Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells
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Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells. / Kaipa, Jagan Mohan; Krasnoselska, Ganna; Owens, Raymond J.; van den Heuvel, Joop.
In: Biomolecules, Vol. 13, No. 5, 817, 2023.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells
AU - Kaipa, Jagan Mohan
AU - Krasnoselska, Ganna
AU - Owens, Raymond J.
AU - van den Heuvel, Joop
N1 - Publisher Copyright: © 2023 by the authors.
PY - 2023
Y1 - 2023
N2 - 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® 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.
AB - 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® 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.
KW - Chinese hamster ovary (CHO) cells
KW - fluorescence-detection size-exclusion chromatography
KW - human embryonal kidney (HEK) cells
KW - insect cells
KW - membrane proteins
KW - transient gene expression
UR - http://www.scopus.com/inward/record.url?scp=85160376495&partnerID=8YFLogxK
U2 - 10.3390/biom13050817
DO - 10.3390/biom13050817
M3 - Journal article
C2 - 37238687
AN - SCOPUS:85160376495
VL - 13
JO - Biomolecules
JF - Biomolecules
SN - 2218-273X
IS - 5
M1 - 817
ER -
ID: 353762311