| Espinosa JM, Portal D, Lobo GS, Pereira CA, Alonso GD, Gómez EB, Lan GH, Pomar RV, Flawiá MM, Torres HN.
(2003)
Molecular and Biochemical Parasitology,
131,
35-44 |
| Undefined |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| None |
| 0.0 |
| 00 |
| pRSET |
| [35S]-radiolabeled PZFP1 was obtained by in vitro coupled transcription–translation using the TNT system (Promega, Madison, WI, USA) and the pzfp1 gene cloned into the pBluescript vector as a template (Stratagene, La Jolla, CA, USA). For bacterial expression, a full-length pzfp1 sequence, containing EcoRI and BamHI restriction sites, was generated by PCR and cloned into pRSET A vector (Invitrogen, Carlsbad, USA). |
| Not Stated |
| OD n/a =
n/a |
| Not stated |
| None |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| n/a |
| 8 M urea or 6 M guanidinium, 5% SDS, and 10 mM DTT |
| 50 mM Tris–HCl buffer, pH 7.7, 250 mM NaCl, 10 μM ZnCl2, 20% glycerol, and 5 mM DTT |
| Metal affinity chromatography |
| no |
| 7.7 |
| 0.0 |
| n/a |
| n/a |
| DTT |
| 5 mM |
| The resulting fusion protein, containing a poly-histidine tag at the N-terminus (His6x-PZFP1), was purified under denaturing conditions by metal affinity using Ni2+-NTA columns (Qiagen, Valencia, USA), following the manufacturer’s instructions. The purified protein was renatured by dialysis against 50 mM Tris–HCl buffer, pH 7.7, 250 mM NaCl, 10 μM ZnCl2, 20% glycerol, and 5 mM DTT. The purity was analyzed by SDS–PAGE.
To generate a His6x-PZFP1 antiserum, 1 mg of SDS–PAGE purified fusion protein (21 kDa band) was mixed with 1 mg of complete Freund’s adjuvant, subcutaneously injected into rabbits and boosted one month later using the same dose. The rabbit was bled one month after the second injection and the serum or purified IgGs were used for immunological analysis. |
| Unspecified |
| None |
| Glycerol |
| 20% |
| n/a |
| n/a |
| n/a |