Single‐Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells

Stephen Buxser, Steven Vroegop, Douglas Decker, Jessica Hinzmann, Roger Poorman, Darrell R. Thomsen, Mike Stier, Irene Abraham, Barry D. Greenberg, Nicole T. Hatzenbuhler, Mary Shea, Kim A. Curry, Che‐Shen C. Tomich

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1012-1018
Number of pages7
JournalJournal of Neurochemistry
Volume56
Issue number3
DOIs
StatePublished - Mar 1991
Externally publishedYes

Keywords

  • Human nerve growth factor

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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  • Cite this

    Buxser, S., Vroegop, S., Decker, D., Hinzmann, J., Poorman, R., Thomsen, D. R., Stier, M., Abraham, I., Greenberg, B. D., Hatzenbuhler, N. T., Shea, M., Curry, K. A., & Tomich, CS. C. (1991). Single‐Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells. Journal of Neurochemistry, 56(3), 1012-1018. https://doi.org/10.1111/j.1471-4159.1991.tb02022.x