E coli bl21 strain

The pCold-NPGM-Gly plasmid was transformed into E. coli BL21 strain (GE Healthcare, Little Chalfont, UK) or E. coli SHuffle strain (New England Biolabs, Ipswich, MA, USA). The transformants were selected on LB agar plates containing 50 μg/ml of ampicillin and grown at 37 °C overnight. The colonies were then grown in LB medium containing ampicillin at 37 °C overnight. An aliquot of the pre-culture solution was diluted with 200 ml of fresh LB medium and incubated at 37 °C. When the cells reached an optical density (OD)₆₀₀ of 0.5, the culture was refrigerated at 15 °C for 30 min. The culture solution was added with isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM and continued with shaking at 15 °C for 24 h. Cells were collected by centrifugation and frozen at −80 °C until further use.
The expression of the target protein in soluble and in insoluble fractions was confirmed by SDS–PAGE. An aliquot of the cell pellet was lysed with BugBuster Protein Extraction Reagent (Novagen, Madison, WI, USA) and separated by centrifugation. Each sample was dissolved in SDS sample buffer and subjected to 15% SDS–PAGE. The gels were stained with 2D-SILVER STAIN II (Cosmobio, Tokyo, Japan).