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  • 1
    Electronic Resource
    Electronic Resource
    Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells (1995)
    Springer
    The protein journal 14 (1995), S. 73-80 
    Details
    ISSN: 1573-4943
    Keywords: Cholesteryl ester transfer protein ; cynomolgus ; lipoprotein ; purification ; recombinant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2Μg/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band ofM r , ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01888364
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  • 2
    Electronic Resource
    Electronic Resource
    Stable production of an analog of human tissue plasminogen activator from culturedDrosophila cells (1992)
    Springer
    Cytotechnology 10 (1992), S. 157-167 
    Details
    ISSN: 1573-0778
    Keywords: Drosophila cell culture ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have studied the expression of an analog of human tissue plasminogen activator, FK2P, inDrosophila Schneider 2 cells. A number of promoters were tested, including theDrosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE),Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1α promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 μg FK2P/106 cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 μg FK2P/106 cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominately as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00570892
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  • 3
    Electronic Resource
    Electronic Resource
    The Phosphomannosyl Recognition System for Intracellular and Intercellular Transport of Lysosomal Enzymes (1982)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 18 (1982), S. 67-85 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.1982.240180107
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  • 4
    Electronic Resource
    Electronic Resource
    Comparative kinetics of phosphomannosyl receptor-mediated pinocytosis of fibroblast secretion acid hydrolases and glycopeptides prepared from them (1983)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 22 (1983), S. 69-86 
    Details
    ISSN: 0730-2312
    Keywords: phosphomannosyl receptor ; pinocytosis ; fibroblast secretions ; glycopeptides ; acid hydrolases ; lysosomotropic amines ; β-hexosaminidase B ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a previous report we demonstrated that phosphorylated oligosaccharides isolated from acid hydrolases were subject to pinocytosis by phosphomannosyl receptors present on the cell surface of human fibroblasts [9]. However, limiting quantities of oligosaccharides precluded detailed comparison of the kinetics of pinocytosis of these phosphorylated oligosaccharides to those of the acid hydrolases from which they were derived. In this report we present studies comparing the kinetics of pinocytosis of acid hydrolases from NH4Cl-induced fibroblast secretions with those of concanavalin A-binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125I-labeled glycopeptides was linear for at least 3 hr, saturable, inhibited competitively by mannose 6-phosphate, and destroyed by prior treatment of the ligand with alkaline phosphatase. The inhibition constants of excess unlabeled glycopeptide for the uptake of 125I-labeled glycopeptides (Ki of 1.5 × 10-6 M) and for the uptake of secretion acid hydrolases (Ki of 2.2 × 10-6 M) were remarkably similar. Furthermore, the Ki for mannose 6-phosphate inhibition of pinocytosis of glycopeptide uptake (3 × 10-5 M) compares closely to that previously determined for the pinocytosis of intact “high-uptake” acid hydrolases (3-6 × 10-5 M).“High-uptake” fractions of both ligands were prepared and quantified by affinity chromatography on immobilized phosphomannosyl receptors purified from bovine liver. Only 10% of the concanavalin A-binding glycopeptides bound to the immobilized phosphomannosyl receptors, while 80% of the acid hydrolases from which they were prepared bound and were eluted with 10 mM mannose 6-phosphate. However, the fraction of each type of ligand that binds to the immobilized phosphomannosyl receptors accounts for all the uptake activity of that ligand. The pinocytosis rates (% of added ligand internalized/mg protein/hr) of the “high-uptake” fraction of both intact acid hydrolase (12%/mg/hr) and glycopeptide (6%/mg/hr) differed by only twofold. The apparent Kuprake for both ligands was of the same order of magnitude. The similarity in the kinetics of pinocytosis of the secreted acid hydrolases and of the phosphomannose-bearing glycopeptides prepared from them suggests that the structural information which confers high-affinity binding to the phosphomannosyl receptor is contained in the glycopeptide units themselves. No additional information from the intact protein backbone appears essential for phosphomannosyl receptor-mediated pinocytosis.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240220202
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