Monday, July 25, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
The L-arabinose (Ara)-controlled T7 expression system was previously constructed by creation of an Escherichia coli BL21(BAD) strain. The production of recombinant proteins in this stain was stringently regulated and reached a very high level upon induction with Ara. Nevertheless, this system is still associated with inherent problems of interference with glucose and of the all-or-nothing induction profile at a subsaturating level of Ara. In this study, these problems were circumvented. This was approached by deletion of ptsG gene and the araFGH and araBAD operon. In addition, the constitutive expression of genomic araE was enhanced to obtain a strain, designated BAD-5. By expression of the faster degrader GFP(LAA), 80% of BAD-5 strain was found visually bright at a subsaturating level of Ara irrespective of glucose. In marked contrast, less than 10% of BL21(BAD) strain exhibited visual brightness and the strain remained dark in the presence of glucose. Moreover, a saturated level of luciferase from Renilla reniformis (Rluc) could be readily obtained in BAD-5 strain at 20 μM Ara regardless of glucose. Overall, it indicates the promising use of this newly constructed system for efficient and homogenous production of recombinant proteins.