G protein-coupled receptors (GPCRs) are a class of proteins, involved in cellular signaling cascades, which are composed of seven transmembrane (TM) domains connected by intra- and extracellular loops (IL and EL, respectively). Structural information regarding these proteins is extremely valuable, but characterization of these proteins is difficult due to their hydrophobicity, flexibility and large size. Smaller fragments of GPCRs are useful for studying membrane protein folding and NMR structural analysis. Our group has been focusing on NMR analysis of fragments of the yeast a-factor receptor, Ste2p. We have recently published an NMR structure for a fragment containing the first two TMs of the receptor in LPPG micelles and have also conducted biophysical analysis of this 80-residue peptide in TFE/water mixtures [1,2]. Here, we report the cloning, expression and purification of two larger fragments of Ste2p for biophysical investigations. A 3TM fragment containing 130 residues of Ste2p, G31-R161, including 19 residues from the N-terminal domain, the first TM through the third TM with connecting loops and five residues of the second IL was cloned downstream of the TrpALE fusion protein. Similarly, a 5TM fragment containing 212 residues of Ste2p, I128-L340, including 13 residues of the first EL, the third TM through the seventh TM with connecting loops, and 40 residues of the C-terminal domain was cloned into the same plasmid. The 3TM fragment was chosen because it may prove to be more stable than the TM1-TM2 fragment. An unsatisfied Arg residue in the TM1-TM2 fragment may form a salt bridge with a Glu residue in the TM1-TM3 fragment. The 5TM fragment was chosen because we wish to mix the TM1-TM2 fragment with the unlabeled 5TM fragment and perform NMR experiments on the reconstituted receptor. Expression for both constructs was optimized in E. coli and CNBr and thrombin cleavages and purification of the target peptides from the fusion tag were attempted. The 3TM peptide has been isolated, characterized by MS and biophysical analysis has begun. The 5TM fusion protein has proven to be more challenging and attempts at purification are in progress.
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