| Ligand gated ion channels (LGICs) are a superfamily of transmembrane proteins that mediate synaptic signaling in eukaryotic nervous systems. Sequence profile searches have demonstrated the existence of homologous LGICs in several prokaryotic species, which share general structural and mechanistic properties. This graduate research involves the expression and isolation of the N-terminal extramembranous domain of a putative LGIC homolog from the cyanobacterial species Nostoc punctiforme. The target gene sequence was successfully cloned into MBP-27b(+) and pETpHSUL expression vectors. Protein was overexpressed from each construct; however, cellular fractionation studies proved them to be insoluble. Extraction and solubilization was only successful with SDS, suggesting improper protein folding and insertion. Affinity chromatography was attempted, but fusion protein was unable to bind to the column due to an inaccessible histidine tag. An improved sonication method led to a small amount of soluble fusion protein. Future research should focus on solubility optimization to reduce the aggregation of protein in inclusion bodies. Structural and functional characterization could lead to the first model of a prokaryotic LGIC heteropentamer. |
