using this strategy, we identified suppressors that mapped to two regions of the E. coli chromosome. The suppressor activity of one of the chromosomal regions was localized to the rssB gene, a response regulator gene known to be involved in the turnover of the stationary-phase sigma factor, RpoS. The second suppressor maps to min 55.4 of the E. coli chromosome, and the factor responsible for the suppressor activity appears to be a novel RNA or protein.