The effect of temperature on the folding of vertebrate glycoproteins in insect cells

M.M. van Oers, L. Xin, M.C. Hikke, S.W.H. Metz, J.M. Vlak, I. Braakman, G.P. Pijlman

Research output: Contribution to conferenceAbstractAcademic

Abstract

Insects are poikilothermic organisms able to survive in fluctuating ambient temperatures. As their body temperature varies with the temperature of the environment, it is likely that these invertebrates have properties that allow correct folding of glycoproteins within a broad temperature range. To test this concept, we studied the folding ability of the influenza virus hemagglutinin (HA) expressed in Sf9 insect cells growing at 27°C and compared with human HeLa cells growing at 37°C, the optimal temperature for HA. HA folding was demonstrated to be more efficient in insect cells than in HeLa cells and remained fast over a broad temperature range, from 24°C to 45°C. An HA mutant that only folds below 27°C, however, showed identical temperature-sensitivity for folding in insect cells: it did not fold at 37°C. We also tested a protein with a low natural optimal temperature. Therefore, we expressed the structural polyprotein of salmonid alphavirus (SAV) in Sf9 cells, using a baculovirus vector. A similar strategy had previously resulted in immunogenic and correctly processed enveloped virus-like particles (eVLPs) of the SAV-related chikungunya virus. However, at 27°C, no SAV eVLPs were formed. The SAV envelope glycoprotein E2 was not transported from the endoplasmic reticulum (ER) to the Golgi due to incorrect folding, despite the presence of E1. SAV normally replicates between 10°C and 15°C. We therefore lowered the temperature during the glycoprotein production phase to 15°C and found that E2 transport to the cell surface recovered and eVLPs were secreted into the culture fluid. We concluded that protein-intrinsic properties may limit the temperature range for correct folding, but that insect cells — in contrast to mammalian cells — have the ability to fold glycoproteins at a broad temperature range. This quality makes insect cells highly suitable for expression of both mammalian and fish virus glycoproteins.
Original languageEnglish
Publication statusPublished - 2015
Event5th spring meeting ISBioTech, Washington, USA - , United States
Duration: 8 Mar 201511 Mar 2015

Conference

Conference5th spring meeting ISBioTech, Washington, USA
CountryUnited States
Period8/03/1511/03/15

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