Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing.

Human BMP-2, a homodimeric protein that belongs to the TGF- ß family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL.

The human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 µg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF-MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

Synthesis of human bone morphogenetic protein-2 (hBMP-2) in E. coli periplasmic space: its characterization and preclinical testing

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis.

Prolactin (PRL) is a hormone produced by the pituitary gland with innumerable functions, such as lactation, reproduction, osmotic and immune regulation. The present work describes the synthesis of hPRL in human embryonic kidney (HEK293) cells, transiently transfected with the pcDNA-3.4-TOPO® vector carrying the hPRL cDNA. A concentration of ~ 20 mg/L, including glycosylated (G-hPRL) and non-glycosylated (NG-hPRL) human prolactin, was obtained, with ~ 19% of G-hPRL, which is higher than that observed in CHO-derived hPRL (~ 10%) and falling within the wide range of 5-30% reported for pituitary-derived hPRL. N-Glycoprofiling analysis of G-hPRL provided: (i) identification of each N-glycan structure and relative intensity; (ii) average N-glycan mass; (iii) molecular mass of the whole glycoprotein and relative carbohydrate mass fraction; (iv) mass fraction of each monosaccharide. The data obtained were compared to pituitary- and CHO-derived G-hPRL. The whole MM of HEK-derived G-hPRL, determined via MALDI-TOF-MS, was 25,123 Da, which is 0.88% higher than pit- and 0.61% higher than CHO-derived G-hPRL. The main difference with the latter was due to sialylation, which was ~ sevenfold lower, but slightly higher than that observed in native G-hPRL. The "in vitro" bioactivity of HEK-G-hPRL was ~ fourfold lower than that of native G-hPRL, with which it had in common also the number of N-glycan structures.

Suppression of Prolactin Secretion Partially Explains the Antidiabetic Effect of Bromocriptine in ob/ob Mice.

Previous studies have shown that bromocriptine mesylate (Bromo) lowers blood glucose levels in adults with type 2 diabetes mellitus; however, the mechanism of action of the antidiabetic effects of Bromo is unclear. As a dopamine receptor agonist, Bromo can alter brain dopamine activity affecting glucose control, but it also suppresses prolactin (Prl) secretion, and Prl levels modulate glucose homeostasis. Thus, the objective of the current study was to investigate whether Bromo improves insulin sensitivity via inhibition of Prl secretion. Male and female ob/ob animals (a mouse model of obesity and insulin resistance) were treated with Bromo and/or Prl. Bromo-treated ob/ob mice exhibited lower serum Prl concentration, improved glucose and insulin tolerance, and increased insulin sensitivity in the liver and skeletal muscle compared with vehicle-treated mice. Prl replacement in Bromo-treated mice normalized serum Prl concentration without inducing hyperprolactinemia. Importantly, Prl replacement partially reversed the improvements in glucose homeostasis caused by Bromo treatment. The effects of the Prl receptor antagonist G129R-hPrl on glucose homeostasis were also investigated. We found that central G129R-hPrl infusion increased insulin tolerance of male ob/ob mice. In summary, our findings indicate that part of Bromo effects on glucose homeostasis are associated with decrease in serum Prl levels. Because G129R-hPrl treatment also improved the insulin sensitivity of ob/ob mice, pharmacological compounds that inhibit Prl signaling may represent a promising therapeutic approach to control blood glucose levels in individuals with insulin resistance.

Evaluation of ions and metals in the blood of GRMD dogs submitted to hASCs therapy by NAA and XRF techniques.

The elements Br, Ca, Cl, Cr, Fe, K, Mg, Na, P, Rb, S, and Zn were investigated in the whole blood samples of Golden Retriever dogs submitted to cell therapy (hASCs). These analyses were performed over 2 years using Neutron Activation Analysis and X-Ray Fluorescence techniques. The results were compared with control and untreated dog's. A significant increase was observed in K blood levels. There was also variation in blood levels of Br, Cr, Fe, Rb, S, and Zn.

Determination of recombinant Interferon-α2 in E. coli periplasmic extracts by reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to analyze Interferon α-2 (IFN-α2) as a pure protein or as a pharmaceutical preparation: a method for analyzing periplasmic IFN-α2 directly in osmotic shock extract has, however, never been reported. This work describes an RP-HPLC methodology for the qualitative and quantitative analysis of human IFN-α2a and IFN-α2b directly in bacterial periplasmic extracts or in purified preparations. The analytical method has been set up and validated for accuracy, precision, linearity, sensitivity and specificity. A recovery test indicated an average bias of ∼1%, intra-day and inter-day quantitative determinations presented relative standard deviations always≤5%, while the working sensitivity was of ∼0.3µg of IFN-α2 (RSD=5%). The method proved to be suitable for detecting and quantifying also glycosylated and oxidized forms and N-methionylated IFN-α2 molecules, it was, however, not able to distinguish between IFN-α2a and IFN-α2b. This rapid methodology allows the application of RP-HPLC as a powerful tool to monitor the production yield and quality of IFN-α2 in osmotic shock fluids, right after, or even during the fermentation process.

Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space.

The human growth hormone receptor antagonist G120R-hGH precludes dimerization of GH and prolactin receptors and consequently JAK/STAT signaling. Some modifications in this antagonist resulted in a drug specific for the GH receptor, called Pegvisomant (Somavert®). However, the original G120R-hGH is usually synthesized in bacterial cytoplasm as inclusion bodies, not being a commercial product. The present work describes the synthesis and characterization of G120R-hGH secreted into bacterial periplasm and obtained with a vector based on a constitutive lambda-PL promoter. This antagonist can be useful for studies aiming at investigating the effects of a simultaneous inhibition of GH and prolactin signaling, as a potential anti-tumoral or anti-diabetic compound. G120R-hGH, synthesized using the W3110 E. coli strain, showed a yield of 1.34 ± 0.24 µg/ml/A600 (∼0.79 mg G120R-hGH/g of wet weight cells) after cultivation at 30 °C up to 3 A600 units and induction at 37 °C, for 6 h, with final 4.3 ± 0.3 A600. A laboratory scale purification was carried out using three chromatographic steps with a total yield of 32%, reaching 98% purity. The obtained protein was characterized by SDS-PAGE, Western Blotting, Mass spectrometry, RP-HPLC, HPSEC and in vitro proliferation bioassay. The proliferation assay, based on Ba/F3-LLP cells, shows that G120R-hGH (100 ng/ml) significantly inhibited (64%) the proliferative action of hGH (1 ng/ml). This is the first time that G120R-hGH is synthesized in bacterial periplasmic space and therefore correctly folded, without the initial methionine. The reasons for a divergent efficacy for antagonizing hGH versus hPRL is currently unknown and deserves further investigation.

Development and biological studies of ¹77Lu-DOTA-rituximab for the treatment of Non-Hodgkin's lymphoma.

The optimization of DOTA-NHS-ester conjugation to Rituximab using different Ab:DOTA molar ratios (1:10, 1:20, 1:50 and 1:100) was studied. High radiochemical yield, in vitro stability and immunoreactive fraction were obtained for the Rituximab conjugated at 1:50 molar ratio, resulting in the incorporation of an average number of 4.9 ± 1.1 DOTA per Rituximab molecule. Labeling with 177Lu was performed in high specific activity with great in vitro stability. Biodistribution in healthy and xenographed mice showed tumor uptake and high in vivo stability as evidenced by low uptake in bone. The properties of 177Lu-DOTA-Rituximab prepared from DOTA-NHS-ester suggest the potential for the application of the 177Lu-labeled antibody in preliminary clinical studies.

N-glycoprofiling analysis in a simple glycoprotein model: a comparison between recombinant and pituitary glycosylated human prolactin.

Human prolactin (hPRL) is a polypeptide hormone occurring in the non-glycosylated (NG-hPRL) and glycosylated (G-hPRL) forms, with MM of approximately 23 and 25kDa, respectively. It has a single, partially occupied N-glycosylation site located at Asn-31, which makes it a particularly simple and interesting model for glycosylation studies. The bioactivity of G-hPRL is lower than that of NG-hPRL (by ca. 4-fold) and its physiological function is not clear. However, carbohydrate moieties generally play important roles in the biosynthesis, secretion, biological activity, and plasma survival of glycohormones and can vary depending on the host cell. The main objective of this study was to determine the N-glycan structures present in native, pituitary G-hPRL and compare them with those present in the recombinant hormone. To obtain recombinant G-hPRL, genetically modified Chinese hamster ovary cells (CHO), adapted to growth in suspension, were treated with cycloheximide, thus increasing the glycosylation site occupancy from 5.5% to 38.3%, thereby facilitating G-hPRL purification. CHO cell-derived G-hPRL (CHO-G-hPRL) was compared to pituitary G-hPRL (pit-G-hPRL) especially with regard to N-glycoprofiling. Among the main differences found in the pituitary sample were an extremely low presence of sialylated (1.7%) and a high percentage of sulfated (74.0%) and of fucosylated (90.5%) glycans. A ∼6-fold lower in vitro bioactivity and a higher clearance rate in mice were also found for pit-G-hPRL versus CHO-G-hPRL. N-Glycan profiling proved to be a useful and accurate methodology also for MM and carbohydrate content determination for the two G-hPRL preparations, in good agreement with the values obtained directly via MALDI-TOF-MS.

Hypermethylation of a New Distal Sodium/Iodide Symporter (NIS) enhancer (NDE) is associated with reduced NIS expression in thyroid tumors.

CONTEXT: In thyroid tumors, reduced radioiodine uptake is associated with worse patient outcome concomitantly with low sodium/iodide symporter (NIS) mRNA expression. Previous studies showed that CpG-island methylation in the NIS proximal promoter does not correlate with mRNA expression. OBJECTIVES: The aim of the study was to identify new CpG-islands within the NIS 5'region and investigate the involvement of their methylation in NIS expression. DESIGN: DNA was obtained from 30 pairs of thyroid samples: tumor (T) and surrounding nontumor (NT) samples. All T samples had reduced NIS mRNA expression compared to NT samples. MAIN OUTCOME MEASURES: Methylation degree was quantified by bisulfite sequencing, and NIS expression by real-time PCR and Western blot. Reporter gene assays were performed to determine CpG-island functionality. Tumor cell cultures were treated with 5-Aza demethylating agent to determine NIS expression, methylation status, and (125)I uptake. RESULTS: We identified a new CpG-island2 with 14 CpG sites, located -2152/-1887 relative to ATG site. CpG-island2 was hypermethylated in T compared to NT samples, in both benign and malignant tumor groups. There was a significant inverse correlation between NIS mRNA expression and degree of CpG-island2 methylation in NT and T samples. This sequence increased the expression of a reporter gene; thus, it was considered a new enhancer. Cell culture treatments with 5-Aza reduced CpG-island2 methylation levels concomitantly with restoration of NIS mRNA and protein expression and (125)I uptake. CONCLUSIONS: We identified a new distal enhancer, NIS distal enhancer, that regulates gene expression through DNA methylation. This enhancer is hypermethylated in T compared to NT samples and is associated with decreased NIS expression in tumors. This epigenetic deregulation may be an early event in tumorigenesis.

Expression, purification, and characterization of authentic mouse prolactin obtained in Escherichia coli periplasmic space.

Prolactin (PRL) is a pleiotropic hormone produced by lactotroph cells of the anterior pituitary gland and is mainly related to lactation control and reproduction. Recombinant mouse prolactin (r-mPRL), never obtained in its authentic form, can be very useful for research and tests in animal models, in which human prolactin (hPRL) is usually employed in a heterologous mode. Synthesis of r-mPRL was carried out here via secretion in Escherichia coli periplasmic space using a plasmid containing mPRL cDNA joined to the DsbA signal peptide sequence under the control of a constitutive major leftward promoter of the bacteriophage λ (λPL). Fermentation in a pilot bioreactor was carried out at 30°C, with 6 H of induction at 37°C, reaching an optical density of 23 A600 units, a specific yield of 0.06-0.1 µg mPRL/(mL A600), and a concentration of up to 2.2 µg/mL. Even with such a low yield and a poor mass fraction, r-mPRL was purified via a three-step laboratory process based on hydrophobic chromatography, reversed-phase high-performance liquid chromatography, and high-performance size-exclusion chromatography (HPSEC). The purified hormone was then characterized using SDS-PAGE, Western blotting, and HPSEC and showed, by Nb2 rat lymphoma cell proliferation assay, a bioactivity of 39.5 IU/mg, determined against the International Standard of recombinant hPRL [World Health Organization (WHO)-97/714].

SJL dystrophic mice express a significant amount of human muscle proteins following systemic delivery of human adipose-derived stromal cells without immunosuppression.

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence of or defective muscular proteins. The murine model for limb-girdle muscular dystrophy 2B (LGMD2B), the SJL mice, carries a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal. The mice show muscle weakness that begins at 4-6 weeks and is nearly complete by 8 months of age. The possibility of restoring the defective muscle protein and improving muscular performance by cell therapy is a promising approach for the treatment of LGMDs or other forms of progressive muscular dystrophies. Here we have injected human adipose stromal cells (hASCs) into the SJL mice, without immunosuppression, aiming to assess their ability to engraft into recipient dystrophic muscle after systemic delivery; form chimeric human/mouse muscle fibers; express human muscle proteins in the dystrophic host and improve muscular performance. We show for the first time that hASCs are not rejected after systemic injection even without immunosuppression, are able to fuse with the host muscle, express a significant amount of human muscle proteins, and improve motor ability of injected animals. These results may have important applications for future therapy in patients with different forms of muscular dystrophies.

Induction of micronuclei by 153Sm-EDTMP in peripheral blood lymphocytes of patients with bone metastases.

The purpose of this study was to evaluate the degree of cytological radiation damage to peripheral blood lymphocytes induced by 153Sm-EDTMP applied for palliation of metastatic bone pain. Blood samples from 16 patients (46-82 years old), 10 without previous radiotherapy and 6 with previous radiotherapy, were collected before and one hour after the administration of a mean activity of 41.7+/-5.8 MBq/kg of 153Sm-EDTMP. Then the lymphocytes were cultured for cytokinesis block micronucleus (MN) assay. The number of MNper binucleated cells (BC) in patients without previous radiotherapy before the treatment was of 0.030 (+/- 0.016) and after one hour 0.035 (+/- 0.013), although we could find inter individual differences. The basal MN/BC of the patients with no previous radiotherapy was similar to the controls. The increment in the percentage of BC with MN was similar in patients with and without previous radiotherapy. The observed mean of MN/BC is equivalent to a dose range of 0.05 to 0.10 Gy of 153Sm-EDTMP in vitro. The relatively low frequency of lymphocyte with micronuclei after the exposure to 153Sm-EDTMP supported the contention that radiation damage in lymphocytes of patients with painful bone metastases is minimal.

Low amounts of the DNA repair XPA protein are sufficient to recover UV-resistance.

DNA integrity is threatened by the damaging effects of physical and chemical agents that can affect its function. Nucleotide excision repair (NER) is one of the most known and flexible mechanisms of DNA repair. This mechanism can recognize and remove damages causing DNA double-helix distortion, including the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine-pyrimidone (6-4) photoproducts, promoted by ultraviolet light (UV). The human syndrome xeroderma pigmentosum (XP) is clinically characterized chiefly by the early onset of severe photosensitivity of the exposed regions of the skin, a very high incidence of skin cancers and frequent neurological abnormalities. The xpa gene seems to be involved during UV damage recognition, in both global genome repair (GGR) and transcription-coupled repair (TCR). The modulation of xpa expression may modify the DNA repair rate in the cell genome, providing a valuable contribution to an understanding of the NER process. The controlled expression of the cDNA xpa in XP12RO deficient cells was achieved through the transfection of a muristerone-A inducible vector, pINXA. The INXA15 clone shows good induction of the XPA protein and total complementation of XP12RO cell deficiency. Overexpression of this protein resulted in UV cell survival comparable to normal control human cells. Moreover, low expression of the XPA protein in these cells is sufficient for total complementation in cellular UV sensitivity and DNA repair activity. These data demonstrate that XPA protein concentration is not a limiting factor for DNA repair.