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To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields. Chemicals/CAS: Ferrous Compounds; Heme, 14875-96-8; lignin peroxidase, EC 1.11.1.-; manganese peroxidase, EC 1.11.1.13; Peroxidases, EC 1.11.1.-; Recombinant Proteins

Filamentous fungi are commonly used in the fermentation industry for large scale production of glycoproteins. Several of these proteins can be produced in concentrations up to 20-40 g per litre. The production of heterologous glycoproteins is at least one or two orders of magnitude lower but research is in progress to increase the production levels. In the past years the structure of protein-linked carbohydrates of a number of fungal proteins has been elucidated, showing the presence of oligo-mannosidic and high-mannose chains, sometimes with typical fungal modifications. A start has been made to engineer the glycosylation pathway in filamentous fungi to obtain strains that show a more mammalian-like type of glycosylation. This mini review aims to cover the current knowledge of glycosylation in filamentous fungi, and to show the possibilities to produce glycoproteins with these organisms with a more mammalian-like type of glycosylation for research purposes or pharmaceutical applications.Chemicals/CAS: Glycoproteins; Oligosaccharides; Recombinant Proteins

BM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated. Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against C1-inactivator, α2-antiplasmin and α1-antitrypsin. During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of α2-antiplasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i.e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher. In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by C1-inactivator, α2-antiplasmin and α1-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form. Chemicals/CAS: alpha 1 antitrypsin, 9041-92-3; alteplase, 105857-23-6; fibrinogen, 9001-32-5; plasmin, 9001-90-5, 9004-09-5; reteplase, 133652-38-7; tissue plasminogen activator, 105913-11-9; proteinase inhibitor, 37205-61-1; BM 06.022; Fibrinolytic Agents; Protease Inhibitors; Recombinant Proteins; Tissue Plasminogen Activator, EC 3.4.21.68

Background and Objective: The purpose of this study was to investigate the effects of chemically modified tetracyclines (CMTs) on the production of gelatinases [matrix metalloproteinase (MMP)-2 and -9] by human periodontal ligament (PDL) cells, and on the activity of recombinant gelatinases. Material and Methods: Human PDL cells were cultured with CMT-1, -3, -5, -7 or -8 in concentrations of 0, 1, 5, 10, 20, 50, 100, 200 and 500 μm. Gelatin zymography was used to determine MMP-2 and -9 production of the cells. The amount of DNA present in the cultures was analyzed using a fluorescent assay. The cytotoxicity of the CMTs was also determined. Recombinant human MMP-2 and -9 were incubated with the CMTs (0-500 μm) and their activity was analyzed using an internally quenched fluorogenic substrate. Results: MMP-2 production was stimulated up to sevenfold by CMT-1, -3, -7 and -8 at low concentrations (10-200 μm). No significant amounts of MMP-9 were produced. In contrast, MMP-2 and -9 activity was reduced by ≈ 10-40-fold at higher concentrations (200-500 μm). CMT-5 had no effect on the production or on the activity of MMP-2 and -9. Only CMT-3 and -8 had cytotoxic effects on the PDL cells at the highest concentrations. Conclusion: Surprisingly, CMTs are able to stimulate MMP-2 production at relatively low concentrations. However, at higher concentrations they exert a much stronger inhibitory effect on gelatinase activity. A possible stimulatory effect of CMTs on MMP production should be considered in their clinical use. © 2006 The Authors. Chemicals / CAS: DNA, 9007-49-2; gelatin, 9000-70-8; gelatinase A, 146480-35-5; gelatinase B, 146480-36-6; DNA, 9007-49-2; Enzyme Inhibitors; Gelatin, 9000-70-8; Matrix Metalloproteinase 2, EC 3.4.24.24; Matrix Metalloproteinase 9, EC 3.4.24.35; Recombinant Proteins; Tetracyclines

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 ± 0.01 h-1, pH 5.4). In cultures supplemented with L-alanine, L-methionine, casamino acids, or peptone, specific glucoamylase (GAM) production rapidly decreased to less than 20% of the initial level. Reducing the pH of the culture to 4.0 resulted in stable GAM production for up to 400 h. Morphological mutants (a light brown and a dark brown mutant) appeared in each fermentation and generally displaced B1. Light brown mutants had higher selection coefficients relative to B1 than dark brown mutants and became the dominant strain in all fermentations except those maintained at pH 4.0. Several mutants isolated from these cultures had reduced ability to produce GAM in batch culture, although few had lost copies of the glaA gene. Some mutants had methylated DNA. © 2000 Academic Press.

The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bipA, the BiP-encoding gene from Aspergillus niger and Aspergillus awamori. As this result could imply that BiP plays a role in protein overproduction, the effect of modulation of bipA gene expression on protein secretion was studied in several recombinant strains expressing glucoamylase (glaA) fusion genes. For overproduction of BiPA in these strains, extra copies of the bipA gene under the control of an inducible promoter were introduced. To allow analysis of the effect of a decreased bipA expression level on protein secretion, replacement of the wild-type gene for a bipA gene driven by the glaA promoter was attempted. However, this endeavour failed because of the lethality of this replacement. Although the final amount of secreted recombinant protein did not change significantly in strains with increased BiPA levels, increased levels of unprocessed fusion protein were detected in the total protein extracts of these strains.

Angiostatin, which consists of the kringle I-IV domains of plasminogen and which is secreted into urine, is an efficient inhibitor of angiogenesis and tumor growth. Because N-terminal apolipoprotein(a) [apo(a)] fragments, which also contain several types of kringle IV domains, are found in urine as well, we evaluated the potential angiostatic properties of these urinary apo(a) fragments and of a recombinant form of apo(a) [r-apo(a)]. We used human microvascular endothelial cell (hMVEC)-based in vitro assays of tube formation in 3-dimensional fibrin matrixes. Purified urinary apo(a) fragments or r-apo(a) inhibited the basic fïbroblast growth factor/tumor necrosis factor-α-induced formation of capillary-like structures. At concentrations varying from 0.2 to 10 μg/mL, urinary apo(a) fragments inhibited tube formation by as much as 70%, whereas there was complete inhibition by r-apo(a). The highest concentrations of both inhibitors also reduced urokinase plasminogen activator production of basic fibroblast growth factor-induced hMVEC proliferation. The inhibitors had no effect on plasminogen activator inhibitor-1 expression. If our in vitro model for angiogenesis is valid for the in vivo situation as well, our data point toward the possibility that apo(a) may also be physiologically operative in modulating angiogenesis, as the concentration of free apo(a) found in humans exceeds that tested herein. Chemicals/CAS: Angiogenesis Inhibitors; Apolipoproteins A; Fibroblast Growth Factor 2, 103107-01-3; Peptide Fragments; Plasminogen Activator Inhibitor 1; Tumor Necrosis Factor-alpha; Urinary Plasminogen Activator, EC 3.4.21.73

We have characterized growth and protein processing characteristics of Aspergillus niger strains carrying a disrupted allele of the previously cloned and characterized kexB gene [Appl. Environ. Microbiol. 66 (2000) 363] encoding a furin-type endoprotease. Deletion of the single-copy gene confirms it to be non-essential but disruptant strains exhibit a morphologically distinct phenotype characterized by hyperbranching. Processing of homologous pro-proteins and fusion proteins comprised of a heterologous protein fused down-stream of glucoamylase and separated at the fusion junction by an endoproteolytic cleavage site was compared in wildtype and mutant strains of A. niger. We show that maturation of the native glucoamylase requires KexB, whereas maturation of aspergillopepsin does not. The processing of fusion proteins carrying Lys-Arg requires KexB, although alternative endoproteases are capable of cleaving protein fusions at sites adjacent to Lys-Arg. © 2003 Elsevier B.V. All rights reserved.

Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 μg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs. © 2015 American Society for Clinical Pharmacology and Therapeutics. Chemicals/CAS: alkaline phosphatase, 9001-78-9; carbon, 7440-44-0; Alkaline Phosphatase; alkaline phosphatase, placental; Carbon Radioisotopes; GPI-Linked Proteins; Isoenzymes; Recombinant Proteins

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L-1 in batch culture and 373 ± 9 mg GAM L-1 in maltodextrin- limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (q(p)) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (q(p), 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 ± 12 to 496 ± 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 ± 0.4 to 16.4 ± 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar glucoamylase (GAM) production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of GAM gene copies in the later strain was reduced, which could explain its reduced GAM production. Shake-flask cultures of morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains. The physiological changes in these morphological mutants which contribute to this decreased level of GAM production were shown

To identify the residues in the carboxyl-terminal region 260-299 of human apolipoprotein E (apoE) that contribute to hypertriglyceridemia, two sets of conserved, hydrophobic amino acids between residues 261 and 283 were mutated to alanines, and recombinant adenoviruses expressing these apoE mutants were generated. Adenovirus-mediated gene transfer of apoE4-mut1 (apoE4 (L261A, W264A, F265A, L268A, V269A)) in apoE-deficient mice (apoE-/-) corrected plasma cholesterol levels and did not cause hypertriglyceridemia. In contrast, gene transfer of apoE4-mut2 (apoE4 (W276A, L279A, V280A, V283A)) did not correct hypercholesterolemia and induced mild hypertriglyceridemia. ApoE-induced hyperlipidemia was corrected by co-infection with a recombinant adenovirus expressing human lipoprotein lipase. Both apoE4 mutants caused only a small increase in hepatic very low density lipoprotein-triglyceride secretion. Density gradient ultracentrifugation analysis of plasma and electron microscopy showed that wild-type apoE4 and apoE4-mut2 displaced apoA-I from the high density lipoprotein (HDL) region and promoted the formation of discoidal HDL, whereas the apoE4-mut1 did not displace apoA-I from HDL and promoted the formation of spherical HDL. The findings indicate that residues Leu-261, Trp-264, Phe-265, Leu-268, and Val-269 of apoE are responsible for hypertriglyceridemia and also interfere with the formation of HDL. Substitutions of these residues by alanine provide a recombinant apoE form with improved biological functions. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Chemicals / CAS: alanine, 56-41-7, 6898-94-8; amino acid, 65072-01-7; leucine, 61-90-5, 7005-03-0; lipoprotein lipase, 83137-80-8, 9004-02-8; phenylalanine, 3617-44-5, 63-91-2; tryptophan, 6912-86-3, 73-22-3; valine, 7004-03-7, 72-18-4; Apolipoproteins E; Cholesterol, 57-88-5; Lipoproteins, HDL; Lipoproteins, VLDL; Peptide Fragments; Recombinant Proteins; Triglycerides; very low density lipoprotein triglyceride

Tolerance induction to prevent activation of a naïve T-cell repertoire has been well documented in rodents and can be readily achieved by intravenous, oral or intranasal administration of antigen in the absence of adjuvants. In autoimmune diseases such as multiple sclerosis (MS) the presence of an established memory/effector T-cell repertoire against self-antigens is likely to be more relevant than the potential reactivity of naive T cells. Methods to eliminate such an established T-cell response are less well understood. In this study, we explored the effectiveness of intravenous soluble antigen to eliminate a pre-existing T-cell response against αB-crystallin, a candidate autoantigen in MS. We used mice that are deficient for the target antigen. This condition allowed for a vigourous T-cell and antibody response to develop upon immunization, and eliminated all possible endogenous mechanisms of tolerance for αB-crystallin that are found in normal rodents. When applied 3 weeks after priming with αB-crystallin, intravenous administration of soluble antigen almost completely abrogated the established T-cell response in a dose-dependent manner as evidenced by T-cell non-responsiveness in tolerized animals to a re-challenge with antigen in complete Freund's adjuvant. Evaluating delayed-type hypersensitivity responses after tolerance induction revealed that the tolerizing effect was achieved within 24 hr. Furthermore, the tolerizing effect was found to be antigen-specific and long lasting. In contrast, serum antibody levels were markedly increased. Our data clarify that in the absence of any natural form of immune regulation, antigen-specific memory/effector T cells can be effectively silenced by intravenous antigen. © 2007 Blackwell Publishing Ltd.

αB-crystallin (CRYAB) is the most abundant gene transcript present in early active multiple sclerosis lesions, whereas such transcripts are absent in normal brain tissue. This crystallin has anti-apoptotic and neuroprotective functions. CRYAB is the major target of CD4+ T-cell immunity to the myelin sheath from multiple sclerosis brain. The pathophysiological implications of this immune response were investigated here. We demonstrate that CRYAB is a potent negative regulator acting as a brake on several inflammatory pathways in both the immune system and central nervous system (CNS). Cryab-/- mice showed worse experimental autoimmune encephalomyelitis (EAE) at the acute and progressive phases, with higher Th1 and Th17 cytokine secretion from T cells and macrophages, and more intense CNS inflammation, compared with their wild-type counterparts. Furthermore, Cryab-/- astrocytes showed more cleaved caspase-3 and more TUNEL staining, indicating an anti-apoptotic function of Cryab. Antibody to CRYAB was detected in cerebrospinal fluid from multiple sclerosis patients and in sera from mice with EAE. Administration of recombinant CRYAB ameliorated EAE. Thus, the immune response against a negative regulator of inflammation, CRYAB, in multiple sclerosis, would exacerbate inflammation and demyelination. This can be countered by giving CRYAB itself for therapy of ongoing disease. ©2007 Nature Publishing Group.

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-staterelated physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilmreactor for the production of a Gla::green fluorescent protein (GFP) fusion protein by Aspergillus oryzae. The biofilmreactor comprises a metal structured packing allowing the attachment of the fungal biomass. Since the production of the target protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, the biofilm mode of culture is expected to enhance the global productivity. Although production of the target protein was enhanced by using the biofilm mode of culture, we also found that fusion protein production is also significant when the submerged mode of culture is used. This result is related to high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2-D gel electrophoresis highlights the preservation of fusion protein integrity produced in biofilm conditions. Two fungal biofilm reactor designs were then investigated further, i.e.with full immersion of the packing or with medium recirculation on the packing, and the scale-up potentialities were evaluated. In this context, it has been shown that full immersion of the metal packing in the liquid medium during cultivation allows for a uniformcolonization of the packing by the fungal biomass and leads to a better quality of the fusion protein.

Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypoosmotic shock and high external calcium) were found transiently to increase [Ca 2+]c. Each of the calcium signatures associated with these physico-chemical treatments was unique, suggesting the involvement of three distinct calcium-mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca2+]c responses to hypo-osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca 2+]c responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca 2+]c levels.