Design and expression of human α7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties

Creating an α7 Nicotinic Acetylcholine Recognition Domain from the Acetylcholine-binding Protein

“…Assays with ␣-Ctx ArIB[V11L,V16D] show the largest increase in affinity to match those values of the h␣7-nAChR, whereas ␣-Btx also significantly increases affinity but is still 100-fold off of the h␣7-nAChR values determined. However, the values determined for ␣-Btx did match those previously reported (60 -70 nM) for an aggregating extracellular domain of h␣7-nAChR (14). (Ϫ)-Lobeline loses 10,000-fold affinity to more closely resemble that of h␣7-nAChR with a 5-10-fold affinity difference remaining.…”

“…Using mutagenesis and affinity labeling, non-neighboring residues in the primary sequence of the protein were identified as important in ligand binding (4 -8). Several studies have shown expression of ␣7 nAChR extracellular domains (9 -11) or variants (12)(13)(14), yet none have resulted in crystal structures. Studies involving the Lymnaea stagnalis (Ls) and Aplysia californica (Ac) acetylcholine-binding proteins (AChBP) have yielded many structural analyses of the binding site for these mollusk homologues (15)(16)(17)(18)(19)(20)(21).…”

Expression of Water-soluble, Ligand-binding Concatameric Extracellular Domains of the Human Neuronal Nicotinic Receptor α4 and β2 Subunits in the Yeast Pichia pastoris

“…On the basis of our previous studies (28,30,33), we replaced the Cys-loop (Cys-133-Cys-147 in ␣4 and Cys-130 -Cys-144 in ␤2) by the corresponding, more hydrophilic one from the LsAChBP (Cys-123-Cys-136); the expressed mutants were also isolated in the form of microaggregates and multimers, but with a much higher yield (0.3 and 2.5 mg/liter, respectively). More specifically, the ␣4-mECD was mainly eluted in the form of microaggregates and multimers, with respective masses of ϳ1100 and 480 kDa (Fig.…”

“…Next, we expressed mutated ␣4-and ␤2-ECDs, in which the hydrophobic region of the Cys-loop domain was replaced by the corresponding, more hydrophilic region of the LsAChBP. We have used this replacement previously in the expression of the human ␣7-ECD, and this led to a significant improvement in the solubility and ligand binding properties of the recombinant molecule (28,30,33). This mutation again led to a significant increase in the expression yield of the singly expressed ECDs but not to a large improvement in their solubility (Fig.…”

“…Yeast Transformation and Screening for Positive ClonesLinearized plasmids were transformed by electroporation into the X33 strain of P. pastoris, and positive clones were selected for small scale cultivation according to the manufacturer's instructions (Invitrogen) and in detail described elsewhere (28,30). The culture supernatants were tested for expression of the recombinant ECDs by dot-blot analysis using anti-Myc 9E.10 mAb (ATCC) or anti-His 6 C terminus antibodies (Invitrogen) and horseradish peroxidase-conjugated polyclonal goat antimouse IgG antibodies (Dako) or using horseradish peroxidaseconjugated anti-FLAG M2 mAb (Sigma), depending on the tag carried by the recombinant protein.…”