I am working with two proteins and I need to increase their affinity in mammalian cell lines. I am trying to find a pair of small proteins/peptides that have high affinity (ideally, in a low nanomolar range or better) to each other and not to proteins present in mammalian cells (e.g. the interaction would be of either bacterial or viral origin). The idea is to introduce these interacting peptide sequences into my proteins of interest and thus promote their interaction with each other. Any advice would be very much appreciated. Thanks.
-David
The zip domain (leucine zipper motif from the yeast protein, GCN4) used in the positive control of the bacterial adenylate cyclase two hybrid system would work. That, or any other positive control domains from a similar two-hybrid assay.
Karimova, G., Pidoux, J., Ullmann, A. & Ladant, D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America 95, 5752–6 (1998).
The zip domain (leucine zipper motif from the yeast protein, GCN4) used in the positive control of the bacterial adenylate cyclase two hybrid system would work. That, or any other positive control domains from a similar two-hybrid assay.
Karimova, G., Pidoux, J., Ullmann, A. & Ladant, D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America 95, 5752–6 (1998).
Of course, you can do or salting out (by Mg+2 for example, Biochemistry, 1984, 23 (25), pp 5912–5923) or salting in (J. Phys. Chem. B, 2013, 117 (20), pp 6116–6128)
To be more specific; is your protein hydrophobic (membrane protein) or hydrophylic (soluble)?. Depending on this you may outline a couple of strategies. I presume you don´t wanna precipitate it, just to agregate. Take your time to read the last reference.
I know by experience that peptide interactions have been found empirically during electrophoresis or electrofocusing, Where, a single spot of protein dissociate dissociate in two by focusing a previous gel. Of course these gels use to contain EDTA to get rid of Cations= Mg+2. After you find the peptide complex, you scale up to a column. How to find which peptide is?
Peptide database base on mass spectra stored at http://peptide.nist.gov/.
Look also at http://www.switchlab.org/bioinformatics/pepx, database
Look at the works of Bayer EA on the cellulosome. Cohesion-Dockerin interaction
Why don't you just make a construct with the two of them covalently linked?
Carbon content and distribution at the binding sites are responsible for affinity. A mutation at active site that increase the carbon content will improve the affinity. While doing so, make sure the carbon distribution is maintained locally. Otherwise that might interact with other proteins as well.
There's a cute trick for pharmacologically increasing the affinity of two recombinant proteins expressed in cell cultures. Basically you tag each of your proteins of interest with domains which bind to rapamycin (FKBP on one, FRB on the other). Following reference:
Banaszynski et al. 2005 Characterization of the FKBP.rapamycin.FRB ternary complex
I've seen this technique successfully used to induce synaptic translocation of CaM kinase II by having it bind to PSD-95. Following reference:
Bingol et al 2011Autophosphorylated CaMKIIa Acts as a Scaffold to Recruit Proteasomes to Dendritic Spines.
I've also been told that this technique, or another like it, can work with a rapamycin analog that won't inhibit important intracellular pathways like mTOR.
Perhaps this could interest you:
Voss, S. & Wu, Y.-W. Tandem Orthogonal Chemically Induced Dimerization. ChemBioChem 14, 1525–1527 (2013).
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