Stable, long-term bacterial production of soluble, dimeric, disulfide-bonded protein pharmaceuticals without antibiotic selection.

Numerous biopharmaceuticals and other recombinant biotechnology products are made in prokaryotic hosts. However, bacterial production of native, biologically active eukaryotic proteins is rarely possible for disulfide-bonded and/or multisubunit proteins. We previously described the production of soluble, native disulfide-bonded dimeric proteins in the Escherichia coli cytoplasm (Miele et al., 1990; Mantile et al., 1993). Native, biologically active proteins with up to six disulfide bonds have been produced with our expression system (Garces et al., 1997). However, plasmid instability during induction limited its usefulness. We now report the stable, high-level expression of soluble, disulfide-bonded human uteroglobin without antibiotic selection. We designed a new vector containing a multifunctional stabilization region that confers complete plasmid stability and increased protein yields without copy number increases. Recombinant expression remains fully inducible after long-term continuous culture in nonselective liquid medium (at least 260 generations). This system may significantly expand the applications of bacterial expression to recombinant production of soluble, bioactive proteins for biochemical studies and biopharmaceutical/industrial purposes. As a result of the very broad activity spectrum of the stabilization region we selected, its use could be extended to bacterial hosts other than enterobacteria.

Adenovirus-mediated gene transfer of the human tissue inhibitor of metalloproteinase-2 blocks vascular smooth muscle cell invasiveness in vitro and modulates neointimal development in vivo.

BACKGROUND: Endovascular injury induced by balloon withdrawal leads to the increased activation of matrix metalloproteinases (MMPs) in the vascular wall, allowing smooth muscle cells (SMCs) to digest the surrounding extracellular matrix (ECM) and migrate from the media into the intima. The objective of this study was to examine the effects of a replication-deficient adenovirus carrying the cDNA for human tissue inhibitor of metalloproteinase-2 (AdCMV.hTIMP-2) on SMC function in vitro and neointimal development in the injured rat carotid artery. METHODS AND RESULTS: Infection of cultured rat aortic SMCs at a multiplicity of infection of 100 with AdCMV.hTIMP-2 resulted in high-level expression of hTIMP-2 mRNA and protein secretion into the medium. Conditioned media (CM) from AdCMV. hTIMP-2-infected but not control virus (AdCMV.null or AdCMV. betagal)-infected SMCs inhibited MMP-2 activity on gelatin zymograms as well as the chemoattractant-directed migration of SMCs across reconstituted basement membrane proteins in the Boyden chamber assay. In contrast, AdCMV.hTIMP-2 CM had no effect on chemoattractant-directed migration of SMCs occurring in the absence of an ECM barrier or on the proliferation of cultured neointimal SMCs. Delivery of AdCMV.hTIMP-2 (2.5x10(9) pfu) to the carotid artery wall at the time of balloon withdrawal injury inhibited SMC migration into the intima by 36% (P<0.05) at 4 days and neointimal area by 53% (P<0.01) at 8 days and by 12% (P=NS) at 21 days after injury. AdCMV.hTIMP-2 had no effect on medial area. CONCLUSIONS: Adenovirus-mediated hTIMP-2 gene transfer inhibits SMC invasiveness in vitro and in vivo and delays neointimal development after carotid injury.

Resonance assignment and secondary structure determination and stability of the recombinant human uteroglobin with heteronuclear multidimensional NMR.

Human uteroglobin (h-UG) or Clara cell 10kDa (cc10kDa) is a steroid-dependent, 17 kDa homodimeric, secretory protein with potent anti-inflammatory/immunomodulatory properties. However, the exact physiological role still remains to be determined. It has been hypothesised that its activity is exerted through the binding of a specific target represented by a small molecule (still unknown), and that the binding is regulated by the formation/disruption of two cysteine bonds. The binding properties of the reduced UG have been proved in vitro for several different molecules, but no in vivo data are available to date. However, binding has been observed between reduced rabbit UG and a protein of an apparent molecular mass of 90 kDa and, more recently, we found an h-UG-binding protein

Recombinant human uteroglobin suppresses cellular invasiveness via a novel class of high-affinity cell surface binding site.

The mechanism(s) that regulates invasion of trophoblasts through the uterine epithelium during embryo implantation and nidation in hemochorial placental mammals is poorly understood. While limited trophoblast invasion is essential for the establishment of normal pregnancy, dysregulation of this process may contribute to the pathogenesis of choriocarcinoma, a highly invasive and lethal form of cancer arising from the trophoblasts. We have previously demonstrated that rabbit uteroglobin (UG), a cytokine-like, antiinflammatory protein, produced by the endometrial epithelium during pregnancy, has a potent antichemotactic effect on neutrophils and monocytes in vitro. Here, we report that recombinant human UG (hUG) dramatically suppresses invasion of human trophoblasts and NIH 3T3 cells through an artificial basement membrane (Matrigel) in vitro but has no effect on that of human choriocarcinoma cells. We identified a previously unreported high-affinity, high molecular weight (approximately 190 kDa), nonglycosylated hUG-binding protein, readily detectable on human trophoblasts and NIH 3T3 cells but totally lacking on choriocarcinoma cells. Taken together, these results raise the possibility that (i) hUG plays a critical role in regulating cellular invasiveness, at least in part, via its previously unrecognized cell surface binding site, and (ii) some of the numerous biological activities of proteins of the UG family, reported so far, may be mediated via this binding site.

Crystallization and characterization of the recombinant human Clara cell 10-kDa protein.

Crystals of recombinant human Clara cell 10-kDa protein were grown both from ammonium sulfate and polyethylene glycol (PEG) solutions. Crystals grown from ammonium sulfate solution have been characterized by X-ray diffraction studies as monoclinic with the space group C2 and lattice constants a = 69.2 A, b = 83.0 A, c = 58.3 A, and beta = 99.7 degrees. The monoclinic crystals diffract to beyond 2.5 A. Some of the crystals grown from PEG were of a similar habit to those grown from ammonium sulfate, but others were triclinic with the space group P1 and cell constants a = 40.3 A, b = 46.3 A, c = 51.3 A, alpha = 117.7 degrees, beta = 102.3 degrees, and gamma = 71.4 degrees. These crystals diffract to beyond 3.2 A.

Recombinant human uteroglobin inhibits the in vitro invasiveness of human metastatic prostate tumor cells and the release of arachidonic acid stimulated by fibroblast-conditioned medium.

Uteroglobin (UG) is a potent immunomodulatory and antiinflammatory secretory protein with high levels detected in human prostate tissue. We used three human prostate cancer cell lines (DU-145, PC3-M, and LNCaP) to test the hypothesis that UG may modulate invasiveness of prostatic carcinoma cells in the Boyden chamber assay for invasion through a reconstituted basement membrane preparation. Fibroblast-conditioned medium was used as the chemoattractant. The most invasive cell line was DU-145, followed by PC3-M, whereas the androgen-dependent LNCaP cell line exhibited extremely low invasive potential. Pretreatment of DU-145 and PC3-M cells for 24 h with 0.01, 0.1, or 1.0 microM recombinant UG had no effect on basal invasiveness but inhibited fibroblast-conditioned medium-stimulated invasion in a dose-dependent manner, reaching up to 60.2 and 87.9% inhibition of DU-145 and PC3-M, respectively. UG had no effect on either cell-reconstituted basement membrane adhesion or simple chemotaxis in the absence of reconstituted basement membrane. UG also strongly inhibited the biphasic release of [14C]-labeled arachidonic acid from fibroblast-conditioned medium-stimulated DU-145 cells. These results suggest that UG may modulate prostate tumor cell invasiveness and that the mechanism may include inhibition of the arachidonic acid signal cascade.

Human Clara cell 10-kDa protein is the counterpart of rabbit uteroglobin.

Human Clara cell 10-kDa protein has been suggested to be a counterpart of rabbit uteroglobin, an immunomodulatory and antiinflammatory secretory protein. Since this human protein is not readily available in substantial quantity for detailed characterization of its biochemical, biological, and pharmacological properties, we sought to express it in Escherichia coli in order to study its structure-function relationship. However, bacterial overproduction of homodimeric proteins with interchain disulfide bonds, such as Clara cell 10-kDa protein, was thought to be impossible until we achieved expression of native uteroglobin (Miele, L., Cordella-Miele, E., and Mukherjee, A.B. (1990) J. Biol. Chem. 265, 6427-6435). Here, we report high level production of recombinant native dimeric human Clara cell 10-kDa protein in E. coli and its characterization. Recombinant human Clara cell 10-kDa protein forms its disulfide bonds within the bacterial cytoplasm. The purified protein possesses two of the most important activities characteristic of uteroglobin: (i) it is an excellent substrate of transglutaminase, and (ii) it is a potent inhibitor of porcine pancreatic and, more importantly, human synovial phospholipase A2. These results demonstrate that human Clara cell 10-kDa protein and rabbit uteroglobin have very similar biochemical properties. Our data suggest that this protein may possess immunomodulatory and antiinflammatory activities of potential physiological and pharmacological importance.

Receptor-mediated endocytosis of galactose and mannose exposing ligands: an electron microscopic study on adult and neonatal cultured rat hepatocytes.

We compared the receptor-mediated endocytosis for galactose and mannose exposing ligands in primary cultures of hepatocytes from newborn and adult rats. The endocytic pathway was revealed ultrastructurally using colloidal gold particles coupled to lactosylated bovine serum albumin and invertase. The binding activity on the cell surfaces is observed by keeping the cells at 4 degrees C. For both ligands used, the binding capacity for hepatocytes from adult rats was greater than for neonatal cultured cells. Increasing the temperature to 37 degrees C, we observed that the protein-gold complexes entered the intracellular endocytic organelles. Within 5-15 min, the marker was confined in vesicles close to the cell surface and in the endosome, while after 60 min, the marker is found in lysosome-like compartments. We found that the process of endocytosis is similar for galactose and mannose exposing ligands. The organelles involved in the process of endocytosis in newborn cultured hepatocytes are not different in shape from those of cultured cells of adult rats, but the process of internalization is slower.