期刊
BIOMOLECULES
卷 13, 期 5, 页码 -出版社
MDPI
DOI: 10.3390/biom13050817
关键词
membrane proteins; transient gene expression; insect cells; human embryonal kidney (HEK) cells; Chinese hamster ovary (CHO) cells; fluorescence-detection size-exclusion chromatography
This study compares the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The choice of expression systems has a significant impact on the yield and quality of the target proteins. Virus-free transient gene expression in insect High Five cells combined with solubilization in dodecylmaltoside plus cholesteryl hemisuccinate generated the most homogeneous samples for all six targets. TGE in High Five insect cells offers a fast and economically attractive alternative for the production of integral membrane proteins.
Membrane proteins are difficult biomolecules to express and purify. In this paper, we compare the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The target proteins were C terminally fused to the green fluorescent marker protein GFP to enable sensitive monitoring. We show that the choice of expression systems makes a considerable difference to the yield and quality of the six selected membrane proteins. Virus-free transient gene expression (TGE) in insect High Five cells combined with solubilization in dodecylmaltoside plus cholesteryl hemisuccinate generated the most homogeneous samples for all six targets. Further, the affinity purification of the solubilized proteins using the Twin-Strep((R)) tag improved protein quality in terms of yield and homogeneity compared to His-tag purification. TGE in High Five insect cells offers a fast and economically attractive alternative to the established methods that require either baculovirus construction and the infection of the insect cells or relatively expensive transient gene expression in mammalian cells for the production of integral membrane proteins.
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