![]() ![]() (a) The panel shows schematic representations of the narG CC(−40.5), CC(−40.5), and CC(−41.5) promoter fragments. Error bars represent SDĮxpression analysis of the narG CC(−40.5) promoter. β‐Galactosidase activities are expressed as nmol ONPG hydrolyzed/min/mg dry cell mass and represent the average of three independent experiments. ![]() Cells were grown in LB medium (LB), minimal salts media (MS), and M9 minimal medium (M9) supplemented with 20 mM sodium nitrate, where indicated. (d) The panel shows measured β‐galactosidase activities in wild‐type JCB387 cells, carrying the ogt104167 promoter fragment cloned into pRW50. Cells were grown in minimal salts media supplemented with 20 mM sodium nitrate, where indicated. (c) The panel shows measured β‐galactosidase activities in wild‐type JCB387 cells, carrying ogt1041, ogt104167, and ogt1052 promoter fragments cloned into the pRW50 lacZ expression vector. At the ogt1041, ogt104167, and ogt1052 promoters, the single DNA sites for NarL are located at positions −77.5, −66.5, and −44.5, respectively, and thus are 65, 55, and 32 bp, respectively, upstream from the corresponding promoter −10 element. Hence, position −77.5 is located between base pairs 77 and 78, upstream from the transcript start. The location of each DNA site for NarL is labeled, according to convention, by the position of the center of the 7–2–7 sequence. The NarL‐binding sites are shown as inverted arrows, −35 and −10 promoter elements are shown as rectangles, and transcript start sites (+1) are indicated by bent arrows. (b) The panel shows schematic representations of the ogt1041, ogt104167 and ogt1052 promoter fragments. The presence of nitrate in the growth medium leads to the phosphorylation of the NarL transcription factor, enabling it to bind to target promoters and control transcript initiation by RNAP (Constantinidou et al., Darwin & Stewart, Santos‐Zavaleta et al., Stewart, 2003). (a) Control of gene expression by nitrate and the NarL transcription activator protein. Biotechnology and Bioengineering published by Wiley Periodicals LLC.Įxpression analysis of the ogt promoter fragments used in this study. As nitrate levels are high in many commercial fertilizers, we demonstrate that controlled RPP can be achieved using readily available and inexpensive garden products.Įscherichia coli biopharmaceuticals recombinant protein production transcription regulation. We show that target proteins, such as green fluorescent protein, human growth hormone, and single-chain variable region antibody fragments can be expressed to high levels using our promoter systems. To simplify this and to cut the cost of RPP, we have developed vectors controlled by the Escherichia coli nitrate-responsive NarL transcription activator protein, which use nitrate, a cheap, stable, and abundant inorganic ion, to induce high-level controlled RPP. However, these can be expensive and, sometimes, chemically unstable. Most Escherichia coli overexpression vectors used for recombinant protein production (RPP) depend on organic inducers, for example, sugars or simple conjugates. ![]()
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