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1.
BMC Biotechnol ; 15: 87, 2015 Sep 18.
Article in English | MEDLINE | ID: mdl-26382581

ABSTRACT

UNLABELLED: BACKGROUND & METHODS: Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & DISCUSSION: The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. CONCLUSIONS: From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.


Subject(s)
Factor VIIa/chemistry , Factor VIIa/metabolism , Glycosylation , Animals , CHO Cells , Carbohydrate Sequence , Cricetinae , Cricetulus , HEK293 Cells , Humans , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
3.
Free Radic Biol Med ; 40(5): 897-906, 2006 Mar 01.
Article in English | MEDLINE | ID: mdl-16520241

ABSTRACT

Although oxygen is essential for aerobic organisms, it also forms potentially harmful reactive oxygen species. For its simplicity, easy manipulation, and cultivation conditions, yeast is used as an attractive model in oxidative stress research. However, lack of polyunsaturated fatty acids in yeast membranes makes yeast unsuitable for research in the field of lipid peroxidation. Therefore, we have constructed a yeast strain expressing a Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This yeast strain expresses the heterologous desaturase in an active form and, consequently, produces Delta9/Delta12 polyunsaturated fatty acids under inducing conditions. The functional expression of the heterologous desaturase did not affect cellular morphology or growth, indicating no general adverse effect on cellular physiology. However, the presence of polyunsaturated fatty acids changed the yeast's sensitivity to oxidative stress induced by addition of paraquat, tert-butylhydroperoxide, and hydrogen peroxide. This difference in sensitivity to the latter was followed by the formation of 4-hydroxy-2-nonenal, one of the end products of linoleic fatty acid peroxidation, which is known to play a role in cell growth control and signaling. Here we show that this yeast strain conditionally expressing the Delta12 desaturase gene provides a novel and well-defined eukaryotic model in lipid peroxidation research. Its potential to investigate the molecular basis of responses to oxidative stress, in particular the involvement of reactive aldehydes derived from fatty acid peroxidation, especially 4-hydroxy-2-nonenal, will be addressed.


Subject(s)
Fatty Acid Desaturases/metabolism , Fatty Acids, Unsaturated/biosynthesis , Lipid Peroxidation , Oxidative Stress , Plant Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Aldehydes/metabolism , Amino Acid Sequence , Cloning, Molecular , Fatty Acid Desaturases/genetics , Free Radicals/toxicity , Hevea/enzymology , Hevea/genetics , Hydrogen Peroxide/toxicity , Molecular Sequence Data , Paraquat/toxicity , Plant Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolism , tert-Butylhydroperoxide/toxicity
4.
FEBS Lett ; 577(3): 501-6, 2004 Nov 19.
Article in English | MEDLINE | ID: mdl-15556636

ABSTRACT

S-adenosyl-L-homocysteine hydrolase (Sah1p, EC 3.3.1.1.) is a key enzyme of methylation metabolism. It catabolizes S-adenosyl-L-homocysteine, which is formed after donation of the activated methyl group of S-adenosyl-L-methionine (AdoMet) to an acceptor, and which acts as strong competitive inhibitor of all AdoMet-dependent methyltransferases. Sah1p is an essential enzyme in yeast and one of the most highly conserved proteins with up to 80% sequence homology throughout all kingdoms of life. SAH1 expression in yeast is subject to the general transcriptional control of phospholipid synthesis. Profound changes in cellular lipid composition upon depletion of Sah1p support the notion of a tight interaction between lipid metabolism and Sah1p function.


Subject(s)
Hydrolases/metabolism , Phospholipids/biosynthesis , S-Adenosylhomocysteine/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Consensus Sequence , Conserved Sequence , Gene Deletion , Gene Expression Regulation, Fungal , Genes, Fungal , Genetic Markers , Methylation , Models, Biological , Molecular Sequence Data , Promoter Regions, Genetic , S-Adenosylhomocysteine/chemistry , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid
5.
Br J Haematol ; 125(5): 629-37, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15147379

ABSTRACT

The C2 domain of factor VIII (FVIII) is important for FVIII-phospholipid (PL) and FVIII-von Willebrand factor (VWF) interactions. A FVIII structural model, derived by electron crystallography, suggests four hydrophobic loops at the FVIII C2 domain-PL interface. Within loop four, the solvent-exposed amino acid, Trp(2313), is believed to contribute to FVIII-PL binding. To analyse this interaction, the amino-acid exchange Trp(2313) to Ala (W2313A) was introduced into the C2 domain of B-domain-deleted FVIII (dBFVIII). Both proteins, dBFVIII and W2313A, were expressed in a mammalian expression system. Labelling experiments showed that the mutation W2313A resulted in reduced secretion but did not affect intracellular synthesis of the protein. Specific activity, kinetic parameters, binding to VWF and haemostatic potential in a murine model of haemophilia A were found to be similar for both proteins. Binding studies to synthetic 4% phosphatidyl-l-serine vesicles showed, however, a 28-fold higher K(D) for W2313A, indicating the important role of Trp(2313) in the FVIII-PL interaction. In conclusion, the C2-domain-surface-exposed residue Trp(2313), is critical for secretion of the protein. The W2313A mutation weakens binding to phosphatidyl-l-serine vesicles but the mutant protein has the same effector function as dBFVIII in vitro and in vivo.


Subject(s)
Factor VIII/genetics , Mutation/genetics , Alanine/genetics , Blotting, Western , Cell Line , Electrophoresis, Polyacrylamide Gel , Factor VIII/chemistry , Factor VIII/pharmacokinetics , Factor Xa/metabolism , Humans , Protein Binding , Protein Structure, Tertiary , Tryptophan/genetics , von Willebrand Factor/metabolism
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