Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.     

Structural analysis of the glycosylation of gene-activated erythropoietin (epoetin delta, Dynepo)

Recently, a novel recombinant human erythropoietin (epoetin delta, Dynepo) has been marketed in the European Union for the treatment of chronic kidney disease, cancer patients receiving chemotherapy, and so forth. Epoetin delta is engineered in cultures of the human fibrosarcoma cell line HT-1080 by homologous recombination and “gene activation.” Unlike recombinant erythropoietins produced in other mammalian cells, epoetin delta is supposed to have a human-type glycosylation profile. However, the isoelectric focusing profile of epoetin delta differs from that of endogenous erythropoietin (both urinary and plasmatic). In this work, structural and quantitative analysis of the O- and N-glycans of epoetin delta was performed and compared with glycosylation from recombinant erythropoietin produced in Chinese hamster ovary (CHO) cells. From the comparison, significant differences in the sialylation of O-glycans were found. Furthermore, the N-glycan analysis indicated a lower heterogeneity from epoetin delta when compared with its CHO homologue, being predominantly tetraantennary without N-acetyllactosamine repeats in the former. The sialic acid characterization revealed the absence of N-glycolylneuraminic acid. The overall sugar profiles of both glycoproteins appeared to be significantly different and could be useful for maintaining pharmaceutical quality control, detecting the misuse of erythropoietin in sports, and establishing new avenues to link glycosylation with biological activity of glycoproteins.     

Effect of preservatives on IgG aggregation, complement-activating effect and hypotensive activity of horse polyvalent antivenom used in snakebite envenomation.

Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4 degrees C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms.     

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation. Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997     

Heritability of running economy: a study made on twin brothers

Running economy (RE), defined as the steady-state of oxygen uptake (V˙O₂) for a given running velocity, is a factor of sports performance the genetic component of which has seldom been reported to date. We studied this component using a heritability index (HI) in a group of 32 male twins, 8 monozygotic (MZ) and 8 dizygotic (DZ) pairs, all sportsmen with similar perinatal and environmental backgrounds. Zygocity was determined by the identity of erythrocytic antigenic, protein and enzymatic polymorphism, and human leucocyte antigen serologic types between co-twins. The subjects exercised twice on a treadmill, once until exhaustion and again at submaximal intensities. Pulmonary gas exchange was measured continuously using an automatic analyser system during both tests. Blood samples were obtained during the recovery period to determine lactate concentrations. No significant differences were observed between MZ and DZ, in respect of RE at any speed or in maximal V˙O₂ relative to body mass. Nevertheless, significant HI (P < 0.05) was found in maximal lactate concentrations (HI = 0.75) and in respiratory equivalent for oxygen at two speeds, 7 km · h⁻¹ (HI = 0.71) and 8 km · h⁻¹ (HI = 0.79), differences which probably suggest that there are differences in RE. In conclusion, we did not detect a genetic component in RE or in maximal oxygen uptake, but a genetic component for markers of anaerobic metabolism was present. Accepted: 5 November 1997     

Analysis of respiratory activity and carbon usage of a mutant of <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> impaired in poly-β-hydroxybutyrate synthesis

In this study, the respiratory activity and carbon usage of the mutant strain of A. vinelandii AT6, impaired in poly-β-hydroxybutyrate (PHB) production, and their relationship with the synthesis of alginate were evaluated. The alginate yield and the specific oxygen uptake rate were higher (2.5-fold and 62 %, respectively) for the AT6 strain, compared to the control strain (ATCC 9046), both in shake flasks cultures and in bioreactor, under fixed dissolved oxygen tension (1 %). In contrast, the degree of acetylation was similar in both strains. These results, together with the analysis of carbon usage (% C-mol), suggest that in the case of the AT6 strain, the flux of acetyl-CoA (precursor molecule for PHB biosynthesis and alginate acetylation) was diverted to the respiratory chain passing through the tricarboxylic acids cycle, and an important % C-mol was directed through alginate biosynthesis, up to 25.9 % and to a lesser extent, to biomass production (19.7 %).     

Immobilization of native and dextran-free dextransucrases from Leuconostoc mesenteroides NRRL B-512F for the synthesis of glucooligosaccharides

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was immobilized using two different methods: covalent attachment to activated silica and entrapment in calcium alginate. For immobilization on silica, native enzyme and dextran-free enzyme were compared. However, the entrapment in calcium alginate beads gave the best results in terms of immobilization yield and stability. This biocatalyst was employed in the acceptor reaction with maltose showing similar glucooligosaccharide production than the native enzyme but increased operational stability.