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Protein gag p24 is a structural protein made by the gag gene in HIV. The gag p24 protein is a capsid protein in the HIV virus and is found in all know strains of HIV. It is a protomer that polymerizes, making the protein coat of the HIV virus, upon a scaffold made of p17, another gag protein. There are approximately 2000 molecules of gag p24 per viral particle. The molecular weight of the p24 protein is ~24 kDa.
Gag p24 has many “sticky” regions that aid in the polymerization of the protomer and the formation of the viral capsid. (1) An aptamer or aptamers that bind to the “sticky” regions of the protein would interfere with the formation of the viral capsid at a later stage in the cellular development of the virus. An aptamer that binds to the terminus of p24 that allows it to form the CA(146-231) capsid subunits would also hinder the formation of the viral capsid (Figure 1. (2) =>) The retention of the p24 protein in all strains of HIV suggests that it is an essential protein, which has function not easily circumvented by random mutations. A battery of multiple aptamers would be best in halting the formation of the virus, not only because multiple functional regions would be blocked, but also because, the virus would not be able to mutate around the obstruction of capsid formation.
I will conduct two simultaneous bead based selections to develope an aptamer that binds to gag p24. However, in order to perform these bead based selections, the protein will have to be functionalized with a functional group that will allow it to be attached to the beads. Through these two selections, I will find multiple strong and weak binding aptamers to multiple functional regions on the p24 protein.
References:
(1) Berthet-Colominas et al. (1999) Head-to-tail dimmers and interdomain flexibility revealed by the crystal structure of HIV-1 capsid protein (p24) complexed with a monoclonal antibody Fab. The EMBO Journal vol. 18 pp.1124-1136
(2) Gamble et al. (1997) Structure of Carboxyl-Terminal Dimerization Domain of the HIV-1 Capsid Protein. Science vol. 278 no. 5339, pp.849-853
Wikipedia sources used pervasive through the abstract:
(3) http://en.wikipedia.org/wiki/Group-specific_antigen
(4) http://en.wikipedia.org/wiki/P24_capsid_protein
Places to Find gag p24 (aka. p24 HIV-1):
RayBiotech- ($628 per .5 mg)
http://www.raybiotech.com/product_view.asp?m=3&ProdID=223669
4 comments:
I found the target HIS tagged on Biocompare for a cheaper total price. I ordered it today from Advanced Targeting Systems. 100ug for $270.
Additionally, I had to do some major editing to the layout. I will address how to do this in class tomorrow but you want to make sure you post looks professional.
Cool target. Especially since it is found across all strains of HIV. I was wondering why your going two run two simultaneous selections. Is it just in hopes to find a wider array of potential aptamers?
Gag-P24 came in from ATS. The Cat# is PRP-149 and we received Lot#74-9q at 2mg/ml. Since it was already resuspended, I aliquoted and stored in the -80C freezer (rack D2 second row back). The protein is 26kDa and it is at 2ug/ul which means it is roughly 76.9uM concentration. I aliquoted some tubes with 2.6ul (200pmol) and other tubes with 1.3ul (100pmol). Use the larger volume for the first few rounds and for binding assays. Use the 100pmol aliquots for rounds ~3 and after unless the data warrants otherwise.
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