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Single-Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells (1991)

Buxser, Stephen ; Vroegop, Steven ; Decker, Douglas ; [et al.]

Oxford, UK : Blackwell Publishing Ltd

Journal of neurochemistry 56 (1991), S. 0

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ISSN: 1471-4159

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Medicine

Notes: Abstract: Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2–3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1–2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transacted into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was 〉 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 ± 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 ± 20 pM and 9.6 ± 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 ± 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from sub-maxillary glands.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1471-4159.1991.tb02022.x

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Structure of O-glycosidically linked oligosaccharides synthesized by the insect cell line Sf9 (1990)

Thomsen, Darrell R. ; Post, Leonard E. ; Elhammer, Åke P.

New York, N.Y. : Wiley-Blackwell

Journal of Cellular Biochemistry 43 (1990), S. 67-79

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ISSN: 0730-2312

Keywords: insect cells ; O-glycosidic glycosylation ; glycosyltransferases ; O-Linked oligosaccharides ; O-glycosylation ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: The O-glycosidically linked Oligosaccharides on the pseudorabies virus (PRV) glycoprotein gp50 synthesized by three different cell lines were studied. The intact membrane protein (gp50) was expressed in Vero cells and in the insect cell line Sf9. In addition, a truncated, secreted form lacking the transmembrane and cytoplasmic domains (gp50T), was expressed in CHO and Sf9 cells. The protein, both in intact and truncated form, synthesized by the two mammalian cells contained only the disaccharide Galβ1-3GalNAc, either unsubstituted or substituted with one or tow sialic acid residues. By contrast, the major O-linked structure on gp50 and gp50T synthesized by Sf9 cells was the monosaccharide GalNAc. The Sf9 cells also linked lower amounts of Galβ1-3GalNAc to gp50 (12%) and gp50T (26%). None of the structures synthesized by Sf9 cells contained sialic acid. Measurements of the two relevant glycosyltransferases revealed that while all three cell lines contain comparable levels of the UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase activity, there is a greater variation in the levels of UDP-Gal: N-acetylgalactosamine, β1-3 galactosyltransferase, with the Sf9 cells containing the lowest level.

Additional Material: 4 Ill.