The E. coli IVTT products were purified using two different methods based on affinity to the N-terminal 6xHIS tag. One method used mini-spin columns with a cellulose acetate filter (Pierce, Rockford, IL) and NI-NTA resin (Qiagen). For that, 200 ml of NI-NTA resin was added to a spin column connected to a collection tube. The column was washed twice with 600 ml of milliQ water and twice with 600 ml of Binding Buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM Imidazole and 8 M Urea pH 8) using centrifugation at 1500 rpm for 2 min. Then 50 ml of IVTT reaction was mixed with 700 ml of Binding Buffer and incubated at 4uC for 20 min in an orbital mixer, to solubilize the inclusion bodies. The solubilized sample was added to a NI-NTA resin column and incubated at 4uC for 30 min in an orbital mixer to maximize protein binding to the resin. The flow-through was removed via centrifugation at 1500 rpm for 2 mins and the column washed three times with 700 ml of Binding Buffer. The recombinant was eluted with 200 ml of Elution Buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM Imidazole and 8 M Urea pH 8) incubated at 4uC for 20 min in an orbital mixer before collection via centrifugation.