Production steps of Est16 as a soluble and stable protein.

(A) 12% SDS-PAGE of the expression fractions and IMAC samples of Est16 from E. coli BL21 (DE3) cells carrying the pET28a-est16 vector, which contained the est16 gene. E. coli cells were grown in LB up to O.D._600nm = 0.5 at 37°C and then induced with 0.1 mM IPTG at 28°C for 20 hours. Lane 1: non-induced cells; Lane 2: induced cells; Lane 3: molecular weight marker (the kDa values are indicated in the picture); Lane 4: soluble extracts; Lane 5: flow-through fraction from the IMAC purification; Lane 6: 10 mM imidazole wash fraction; Lanes 7, 8 and 9: 50 mM, 100 mM and 1 M imidazole elution fractions, respectively. (B) The chromatogram obtained after the size-exclusion chromatography using a HiLoad 16/60 Superdex 200 column (GE Healthcare Bio-Sciences). The insets show the samples related to the peaks loaded into the 12% SDS-PAGE polyacrylamide gels. Lane 1, molecular weight standards; Lane 2: sample before SEC; Lanes 3–4: fractions from the first peak; Lanes 5–12: fractions from the second peak, which were concentrated for further spectroscopic and biochemical analyses.