Toxic Protein Expression in Escherichia Coli Using a Rhamnose-Based Tightly Regulated and Tunable Promoter System

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Abstract
The refinement of tightly regulated prokaryotic expression systems that permit functional expression of toxic recombinant proteins is a continually evolving process. Unfortunately, the current best promoter options are either tightly repressed and produce little protein, or produce substantial protein but lack the necessary repression to avoid mutations stimulated by leaky expression in the absence ofinducer. In this report, we present three novel prokaryotic expression constructs that are tightly regulated by L-rhamnose and D-glucose. These expression vectors utilize the Escherichia coli rhaT promoter and corresponding regulatory genes to provide titratable, high-level protein yield without compromising clone integrity. Together, these components may enable the stable cloning and functional expression of otherwise toxic proteins. Control Freak In order to produce host-toxic recombinant proteins in E. coli, tight regulation of expression is required. One such tightly regulatable vector is pBAD, which relies upon catabolite repression and positive induction; however, this control comes at the cost of production of only a relatively small amount of recombinant protein. Another alternative is a vector system in which the recombinant protein is under the control of T7 polymerase, which is itself expressed from a lactose-inducible promoter located on the bacterial chromosome. In this case, higher-level expression is achieved, but with a disadvantage that the system is prone to leaky expression. A system that combines tight regulation with high-level induction would be enormously useful; on p. 355, that is just what Giacalone et al. describe. The new vectors are induced by L-rhamnose and repressed by D-glucose. The authors show that their pRHA vectors are capable of efficient protein production upon induction and that the level of recomb...
Funding Information
  • Mpex Pharmaceuticals