Recombinant E. coli Glutathione reductase (gor) (a.a. 1-450) [His]

Functional antibody fragments with native disulfide bonds can be expressed in Escherichia coli trxB gor mutant strains having an oxidizing cytoplasm that allows the formation of disulfide bonds. However, expression yields in the cytoplasm are generally lower than those obtained by secretion into the periplasm. We developed a novel methodology for the screening of genomic DNA fragments that enhance expression yields of scFvs in the cytoplasm of trxB gor cells by capitalizing on bacteriophage lambda display. The anti-digoxin 26.10 scFv was displayed on lambda as a fusion to the coat protein gpD. A genomic E. coli library was cloned into lambda gt11 downstream from the lac promoter and used to lysogenize cells transformed with a plasmid encoding the scFv-gpD fusion. Following induction of expression of the cloned gene fragments, phage was prepared and screened for improved functional display via panning against immobilized hapten. Phage exhibiting improved display was isolated after two rounds. One of the isolated clones, encoding the N-terminal domain of the alpha-subunit of RNA polymerase (alpha-NTD), was shown to increase the yield of scFv expressed in soluble form in the cytoplasm. (c) 2007 Elsevier B.V. All rights reserved.

In plants, defence against oxygen free radicals relies on the Asada-Halliwell pathway a series of enzyme-catalysed redox reactions that reduce toxic oxygen species to water at the expense of reductant. The rare limiting step in this cycle is believed to be the reduction of glutathione, by glutathione reductase (GR, EC 1.6.4.2), using NADPH. As a tool to understanding the plant antioxidant system, tobacco (Nicotiana tabacum cv. Samsun) containing the Escherichia coli GR gene (gor) has been engineered by Agrobacterium tumefaciens-mediated transformation. The response of these transformants to light has been examined and data indicates that both plant and E. coli glutathione reductases are subject to diurnal variation. Total GR activity (GRA) in control and transgenic plants increases following exposure to light, but the increases are greater in the transgenic plants. Separation of native and E. coli GR isoforms indicates that this is due to increases in both tobacco and E. coli GRA. Rises in E. coli GRA can be ascribed primarily to a light-mediated post-translational activation. Although E. coli GR transcript and protein levels vary over the photoperiod, the nature and magnitude of the fluctuations do not account for the changes in E. coli GRA, suggesting that E. coli GR is subjected to a light-mediated post-translational activation, While tobacco GR levels could not be assessed, it is likely that tobacco GR is subject to the same post-translational activation.