Figure 6

The properties of secreted proteins. Silk proteins measured in a previous publication are also plotted for reference [6]. Different spider species are shown by different symbols Latrodectus hesperus MaSp1 (blue +), Nephila clavipes Flagelliform (green circles), Bombyx mori cocoon silk (yellow square), Araneus diadematus ADF1 (red X) ADF2 (purple triangle) ADF3 (grey diamonds) and ADF4 (blue triangle). A. Secretion of each protein is plotted versus the expression level. Each point is the average of three independent colonies and error bars represent +/- one standard deviation. B. Secretion efficiency is plotted as a function of protein length. Parallel dotted lines are shown to guide the eye. The number of amino acids noted only corresponds to the length of the silk polypeptide and does not include the 178 amino acid SptP tag and TEV site. C. Secretion efficiency is plotted against hydrophobicity (GRAVY score). D. The secretion efficiency versus net charge for each secreted protein is determined at pH 7.0 and plotted. A guide for the eye is drawn over the data. For each silk protein, the charge was calculated including the SptP tag, TEV linker sequence, and 3× FLAG tag. The ADF3 protein has a native charge of -0.4 (grey diamond) and when a 6× histadine tag is added the net charge at pH 7.0 increases to +10 (green diamond). This modification to ADF3 eliminates secretion (identified by the arrow).