Suitable transmembrane domain significantly increase the surface-expression level of Fc(epsilon)RIalpha in 293T cells.
Evidence showed that the extracellular part of Fc(epsilon)RIalpha (FCR) with its own transmembrane domain (TMF) cannot be expressed as a transmembrane form in CHO cell line. However, FCR could be displayed on cell surface with the transmembrane domain (TM) of human IL2Ralpha (TMI). Theoretical analysis of TMF and TMI using TM prediction methods showed that TMI possessed strong orientation tendency to form "outside to inside" transmembrane mode from N-terminal to C-terminal, while TMF was prone to form "inside to outside" mode. Based on the analyzing results, the TM of Her2 (TMH) was studied and showed similar transmembrane mode as that of TMI, which implied that TMH might be a novel TM to obtain the surface display of FCR. Then, DNA sequences encoding TMH and TMF were fused to 3'-end of FCR gene, respectively. Fluorescent microscope observation indicated that FCR_TMH seemed to be located mainly on cell surface, while FCR_TMF appeared in endochylema. Flow cytometry analysis and Western blot also showed that the surface expression of FCR was enhanced significantly by TMH, while FCR_TMF could not be surface displayed in 293T cell. The experimental results were consistent with the theoretical predictions and demonstrated that the orientation tendency of TM may be very important in subcellular location of proteins.
Qiao
Chunxia
C
Laboratory of Molecular Immunology, Institute of Basic Medical Sciences, Beijing, China.
Guo
Leiming
L
Sun
Jian
J
Li
Yan
Y
Lin
Zhou
Z
Lv
Ming
M
Feng
Jiannan
J
Shen
Beifen
B
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism; D008390:Markov Chains; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016571:Neural Networks, Computer; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D017455:Receptors, IgE / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D053645:Interleukin-2 Receptor alpha Subunit; D018719:Erb-b2 Receptor Tyrosine Kinases; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; C508017:IL2RA protein, human