Cloning trap for signal peptide sequences.
Novel secreted and/or type I transmembrane proteins containing N-terminal signal sequences have been successfully cloned using the signal sequence trapping (SST) method. Often this involves random cloning of short 5' cDNA terminal ends into an epitope-tagged expression vector and the detection of expressed recombinant proteins on the cell surfaces of transfected cells with an antibody to the tagged epitope. Here, we report a novel cloning system for the detection of secreted proteins also using SST. In this method, we used the human immunodeficiency virus (HIV-1) p24 as the epitope for tagging. To test the system, two constructs were created. The 5' terminal end of a human beta-chemokine (which was regulated upon activation, expressed by normal T cells and presumably secreted [RANTES]) and the 5' end of a human CD4 receptor were cloned upstream of and in-frame with the p24 cDNA. Secreted p24 was detectable in the culture media two days after transfection of either DNA construct into the human cell lines, HeLa and 293T. When the chimeric p24 expression constructs were transfected at a ratio of 1:100 to the vector pcDNA3.1(+), p24 could still be detected in cell supernatants. The use of a secreted viral antigen like HIV-1 p24 (or of any noncellular protein) as a marker in SST cloning approaches is likely to be advantageous because it reduces the background noise in detection and also renders this system suitable for high-throughput screening.
Lim
S P
SP
Institute of Molecular and Cell Biology, Singapore. mcblimsp@mcbsgs1.imcb.nus.edu.sg
Garzino-Demo
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D000956:Antigens, Viral / Q000235:genetics; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D019742:Chemokines, CC / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005434:Flow Cytometry; D005819:Genetic Markers; D005820:Genetic Testing; D016655:HIV Core Protein p24 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics*; D014162:Transfection
D000956:Antigens, Viral; D015704:CD4 Antigens; D019742:Chemokines, CC; D000939:Epitopes; D005819:Genetic Markers; D016655:HIV Core Protein p24; D021382:Protein Sorting Signals