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 μg/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 K(d) of 0.5 μM. 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 V(max) of 11 nmol·min·mg of kinase and an apparent K(m) of 270 μM. Purified protein was crystallized using the hanging drop method with seeding. Crystalline ALBP was orthorhombic with cell dimensions of a = 34.4 Å, b = 54.8 Å, and c = 76.3 Å. The space group was P212121, and there was one molecule per asymmetric unit.
|Original language||English (US)|
|Number of pages||4|
|Journal||Journal of Biological Chemistry|
|State||Published - 1991|