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The effect of protein synthesis inhibitors on the glycosylation site occupancy of recombinant human prolactin

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Cell Culture Engineering IV

Part of the book series: Current Applications of Cell Culture Engineering ((CACC,volume 1))

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Abstract

The relationship between synthesis and N-linked glycosylation site occupancy of recombinant human prolactin produced from C127 cells was studied with the aid of a battery of protein synthesis inhibitors. Non-lethal concentrations of sodium fluoride, gougerotin, puromycin, anisomycin, and emetine did not alter site occupancy, but low concentrations (<10μg ml-1) of cycloheximide increased the fraction of secreted prolactin bearing oligosaccharide from 20% to 80% of the total. Cycloheximide is an inhibitor of the elongation step of protein synthesis. The observed increase in glycosylation site occupancy upon addition of cycloheximide is consistent with the current opinion that the initial glycosylation event occurs cotranslationally during a limited time period. Cycloheximide may extend this time period by reducing elongation rate. However, the absence of any effect from treatment with other inhibitors of elongation suggests that cycloheximide is unique in its behavior on this system.

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Abbreviations

clp-PRL:

clipped form of prolactin

DMEM/F12:

1:1 Dulbecco’ Modified Eagle’ Medium/Ham’ nutrient mixture F12

G-PRL:

glycosylated (N-linked) fraction of prolactin

NG-PRL:

prolactin fraction without N-linked glycosylation

PMSF:

phenylmethylsulfonylfluoride

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Authors and Affiliations

  1. Departments of Chemical Engineering, Massachusetts Institute of Technology, 18 Vassar St., 20A-207, Cambridge, Massachusetts, 02139-4308

    Marc Shelikoff & Gregory Stephanopoulos

  2. Departments of Biology and the Biotechnology Process Engineering Center, Massachusetts Institute of Technology, 18 Vassar St., 20A-207, Cambridge, Massachusetts, 02139-4308

    A. J. Sinskey

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  1. Marc Shelikoff
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  2. A. J. Sinskey
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  3. Gregory Stephanopoulos
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Editors and Affiliations

  1. Merck Research Laboratories, Biochemical Process R&D, Rahway, New Jersey, USA

    Barry C. Buckland

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© 1994 Springer Science+Business Media Dordrecht

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Shelikoff, M., Sinskey, A.J., Stephanopoulos, G. (1994). The effect of protein synthesis inhibitors on the glycosylation site occupancy of recombinant human prolactin. In: Buckland, B.C., Aunins, J.G., Bibila, T.A., Hu, WS., Robinson, D.K., Zhou, W. (eds) Cell Culture Engineering IV. Current Applications of Cell Culture Engineering, vol 1. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-0257-5_22

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  • DOI: https://doi.org/10.1007/978-94-011-0257-5_22

  • Publisher Name: Springer, Dordrecht

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