| Srivastava AK, Sharma Y, Chary KV.
(2008)
Protein Expression and Purification,
1,
1 |
| Over expression & Renaturation |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 4 h |
| pET21a |
| Escherichia coli BL21(DE3) cells containing pET21a-Hahellin recombinant plasmid were inoculated from a single colony and grown overnight in Luria–Bertani (LB)1 media containing 100 μg/ml ampicillin at 37 °C by shaking at 225 rpm. After 12 h, 2.5 ml of culture was transferred into 250 ml of LB media with 100 μg/ml ampicillin. After growing the culture until A600 reached 0.6 in LB medium, the heterologous protein synthesis was induced by the addition of IPTG (isopropyl-β-d-thiogalactopyranoside) to a final concentration of 1 mM. After further incubation for 4 h at 37 °C, cells were pelleted by centrifugation at 6000 rpm for 20 min at 4 °C. The cell pellet (1.2 g) was suspended in 5 ml of a lysis buffer (50 mM Tris–Cl, 2 mM EDTA, 100 mM KCl, 1 mM MgCl2 containing 0.5 mM PMSF, pH 8.0). The cell suspension was extensively sonicated (20 × 1 min, Branson sonifier 450) on ice. The resulting cell lysate was centrifuged at 16,000 rpm for 20 min. The supernatant and the pellet were checked for the presence of the protein. The protein predominantly overexpressed in the form of inclusion bodies. Therefore, inclusion bodies were used for protein purification. |
| IPTG |
| OD 0.6 =
600 |
| Sonication |
| None |
| Ion-exchange chromatography |
| insoluble |
| column refolding: ion-exchange chromatography |
| 100 mM Tris–Cl, 5 mM EDTA, 4% Triton X-100, 2 M urea, pH 8.0 |
| 10 mM Tris–Cl, 1 mM EDTA, pH 9.5, containing 8 M urea |
| 10 mM Tris–Cl, 1 mM EDTA, pH 9.5 |
| Ion-exchange chromatography |
| no tag |
| 9.5 |
| 25.0 |
| n/a |
| n/a |
| None |
| n/a |
| Purification and refolding of protein from inclusion bodies
The procedure of on-column refolding was used to refold and purify Hahellin from the inclusion bodies. The inclusion bodies (frozen pellets) were thawed and suspended in 30 ml of a washing buffer (100 mM Tris–Cl, 5 mM EDTA, 4% Triton X-100, 2 M urea, pH 8.0) and washed 4–5 times with the same buffer until a clear supernatant was obtained. Then the inclusion bodies were washed with wash buffer lacking urea and Triton X-100 to remove the denaturant and detergent. The pI of the protein was calculated from amino acid sequence data as 5.2 (Expasy ProtParam tool). The washed inclusion bodies were dissolved in 20 ml of buffer A (10 mM Tris–Cl, 1 mM EDTA, pH 9.5) containing 8 M urea, stirred continuously to solubilize the protein and incubated at 4 °C for 12 h to ensure complete denaturation. The suspension was centrifuged at 16,000 rpm for 30 min at 4 °C to remove insoluble particles to get a clear solution. A Q-Sepharose (Pharmacia) anion exchanger column (1.5 × 10 cm.) was equilibrated with 5 bed volumes of buffer A containing 8 M urea. The protein solubilized in urea was loaded onto the equilibrated column with flow rate 25 ml/hour. The column was washed with one bed volume of buffer A containing 8 M urea. After binding the protein the column was treated with a gradient of urea (8 to 0 M) in two bed volumes of buffer A to gradually remove the denaturant. The column was washed with two bed volumes of buffer A to ensure the complete removal of urea. The bound protein was eluted by multi-step elution, each step of one bed volume, with buffer containing 100 mM, 200 mM and then 300 mM NaCl. Flow rate was 25 ml/h. In such purification procedure the last step was size exclusion chromatography. Thus, we could get pure protein in its native state (i.e. in the absence of any denaturant), as established later by recording a 2D [15N-1H] HSQC. Concentration of the protein was estimated using an extinction coefficient value (at 280 nm) of 5960 M−1 cm−1, which was determined from its amino acid sequence using Expasy ProtParam tool (www.expasy.org). |
| Mass spectrometry |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |