The murine thymic stromal lymphopoietin receptor is partially expressed with an extracellular C-terminus.
The gene encoding the murine thymic stromal lymphopoietin 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 with an N-terminal poly-histidine tag and a C-terminal FLAG-tag in the Spodoptera frugiperda insect cell line Sf9 during viral infection. Flow cytometer analysis of cells infected with the produced recombinant virus FastBacHisB-mdelta1-FLAG demonstrated that a majority of the infected cells expressed the mTSLPR with an extracellular C-terminal end. A similar observation was noticed in COS cells transfected with pSVL-mTSLPR-FLAG. Immunoblotting with monoclonal anti-FLAG or anti-his antibodies indicated that the corresponding receptor protein migrated as an approximately 50 kDa protein. mTSLPR produced in presence of tunicamycin migrated with a molecular weight around 40 kDa. The genetically fused poly-histidine tag was also demonstrated to be functional using a Ni-NTA purification system, indicating this protein otherwise to have normal biochemical properties.
Wikman
Linnea
L
Department of Biology, Abo Akademi University, Artillerigatan 6, Turku FIN-20520, Finland.
Boström
Marina
M
Järnström
Ann-Charlotte
AC
Lundin
Karolina
K
Nordström
Tommy
T
Lindqvist
Christer
C
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D007136:Immunoglobulins; D051379:Mice; D015335:Molecular Probes; D008970:Molecular Weight; D018121:Receptors, Cytokine / Q000737:chemistry* / Q000235:genetics*; D017154:Stromal Cells; D014415:Tunicamycin / Q000494:pharmacology
D007136:Immunoglobulins; D015335:Molecular Probes; D018121:Receptors, Cytokine; C410591:Tslpr protein, mouse; D014415:Tunicamycin