<p>The lack of soluble proteins in the supernatant and non-specific binding of the tag has always been a persistent challenge for us. Under this circumstance, we tried to replace the promoter and protein tag for WB and add a solubilizing protein tag for the original part (See more in Engineering).
Due to time constraints, not all genetic circuits were completed. We successfully built up SUMO-HSP60 circuits in E. coli BL21 and other three fruitful circuits are presented below (Fig29).
We introduced the soluble protein tag SUMO into the HSP60 construct to increase the amount of soluble protein in the supernatant, and we replaced the 6xHis tag with GST to improve antibody binding specificity. Furthermore, we tried to introduce SUMO-HSP60 and LAP fragment into plasmid backbone pBAD to change the promoter, and to replace LAP-6x His with LAP-GST at the same time.</p>
