Expression and Purification of Recombinant Proteins in E coli

5.5.3.1 Expression and Purification of Recombinant GST Fusion Proteins in E. coli 5.5.3.1.1 Screening Recombinants for GST-Fusion Protein Expression in E. coli

The cloned plasmids for the expression of GST fused protein were transformed into BL21 or W3110 E. coli strains using the electroporation. The pGEX recombinants were screened for fusion protein expression as follows. Several colonies of E. coli transformed with the pGEX recombinants were picked and transferred into separate tubes containing 2 ml of dYT medium. The liquid cultures were grown to an OD600 of 0.6-0.8 (3-5 hours) with vigorous agitation at 30-37 °C. The fusion protein

expression was induced in all cultures, except for one (uninduced control) with 1 mM IPTG. The cultures were incubated for 2 hours with vigorous agitation at 30-37 °C. After 2 hours, 1.5 ml of the culture were harvested in eppendorf tubes by centrifugation for 1 minute at 13000 rpm. The cells pellet was resuspended in 300 µl of ice-cold 1x PBS supplemented with 1 mM PMSF. The cell suspension was disrupted using an ultra-sound sonicator on ice 6 times for 10 seconds. 10 µl of the lysate (crude lysate) were analyzed on SDS gel and stained with Coomassie brilliant blue stain. The positive lysate was centrifuged at 13000 rpm for 10 minutes at 4 °C and the supernatant (cleared lysate) was transferred into new tube. 10 µl of the cleared lysate were saved for SDS gel analysis. To the rest of the cleared lysate and 20 µl of 50% slurry of glutathione sepharose 4B (prepared as described by the manufactured) were added and the tubes were mixed gently for 5 minutes at room temperature. The mixture was washed three times with 100 µl of ice-cold 1x PBS and the beads were collected by centrifugation. The supernatant was discarded and the beads were resuspended in 10 µl of protein loading buffer. The purified GST fusion protein in positive lysate was analyzed on SDS gel and stained with Coomassie brilliant blue stain.

5.5.3.1.2 Purification of Recombinant GST-Fusion Proteins

For the purification of the GST-fusion proteins, an affinity chromatography on glutathione-coupled sepharose 4B resin (Amersham Pharmacia) was performed according to the manufacturer’s instructions. A large-scale bacterial sonicate was prepared as follows. A single colony of E. coli cells containing a positive recombinant pGEX plasmid was used to inoculate 100 ml of dYT media and the culture was incubated for 12-15 hours at 37 °C with vigorous shaking. The culture was induced with 1 mM IPTG and the incubation continued for an additional 6 hours. The cells were harvested by centrifugation at 5000 rpm for 5 minutes at 4 °C and the supernatant was discarded. The pellet was completely resuspended in 2.5 ml of ice-cold 1x PBS supplemented with 1 mM PMSF, 0.1% Tween-20 and 1% Lysozym. The suspension was incubated on ice for 30 minutes. The suspended cells were disrupted using an ultra- sound sonicator on ice 6 times for 10 seconds each. The crude lysate was centrifuged for 10 minutes at 13000 rpm for 10 minutes at 4 °C. The supernatant was transferred to a fresh tube and incubated with 100 µl of the 50% slurry of glutathione sepharose 4B and the suspension was mixed gently for 30 minutes at room temperature. The mixture was washed three times with 1 ml of ice-cold 1x PBS and the beads were collected by centrifugation. The supernatant was discarded and the beads were resuspended in 50 µl of glutathione elution buffer and incubated for 15 minutes at room temperature. The suspension was centrifuged for 1 minute at 13000 rpm and the supernatant (the eluate) was transferred into a new tube, and the elution step was repeated twice with a yield averaged between 0.5-10 µg/µl. The eluates were stored at -20 °C till use.

5.5.3.2 Expression and Purification of Recombinant 6x His Fusion Proteins in E. coli 5.5.3.2.1 Screening Recombinants for 6x His-Fusion Protein Expression in E. coli

The cloned plasmids for the expression of 6x His-fused protein were transformed into BL21 or W3110 E. coli strains by electroporation. The pET28a recombinants were screened for positive clones. Several colonies of E. coli transformed with the pET recombinants were picked and transferred several into separate tubes containing 2 ml of dYT medium. The fusion protein expression was induced in all cultures except for one (uninduced control) with 1mM IPTG. The cultures were incubated for 2 hours with vigorous agitation at 30-37 °C. After 2 hours, 1.5 ml of the culture were harvested in eppendorf tubes by centrifugation for 1 minute at 13000 rpm. The cells pellet was resuspended in 300 µl of nickel nitrilotriacetic acid lysis buffer. The cell suspension was disrupted using an ultra-sound sonicator on ice 6 times for 10 seconds. 10 µl of the lysate (crude lysate) were analyzed on SDS gel and stained with Coomassie brilliant blue stain. The positive lysate was centrifuged at 13000 rpm for 10 minutes at 4 °C and the supernatant (cleared lysate) was transferred into new tube. 10 µl of the cleared lysate were saved for SDS gel analysis. To the rest of the cleared lysate, 20 µl of 50% NiNTA®-matrix resin (prepared as described by the manufactured) were added. The tubes were mixed gently for 5 minutes at room temperature. The mixture was washed three times with nickel nitrilotriacetic acid wash buffer and

the beads were collected by centrifugation. The supernatant was discarded and the beads were resuspended in 10 µl of protein loading buffer. The purified 6x His-fusion protein in positive lysate was analyzed on SDS gel and stained with Coomassie brilliant blue stain.

5.5.3.2.2 Purification of Recombinant 6x His-Fusion Proteins

For the purification of the 6x His fusion proteins, an affinity chromatography on NiNTA®-matrix resin (Qiagen) was performed according to the manufacturer’s instructions. A large-scale bacterial sonicate was prepared. A single colony of E. coli cells containing a positive recombinant pET plasmid was used to inoculate 100 ml of dYT medium and the culture was incubated for 12-15 hours at 37 °C with vigorous shaking. The culture was induced with 1 mM IPTG and the incubation continued for an additional 6 hours. The cells were harvested by centrifugation at 5000 rpm for 5 minutes at 4 °C and the supernatant was discarded. The pellet was completely resuspended in 5 ml of nickel nitrilotriacetic acid lysis buffer supplemented with 1% Lysozym and the suspension was incubated on ice for 30 minutes. The suspended cells were disrupted using an ultra-sound sonicator on ice 6 times for 10 seconds each. The crude lysate was centrifuged for 10 minutes at 13000 rpm for 10 minutes at 4 °C. The supernatant was transferred to a fresh tube and incubated with 1 ml of the 50% NiNTA®-matrix resin and the suspension was mixed gently for 1 hour at 4 °C. The mixture was washed three times with 250 µl of nickel nitrilotriacetic acid wash buffer and the beads were collected by centrifugation. The supernatant was discarded and the beads were resuspended in 250 µl Nickel nitrilotriacetic acid elution buffer and incubated for 30 minutes at 4 °C. The suspension was centrifuged for 1 minute at 13000 rpm and the supernatant was transferred into new tube, and the elution step was repeated twice with a yield averaged between 0.5-1 µg/µl. The eluates were stored at -20 °C till use.