The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear receptor supergene family and are considered as key sensors of both lipid and glucose homeostasis. The role of the PPAR␥ isoform in glucose metabolism is illustrated by the fact that antidiabetic thiazolidinediones have been shown to be bona fide PPAR␥ ligands. Here we report the crystal structure of apo-PPAR␥ ligand binding domain (LBD) determined to 2.9-Å resolution. Although the structure of apo-PPAR␥-LBD retains the overall fold described previously for other nuclear receptor LBDs, three distinct structural differences are evident. 1) The core AF-2 activation domain of apo-PPAR␥ LBD is folded back toward the predicted ligand binding pocket similar to that observed in the holo-forms of other nuclear receptors. 2) The proposed ligand binding pocket of apo-PPAR␥-LBD is larger and more accessible to the surface in contrast to other LBDs. 3) The region of the LBD called the -loop is extended in PPAR␥ and contains additional structural elements. Taken together, the apo-PPAR␥-LBD structure is in several aspects different from previously described LBDs. Given the central role of PPAR␥ as a mediator in glucose regulation, the structure should be an important tool in the development of improved anti-diabetic agents.
The monoclonal antibody 5T4, directed against a human tumor-associated antigen, was expressed as a secreted Fab superantigen fusion protein in Escherichia coli. The product is a putative agent for immunotherapy of non-small cell lung cancer. During fermentation, most of the fusion protein leaked out from the periplasm to the growth medium at a level of approximately 40 mg/ liter. This level was notably low compared with similar products containing identical C H 1, C L , and superantigen moieties, and the Fv framework was therefore engineered. Using hybrid molecules, the light chain was found to limit high expression levels. Substituting five residues in V L increased the level almost 15 times, exceeding 500 mg/liter in the growth medium. Here, the substitutions Phe-10 3 Ser, Thr-45 3 Lys, Thr-77 3 Ser, and Leu-78 3 Val were most powerful. In addition, replacing four V H residues diminished cell lysis during fermentation. Thereby the product was preferentially located in the periplasm instead of the growth medium, and the total yield was more than 700 mg/liter. All engineered products retained a high affinity for the tumorassociated antigen. It is suggested that at least some of the identified framework residues generally have to be replaced to obtain high level production of recombinant Fab products in E. coli.
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