Folding of influenza virus hemagglutinin in insect cells

Proteins play crucial roles for life. To be able to function, proteins need to fold into their correct conformation. Proper folding is equally important for foreign proteins that are expressed in a heterologous system. More and more proteins are being expressed in insect cells for various purposes. However, how they are folded in these cells is barely known. Elucidating protein-folding processes is important for understanding and improving the quality and efficiency of protein expression in insect cells.We used influenza virus hemagglutinin (HA) as our model protein and studied its folding in Sf9 cells. We adapted the pulse-chase assay to Sf9 cells, studied folding of HA, and found that its folding was efficient. Yet, HA folding in Sf9 cells showed different kinetics than in HeLa cells. In contrast to HeLa cells, folding of HA in Sf9 cells was efficient at all tested temperatures, ranging from 24°C to 45°C. These suggested that HA folding was faster and more robust in insect cells than in mammalian cells. To determine the cause for the HA folding differences between insect and mammalian cells, we co-expressed mammalian chaperones with HA in Sf9 cells and identified PDI, CNX, and CRT as the chaperones and folding enzymes most likely to be responsible for these differences. Mammalian PDI, CNX, and CRT slowed down HA folding at different steps and that CNX and CRT converted part of the HA folding phenotype in Sf9 cells into that in mammalian cells. HA folding in Sf9 cells showed no IT1 population or temperature sensitivity. In mammalian cells folding of the temperature-sensitive mutant HA C139A is blocked in an IT1-like intermediate at non-permissive temperatures above 26°C. We found that HA C139A did not escape the IT1 folding block and its folding remained temperature sensitive in Sf9 cells, indicating that HA folding was not only determined by the folding environment but also by the characteristics of HA itself. Proteins need to be co-translationally translocated into the ER before folding, which needs mediation by the signal peptide and chaperones that reside in the endoplasmic reticulum (ER), such as BiP. We describe that manipulation of translocation did not affect folding of HA. Changing the signal peptide to that of the HIV-1 Envelope glycoprotein (Env) did not change HA folding despite a change in translation/translocation. We showed that BiP interacted with HA early during folding suggesting that it may help HA translocation, which confirmed our findings in chapter 3. Prolonging the interaction by either mutation of HA or BiP did not affect HA folding. Taken together, we found that signal peptide and BiP are involved in HA translation/translocation without a strong effect on folding. HA folding in Sf9 cells was efficient and temperature insensitive. Mammalian PDI, CNX, and CRT slowed down HA folding at different steps in Sf9 cells. Human CNX and CRT introduced part of the mammalian HA folding phenotype into insect cells. Although folding factors did affect HA folding efficiency and rate, the HA protein identity determined pattern and outcome of folding.