Expression of the mouse interleukin-2 receptor gamma chain in insect cells using a baculovirus expression vector--comparison with the human common gamma chain.
The gene encoding the gamma-chain of the mouse Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates prepared from insect cells infected with the produced recombinant virus VL1392-mIL-2R gamma. Kinetic analysis demonstrated that the corresponding protein could be detected as an approximately 50 kDa protein already at 24 h post-infection. Intrinsic labelling with [35S]-methionine/cysteine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma protein could also be determined on the surface of infected insect cells by flow cytometric analysis. Comparison of the molecular weights between baculovirus expressed human and mouse IL-2R gamma chains indicated differences in the glycosylation pattern despite similar numbers of N-linked glycosylation sites.
Stenroos
K
K
Department of Biochemistry, Abo Akademi University, Turku, Finland.
West
A
A
Raivio
E
E
Lindqvist
C
C
D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007915:Lepidoptera; D051379:Mice; D017924:Nucleopolyhedroviruses / Q000235:genetics; D000077183:Occlusion Body Matrix Proteins; D011401:Promoter Regions, Genetic; D015375:Receptors, Interleukin-2 / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins
D000077183:Occlusion Body Matrix Proteins; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus