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1.
Vet Parasitol ; 197(3-4): 571-9, 2013 Nov 08.
Article in English | MEDLINE | ID: mdl-23747105

ABSTRACT

Serodiagnosis of surra, which causes vast economic losses in livestock, is still based on native antigens purified from bloodstream form Trypanosoma (T.) evansi grown in rodents. To avoid the use of laboratory rodents in antigen preparation we expressed fragments of the invariant surface glycoprotein (ISG) 75, cloned from T. brucei gambiense cDNA, and the variant surface glycoprotein (VSG) RoTat 1.2, cloned from T. evansi gDNA, recombinantly in Pichia (P.) pastoris. The M5 strain of this yeast has an engineered N-glycosylation pathway resulting in homogenous Man5GlcNAc2 N-glycosylation which resembles the predominant Man9-5GlcNAc2 oligomannose structures in T. brucei. The secreted recombinant antigens were affinity purified with yields of up to 10mg and 20mg per liter cell culture of rISG 7529-465-E and rRoTat 1.223-385-H respectively. In ELISA, both recombinant proteins discriminated between pre-immune and immune serum samples of 25 goats experimentally infected with T. evansi. The diagnostic potential of rRoTat 1.223-385-H but not of rISG 7529-465-E was confirmed with sera of naturally infected and control dromedary camels. The results suggest that rRoTat 1.223-385-H expressed in P. pastoris requires further evaluation before it could replace native RoTat 1.2 VSG for serodiagnosis of surra, thus eliminating the use of laboratory animals for antigen production.


Subject(s)
Gene Expression Regulation/physiology , Pichia/metabolism , Protozoan Proteins/metabolism , Trypanosoma/metabolism , Animals , Dog Diseases/prevention & control , Dogs , Female , Protozoan Proteins/genetics , Time Factors , Trypanosoma/isolation & purification
2.
Eur J Cancer ; 43(17): 2515-22, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17933516

ABSTRACT

Many promising recombinant cancer medicines are generated by academic research and increasing the number of these products that are translated into the clinic will increase the pipeline of new therapies. Recombinant proteins for use in Phase I/II cancer trials must be produced to standards of Good Manufacturing Practice (GMP) in compliance with EU law. This can be a major obstacle for translating experimental products to clinical reality especially when there is no established process or prior experience with GMP. Here, we illustrate the principals of GMP with a step-by-step guide and we show that GMP can be achieved on a relatively small scale in the researchers own institution. The process is exemplified with an antibody-based therapeutic expressed in the yeast Pichia pastoris. The purified product has been used safely in patients and the principles are applicable to any recombinant protein required for Phase I/II cancer trials.


Subject(s)
Antineoplastic Agents/standards , Diffusion of Innovation , Neoplasms/drug therapy , Recombinant Proteins/standards , Antineoplastic Agents/therapeutic use , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Drug Design , Drug Industry/standards , Education, Pharmacy , Fermentation , Glycosylation , Humans , Professional Role , Quality Control , Recombinant Proteins/therapeutic use , Technology, Pharmaceutical/standards , Yeasts
3.
Int J Biochem Cell Biol ; 32(3): 317-26, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10716629

ABSTRACT

Type II ribosome inactivating proteins (RIP II) are generally known to induce apoptosis in human cells by the inhibition of protein biosynthesis. Recent data from mistletoe RIP II proteins (eg. mistletoe lectin I; ML1) suggest an additional mode of apoptosis induction through the binding of their lectin part to certain cell surface receptors as is known for some human galectins. In order to clarify this possibility, we used highly sensitive flow cytometric apoptosis assays and mistletoe hololectin subunits of proven purity to show that neither human lymphocytes nor Molt-4 cells undergo apoptosis after treatment with isolated lectin-type B-chains. In contrast to earlier investigations, only the hololectin was able to induce apoptosis in these assays. We conclude that direct apoptosis induction by mistletoe lectins occurs only after uptake of the molecules into the cell due to the action of the ribosome inactivating A-chain.


Subject(s)
Apoptosis/drug effects , Lectins/toxicity , Plant Preparations , Plant Proteins , Toxins, Biological/toxicity , Cell Line , Flow Cytometry , Humans , In Vitro Techniques , Lectins/chemistry , Lymphocytes/cytology , Lymphocytes/drug effects , Mistletoe , Plant Lectins , Plants, Medicinal , Protein Structure, Quaternary , Ribosome Inactivating Proteins, Type 2 , Ribosomes/drug effects , Toxins, Biological/chemistry , Toxins, Biological/isolation & purification
4.
Cytometry ; 37(2): 133-9, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10486525

ABSTRACT

BACKGROUND: It is unclear whether expression of newly described mitochondrial Apo2.7 molecules (7A6 antigen) is specific for apoptosis or may also occur in necrosis. METHODS: We incubated human lymphocytes with the apoptosis-inducing mistletoe lectin (ML) I and the cell membrane-permeabilizing viscotoxins (VT), and measured cell death-associated changes by flow cytometry. RESULTS: In ML I-treated lymphocytes, Apo2.7 expression and caspase-3 activation was recognized within 24 h. In VT-treated cells, we observed an Apo2.7 expression with low fluorescence level, while active caspase-3 and DNA fragments (TUNEL) were not detected within 24 h. In these cells, caspase-3 activation was recognized 48 h later. As a major subset of ML-treated cells expressing Apo2.7 molecules did not activated caspase-3, while all caspase-3(+) cells did express Apo2.7, one may suggest that the caspase pathway is activated secondarily to mitochondrial events. CONCLUSIONS: Expression of Apo2.7 is sensitive marker of cell death but may not be specific for apoptosis alone as it can be detected also in cells treated with cell membrane-permeabilizing toxins. On the other hand, this expression may be the consequence of an induction of distinct "death signals" resulting in apoptosis later on.


Subject(s)
Apoptosis , Caspases/metabolism , Lectins/pharmacology , Lymphocytes/drug effects , Membrane Proteins/metabolism , Plant Preparations , Plant Proteins , Toxins, Biological/pharmacology , Caspase 3 , Cells, Cultured , Enzyme Activation , Flow Cytometry , Humans , In Situ Nick-End Labeling , Lymphocytes/metabolism , Lymphocytes/ultrastructure , Microscopy, Electron , Mistletoe , Plant Lectins , Plants, Medicinal , Ribosome Inactivating Proteins, Type 2 , Time Factors
5.
FEBS Lett ; 452(3): 365-70, 1999 Jun 11.
Article in English | MEDLINE | ID: mdl-10386623

ABSTRACT

The human N-acetylglucosaminyltransferase I gene was introduced in the genome of Trichoderma reesei strain VTT-D-80133. Expression was studied after induction from the cellobiohydrolase I promoter. Successful in vivo transfer of GlcNAc was demonstrated by analyzing the neutral N-glycans which were synthesized on cellobiohydrolase I. Final proof of the formation of GlcNAcMan5GlcNAc2 was obtained by NMR analysis.


Subject(s)
N-Acetylglucosaminyltransferases/metabolism , Polysaccharides/biosynthesis , Trichoderma/physiology , Carbohydrate Sequence , Cellulase/genetics , Cellulose 1,4-beta-Cellobiosidase , Cloning, Molecular , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , N-Acetylglucosaminyltransferases/genetics , Oligosaccharides/chemistry , Polysaccharides/chemistry , Polysaccharides/isolation & purification , Promoter Regions, Genetic , Recombinant Proteins/metabolism , Spores, Fungal , Trichoderma/metabolism
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