| Quintas-Granados LI, Orozco E, Brieba LG, Arroyo R, Ortega-López J.
(2008)
Protein Expression and Purification,
1,
1 |
| Protein refolding |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| E coli |
| M15 |
| 37.0 |
| 4 h |
| pQE80L |
| The precursor ppEhCP112 gene was amplified by PCR using the direct and reverse oligonucleotides: ppEhCP112-5′ (5′-AGGTCGGATCCATGAC AGCGATTGTTGTCGCT), ppEhCP112-3′ (5′-AGGCAAAGCTTTTAGATTGTATGATTGTAGAATTG), respectively, and using the plasmid pRSET A-ppEhCP112 as DNA template [2]. The primers contained BamHI and HindIII restriction sites (bold) to allow directional cloning of the amplified DNA into the prokaryotic expression vector pQE80L (Qiagen). Transformed Escherichia coli M15 cells with the pQE80L-ppEhcp112 construct were grown to inoculate 500 ml of terrific broth (TB) (100 μg/ml ampicillin, 30 μg/ml kanamycin) at 37 °C and 200 rpm; when cultures reached an OD600 = 0.8, the recombinant protein was induced by adding 1 mM of IPTG for 4 h. Bacteria were resuspended in 300 mM NaCl, 20 mM Tris–HCl pH 8.0 (1 ml buffer per gram of cells), supplemented with lysozyme (1 mg/ml), incubated on ice for 30 min, and lysated by sonication. The lysate was centrifuged at 16,000g for 30 min at 4 °C to isolate the soluble and insoluble fractions. |
| IPTG |
| OD 0.8 =
600 |
| Sonication |
| Lysozyme |
| Metal affinity chromatography |
| insoluble |
| Column refolding: Size-exclusion chromatography |
| 20 mM Tris–HCl (pH 8.0), 500 mM NaCl, 2% Triton X-100, and 2 M urea. |
| 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 10 mM imidazole, and 8 M urea |
| 5 mM CaCl2, 0.02% SDS, 100 mM Tris–HCl, pH 8.0 |
| Metal affinity chromatography |
| yes |
| 8.0 |
| 25.0 |
| n/a |
| n/a |
| None |
| n/a |
| A volume of 2.5 ml of the purified and unfolded ppEhCP112 (1–2 mg/ml) was applied to a 1.45 × 8 cm PD10 Desalting Column with 8.3 ml of Sephadex G-25 (GE Healthcare Science) pre-equilibrated with refolding buffer (5 mM CaCl2, 0.02% SDS, 100 mM Tris–HCl, pH 8.0). This simple purification step removes the urea and allows the exchange of the protein to the refolding buffer. Therefore, it allows the protein to refold into its native conformation. Processing of ppEhCP112 into an enzymatically active protein (EhCP112) was achieved by incubating 500 μg of the ppEhCP112 in 1 ml refolding buffer supplemented with 10 mM DTT at 25 °C. This activation protocol is similar to protocols successfully employed with other recombinantly expressed CPs of E. histolytica, like EhCP1, EhCP2 and EhCP5 [15], [21] and [22].
To monitor the activation of ppEhCP112, 50 μl samples were taken at 15, 20, 25, 30, 35, 40, and 45 min, and analyzed by SDS–PAGE and gelatin–SDS–PAGE. In addition, a spectrophotometric enzymatic assay using azocasein as substrate was performed to corroborate proteolytic activity.
To determine whether the activation was an autocatalytic process, 20 μg of refolded ppEhCP112 in 500 μl of refolding buffer were incubated at 37 °C for 10 min in the presence of 10 μM E-64, 1 mM PMSF, or 1 μM pepstatin A before the activation process with DTT. These samples were analyzed by SDS–PAGE after 20 and 35 min of activation. |
| Azocasein spectrophotometric assay |
| None |
| None |
| n/a |
| 50 mg per 500ml |
| n/a |
| n/a |