I want to use an enzyme (21kDa) as therapeutics. I want to know what methodologies can be employed to make the protein cross the plasma membrane onto the cytoplasm.
Also, can tagging the protein with HA or other small tags help?
Tagging with HA won't help you much if at all. You need some polybasic sequences in your protein. The most commonly used is the TAT sequence -a basic domain derived from the HIV protein Tat. There are plenty of references documenting this. See, for example:
http://www.jbc.org/content/272/25/16010.long
1-It depends on which protein , amino ac,ds contents etc
2-You can render the peptides less iyonic, soluble salt form and more absorbable?
3-Your target? There is a nice manu concerning amino acid transport to brain"MARIA HÄGGLUND, 2013). please refer to it
You can add small tags to them (like TAT mentioned above) or polyarginine tags (R9 is common). Additionally, you could try transfection methods to get them in, or use serum-free media when you add your enzyme to induce the cells to uptake things from the media.
Thanks everyone for your help.
I have designed the forward primer containing the TAT sequence encoding (YGRKKRRQRRR) followed with four Glycine (for free bond rotation) and then my gene primer starting with ATG. I plan to clone this in pET28 vector. I plan to use this purified proteins in in vivo assays.
Any suggestions?
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