Investigating the role of the IQ67 domain in the interaction between SUN and calmodulin: an endeavor into the treacherous world of protein expression via Escherichia coli
Through thousands of years of domestication, humans have selectively created a varied species of cultivated tomatoes (Solanum lycopersicum). A recently discovered protein, SUN, has been linked to the elongated shape seen in many varieties of S. lycopersicum. The SUN gene contains the IQ67 domain, which contains several IQ motifs. The IQ motif is involved in calmodulin binding. Calmodulin, which binds calcium, is an essential protein in the cell. Calmodulin-binding proteins are regulated by calmodulin in its calcium-bound state. Because SUN contains the IQ motif, it has been hypothesized that SUN binds calmodulin. Initially, expression of the full SUN gene was attempted in two codon specific strains of Escherichia coli: BL21 (DE3) RIPL and BL21 (DE3) RP. This method appeared to be successful and protein purification was attempted by affinity chromatography with the GST fusion protein. However, purification was not successful. Attempts to re-express the protein were also unsuccessful, so a clone containing the IQ67 domain was constructed and inserted into a pGEX vector. In the future, protein expression of the GST-IQ67 fusion protein will be induced with Isopropyl B-D-1-thiogalactopyranoside (IPTG). Following purification, assays may be run to determine if IQ67 binds to calmodulin.
© Copyright 2009 Stephanie Louise Edmisson