I did small scale expression (total culture volume of 1 mL in 24 multi-well plate) of an esterase gene cloned into pQE80L plasmid and transformed into Rosetta-gami B. I induced the cultures grown in LB with 0.1 mM IPTG for 12 hours at 37°C. The cells were harvested by centrifugation and then re-suspended in two types of buffers; potassium phosphate buffer (pH 7) and lysis buffer (20 mM NaH2PO4, 500 mM NaCl, and 20 mM imidazole.HCl, pH 7.4). Both buffers were added with 30% glycerol to aid with the folding. After cell disruption, both supernatant and pellet were analyzed with SDS-PAGE. Colorimetric enzyme assay using p-nitrophenyl acetate as substrate were also done for supernatant samples. Empty vector without inserts underwent all assays was used as control. 

For SDS-PAGE, no bands at intended molecular weight were observed for all samples (potassium phosphate buffer without glycerol; potassium phosphate buffer with 30% glycerol; lysis buffer without glycerol;and lysis buffer with 30% glycerol). For enzyme assay, with potassium phosphate buffer without glycerol, no enzyme activity was detected. The addition of glycerol showed little activity. However, with lysis buffer with or without the addition of glycerol, some enzyme activity was detected (~2.2 U/mL). Assay with an empty vector gave similar result (i.e no enzyme activity in potassium phosphate buffer, little activity with glycerol addition; some activity in lysis buffer with or without glycerol).

I have been trying to express the esterase in E.coli by using different host cells and vectors, changing medium, induction conditions and concentrations, induction temperatures, co-expression with chaperon etc but yet to obtain soluble esterase. I appreciate any suggestion that can help to improve my protein solubility.