Expression, purification, and crystallization of the adipocyte lipid binding protein.
The murine adipocyte lipid binding protein (ALBP/aP2) has been cloned and expressed in Escherichia coli, purified to homogeneity, biochemically characterized, and crystallized for x-ray diffraction study. In the cloning, the ALBP coding region was placed under control of the recA promoter and downstream of the phage T7 g-10 translation enhancer sequence. Nalidixic acid (50 micrograms/ml) induced the expression of ALBP 20-fold over that attained using the pT7 system previously reported (Chinander, L. L., and Bernlohr, D. A. (1989) J. Biol. Chem. 264, 19564-19572). Recombinant ALBP was purified to homogeneity using a combination of pH fractionation, gel filtration, and immobilized metal affinity chromatography. The fluorescent affinity ligand 12-(9-anthroyloxy)oleic acid bound to homogeneous ALBP with an apparent Kd of 0.5 microM. rALBP was devoid of endogenous fatty acid, and oleic acid inhibited cysteine 117 modification by 5,5' -dithiobis-(2-nitrobenzoic acid) indicating integrity of the binding domain. Recombinant ALBP was phosphorylated by the soluble kinase domain of the insulin receptor with a Vmax of 11 nmol.min.mg of kinase and an apparent Km of 270 microM. Purified protein was crystallized using the hanging drop method with seeding. Crystalline ALBP was orthorhombic with cell dimensions of a = 34.4 A, b = 54.8 A, and c = 76.3 A. The space group was P212121, and there was one molecule per asymmetric unit.
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Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455.
Buelt
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Banaszak
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Bernlohr
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D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003460:Crystallization; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005784:Gene Amplification; D005798:Genes, Bacterial; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D011972:Receptor, Insulin; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014961:X-Ray Diffraction
D002352:Carrier Proteins; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D013438:Sulfhydryl Compounds; D004247:DNA; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin