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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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