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

Journal Article · Thu Oct 01 00:00:00 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 (United States)
• OSTI ID: 7242272
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 84:19
• Country of Publication: United States
• Language: English

Related Subjects

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