title: Cloning of murine adipocyte lipid binding protein in Escherichia coli. Its purification, ligand binding properties, and phosphorylation by the adipocyte insulin receptor.
abstract: The murine adipocyte lipid binding protein (ALBP) has been cloned into Escherichia coli, purified from expressing cultures, and its ligand binding and phosphorylation properties studied. In the cloning strategy, the recombinant, pT7-5 rALBP, was transformed into E. coli strain K38 harboring plasmid pGP1-2 which directs the synthesis of T7 RNA polymerase. Upon shifting the temperature from 30 to 42 degrees C to induce T7 RNA polymerase expression, the 14.6-kDa recombinant ALBP (rALBP) was expressed for approximately 2 h and accumulated to about 1% of total E. coli protein. The recombinant ALBP was soluble in E. coli extracts and resistant to bacterial proteolysis. A procedure for purifying rALBP was developed utilizing immuno-chemical detection based upon reactivity with anti-murine ALBP antiserum. A combination of acidic ammonium sulfate fractionation, gel permeation chromatography, and carboxymethyl ion-exchange high performance liquid chromatography separation was used to prepare homogeneous rALBP. Sequence analysis of rALBP indicated that the initiating methionine residue had been removed and the amino-terminal cysteine residue was not blocked. Purified rALBP exhibited stoichiometric, saturable binding of oleic acid (n = 1.0, K0.5 approximately 100 microM) and retinoic acid (n = 1.0, K0.5 approximately 170 microM). Incubation of rALBP with wheat germ agglutinin-purified insulin receptor, ATP, and 100 nM insulin resulted in a 5-fold stimulation of rALBP phosphorylation above the basal state. Kinetic analysis of rALBP phosphorylation by the 3T3-L1 insulin receptor kinase yielded a Michaelis constant (Km) of 50 microM and a maximal velocity of 1 mol of rALBP phosphorylated/min/mol insulin binding sites. Phosphoamino acid analysis indicated that phosphorylation occurred upon tyrosine. These results indicate that murine ALBP has been cloned and expressed in E. coli, purified to homogeneity, and is a substrate for the insulin receptor tyrosyl kinase in vitro.
authors.affiliation: Department of Biochemistry, University of Minnesota, St. Paul 55108.
authors.forename: L L
authors.initials: LL
authors.lastname: Chinander
authors.forename: D A
authors.initials: DA
authors.lastname: Bernlohr
mesh_terms: D000273:Adipose Tissue / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D004247:DNA; D011972:Receptor, Insulin