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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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Probing the structure-function relationship of nerve growth factor (1992)

Vroegop, Steven ; Decker, Douglas ; Hinzmann, Jessica ; [et al.]

Springer

The protein journal 11 (1992), S. 71-82

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ISSN: 1573-4943

Keywords: Nerve growth factor (NGF) ; structure/function comparison ; NGF-receptor ; neurite outgrowth ; biological activation

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED50 for biological activation is approximately 100-fold lower than theK d for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (K d=10−9 M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF: NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.