ABSTRACT
Hypoxia, a condition of reduced oxygen, occurs in a wide variety of biological contexts, including solid tumors and bacterial biofilms, which are relevant to human health. Consequently, the development of chemical tools to study hypoxia is vital. Here we report a hypoxia-activated, small-molecule-mediated gene expression system using a bioreductive prodrug of the inducer isopropyl 1-thio-ß-d-galactopyranoside. As a proof-of-concept we have placed the production of a green fluorescent protein under the control of hypoxia. Our system has the potential to be extended to regulate the production of any given protein of choice.
Subject(s)
Gene Expression/drug effects , Green Fluorescent Proteins/metabolism , Isopropyl Thiogalactoside/analogs & derivatives , Isopropyl Thiogalactoside/pharmacology , Prodrugs/pharmacology , Anaerobiosis/physiology , Cell Line, Tumor , Escherichia coli/genetics , Escherichia coli/metabolism , Green Fluorescent Proteins/genetics , Humans , Isopropyl Thiogalactoside/chemical synthesis , Isopropyl Thiogalactoside/metabolism , Nitrofurans/chemical synthesis , Nitrofurans/metabolism , Oxazines/chemical synthesis , Oxazines/metabolism , Prodrugs/chemical synthesis , Prodrugs/metabolismABSTRACT
[reaction: see text] Addition of isopropyl-beta-d-thiogalactopyranoside (IPTG) to bacterial cultures is often used to induce expression of plasmid-based genes for the production of recombinant proteins under control of the lac promoter, but a simple method to circumvent the inherent instability of this compound has not been addressed experimentally. Herein we report the first synthesis of isobutyl-C-galactoside (IBCG), the C-glycoside analogue of IPTG, and show that IBCG is superior to IPTG in inducing protein expression over long induction times.
Subject(s)
Escherichia coli/genetics , Galactosides/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Isopropyl Thiogalactoside/chemical synthesis , Protein Biosynthesis , DNA/biosynthesis , DNA/genetics , Escherichia coli/drug effects , Escherichia coli/metabolism , Green Fluorescent Proteins , Lac Operon/genetics , Luminescent ProteinsABSTRACT
Production of beta-glucosidase in Aspergillus niger B1 is subjected to catabolic repression by glucose. Aspergillus niger B1 grown on bran as a carbon source secreted beta-glucosidase. The maximum level of the enzyme was reached after 7 d of fermentation. Addition of 1% glucose to the medium suppressed beta-glucosidase production to undetectable levels. In this study, the organic synthesis of a potential inducer of beta-glucosidase production by A. niger B1's reported. Isopropyl-1-thio-beta-D-glucopyranoside (IPTGlc) was synthesized using a two-step organic synthesis protocol. The H-NMR data agreed with those reported previously for the galactoside analog. When IPTGlc was added 24 h after inoculation at a final concentration of 0.4 mM, similar levels of beta-glucosidase were reached 3 to 4 d earlier as compared to fermentation without IPTGlc induction. In practice, this may translate to a more efficient method of producing beta-glucosidase from this fungus.
