The effect of protein synthesis inhibitors on the glycosylation site occupancy of recombinant human prolactin |
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Authors: | Marc Shelikoff A J Sinskey Gregory Stephanopoulos |
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Institution: | (1) Department of Chemical Engineering, Massachusetts Institute of Technology, 18 Vassar St., 20A-207, 02139-4308 Cambridge, Massachusetts, USA;(2) Department of Biology, Massachusetts Institute of Technology, 18 Vassar St., 20A-207, 02139-4308 Cambridge, Massachusetts;(3) Biotechnology Process Engineering Center, Massachusetts Institute of Technology, 18 Vassar St., 20A-207, 02139-4308 Cambridge, Massachusetts |
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Abstract: | The relationship between synthesis and N-liked 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 (<10g 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.Abbreviations clp-PRL
clipped form of prolactin
- DMEM/F12
11 Dulbecco's Modified Eagle's Medium/Ham's nutrient mixture F12
- G-PRL
glycosylated (N-linked) fraction of prolaction
- NG-PRL
prolactin fraction without N-linked glycosylation
- PMSF
phenylmethylsulfonylfluoride |
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Keywords: | Cycloheximide glycosylation inhibition of protein synthesis post-translation processing prolactin |
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