Recombinant protein or peptide? - (May/05/2009 )
I am researching the role of the 18-amino acid cytoplasmic domain of a transmembrane receptor. I want to do a couple things: 1) screen cell lysates to see which proteins associate with this cytoplasmic domain, 2) transduce the cytoplasmic domain into cells via a cell-penetrating TAT sequence to determine if it is inhibitory (by sequestering binding proteins from the endogenous receptor).
I have several questions:
1) Can such a short sequence be successfully expressed as a recombinant protein with, for example, a GST tag? This would be ideal to accomplish #1 above as lysates can simply be run over the affinity column on which the protein was purified.
2) I was working under the assumption that I would need to order custom peptides to accomplish #2 above, but would it be possible to simply express the TAT and target sequence as a single protein, purify with affinity tag, then cleave the tag?
Thanks in advance for any help!
1. no problem to add the GST with your 18 aa. I have done this successfully.
2. I am the peptide guy from Lifetein, a peptide company. If you know the peptide region is the binding site, you should try the synthesized peptides. I do not want to do ads here form my company but your idea of using peptide is sound. Email me if you have any questions: firstname.lastname@example.org