Overproduction and purification of the luxR gene product: transcriptional activator of the Vibrio fischeri luminescence system

Journal Article · Thu Oct 01 04:00:00 EDT 1987 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.84.19.6639· OSTI ID:7242272

Kaplan, H B; Greenberg, E P

Expression of Vibrio fischeri luminescence genes requires an inducer, termed autoinducer, and a positive regulatory element, the luxR gene product. A plasmid containing luxR under control of a tac promoter was engineered to overproduce this gene product. The overproduced luxR gene product was active in vivo, and its apparent monomeric molecular weight was indistinguishable from that of the protein encoded by luxR under control of its own promoter (M/sub r/ 27,000). The new tac-luxR construct directed the synthesis of large quantities of the luxR gene product in induced Escherichia coli cells lacking other lux genes. In the presence of other lux genes, overexpression of the tac-luxR construct was not detected. The overproduced luxR gene product, which formed cytoplasmic inclusion bodies, was purified and used in subsequent studies. Nonequilibrium pH gradient electrophoresis indicated that the protein was basic, and the amino-terminal 15 amino acids were sequenced. DNA-binding activity was detected by membrane filter binding assays; under the conditions used, the binding was not lux DNA-specific. Binding of tritium-labeled autoinducer to the luxR gene product was not detected, and autoinducer enhancement of the binding of the luxR gene product to DNA could not be detected reproducibly.

• Research Organization: Cornell Univ., Ithaca, NY (USA)
• OSTI ID: 7242272
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 84:19; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
DNA SEQUENCING
ELECTROPHORESIS
ESCHERICHIA COLI
GENE REGULATION
GENE REPRESSORS
ISOTOPES
LIGHT NUCLEI
LUMINESCENCE
MICROORGANISMS
MOLECULAR WEIGHT
NUCLEI
NUCLEOPROTEINS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PLASMIDS
PROTEINS
PURIFICATION
RADIOISOTOPES
STRUCTURAL CHEMICAL ANALYSIS
TRANSCRIPTION