Overexpression, purification, and characterization of glutaminase-interacting protein, a PDZ-domain protein from human brain.
A human brain cDNA clone coding for a novel PDZ-domain protein of 124 amino acids has been previously isolated in our laboratory. The protein was termed GIP (glutaminase-interacting protein) because it interacts with the C-terminal region of the human brain glutaminase L. Here we report the heterologous expression of GIP as a histidine-tagged fusion protein in Escherichia coli cells. The induction conditions (temperature and isopropyl beta-d-thiogalactopyranoside concentrations) were optimized in such a way that GIP accounted for about 20% of the total E. coli protein. A simple and rapid procedure for purification was developed, which yielded 17 mg of purified GIP per liter of bacterial cell culture. The apparent molecular mass of the protein by SDS-PAGE was 16 kDa, whereas in native form it was determined to be 28 kDa, which suggests dimer formation. The nature and integrity of the recombinant protein were verified by mass spectrometry analysis. The functionality of the GIP protein was tested with an in vitro activity assay: after being pulled down with glutathione S-transferase-glutaminase, GIP was revealed by Western blot using anti-GIP antibodies. Furthermore, the glutaminase activity in crude rat liver extracts was inhibited by the presence of recombinant purified GIP protein.
Aledo
J C
JC
Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, Málaga, 29071, Spain
Rosado
A
A
Olalla
L
L
Campos
J A
JA
Márquez
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005972:Glutaminase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D008970:Molecular Weight; D018377:Neurotransmitter Agents; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D047908:Intracellular Signaling Peptides and Proteins; D018377:Neurotransmitter Agents; D011993:Recombinant Fusion Proteins; C421249:TAX1BP3 protein, human; D006639:Histidine; D005972:Glutaminase