grown to od600 of 0 5 induced with 0 5 mm isopropyl u03b2 d thiogalactopyranoside iptg and then incubated for 4 h the cells were harvested by centrifugation and resuspended in 4ml of lysis buffer 10 mm tris hcl 150 mm nacl and 1 mm edta ph 8 0 containing 1 mm phenylmethylsulfonyl fluoride pmsf for each 100 ml of culture lysozyme 0 5 mg ml was added and the mixture was incubated on ice for 30 min the suspended cells were disrupted by sonication vibracell 4 times each for 15 sec and centrifuged at 4 u00b0 c the precipitate containing inclusion bodies was solubilized in 6 m guanidine hydrochloride overnight at 4 u00b0 c this solution was loaded on a ni 2 charged histrap column amersham bioscience and the column was washed with buffer containing 6 m urea and 20 mm imidazole several times to remove the nonspecific binding refolding of the bound protein was performed in an fplc bio rad biologic hr using a linear urea reverse gradient 6 m to 0 m the total volume and flow rate of the buffer used in the linear gradient were 30 ml and 0 5 ml min respectively finally the refolded protein was eluted with elution buffer containing 500 mm imidazole the eluted 30k protein was desalted into the distilled water to remove the imidazole using a hitrap desalting column amersham bioscience and concentrated using a lyophilizer the purity of the protein obtained was determined by scanning the 30k protein band on sds page gel using total lab v1 10 nonlinear dynamics the total protein concentration was measured using a modified lowery protein determination kit peterson 39 s.