I have cloned a fragment of a human membrane protein fused with GFP in a lentiviral vector, once virus were produced in HEK293 I infected human endothelial cells, my control (only GFP lentivirus) was perfect after puromycin selection, all the cells green in the cytoplasm, but my construction was poor and showing like fluorescent dots, often aligning with the cell membrane. I have already infected with the whole protein fused to GFP and I did not have the same result. It is a problem with lysosomal aggregation, or a problem with my construction?
GFP fusion proteins upon overexpression in infected cells exhibit mislocalization due to overproduction. Further, the localization varies among cell population. Therefore, you need to analyze up to 300 cells to assess the quality of your protein. If the fusion protein sequence was correct and the protein was made using the correct reading frame, some optimization may help.
your protein of interest in the construct might be a cell membrane receptor protein
Thank you for your answers, yes my fusion protein is a membrane protein, in fact it is a fragment of the protein. I will check every thing and repeat experiment becasuse the cells do not survive very well.
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