Dorion S, Parveen, Jeukens J, Matton D.P and Rivoal J
(2005)
Plant Science,
168,
183-194 |
Cloning and expression |
N-terminal hexahistidine tag |
Protein recombinantly expressed as and refolded from inclusion bodies. |
Escherichia coli |
DH5α |
37.0 |
00 |
pProEx-HTb |
We took advantage of the fact that the untranslated 5′ sequence of the TPI cDNA contained a BglII restriction site. pBKCMV carrying the cTPI cDNA was digested with BglII and XhoI and the resulting restriction fragment carrying the cDNA was cloned into BamHI/XhoI digested pProEx HTb (Invitrogen Canada Inc., Burlington, ON, Canada). The ligated plasmid was used to transform competent E. coli cells (DH5α strain). Restriction digestions were used to confirm the presence and correct orientation of the insert. In addition, induced cells carrying the construct for the recombinant protein had elevated TPI activity (>100-fold) compared to induced control cells carrying the empty plasmid. The resulting expression plasmid carried the entire coding sequence of S. chacoense cTPI in frame with a 44 amino acids extension at the N-terminus. This extension was encoded partly by the sequence of the (6 × His) tag on the expression vector and partly by the sequence of the untranslated 5′ end of the cDNA. The deduced amino acid sequence of the construct for the resulting recombinant protein was 298 amino acids in length and encoded a 32,422.86 Da product. The transformed E. coli clone carrying pProEx HTb with the construct encoding recombinant cTPI was grown in Luria-Bertani broth medium (1 L total volume) at 37 °C to an A600 of 0.5. Isopropyl ß-D-thiogalactoside was then added to the culture at a final concentration of 0.6 mM. Bacteria were grown as described above until the culture reached an A600 of 0.9–1. Cells were harvested by centrifugation (10 min, 5000 × g) and the pellets frozen at −80 °C until used. |
IPTG |
OD 0.5 =
600 |
French Press |
None |
not specified |
insoluble |
Column refolding: Nickel-chelating chromatography |
100 mM NaH2PO4, 10 mM Tris–Cl, 1 mM PMSF, 6 M urea pH 6.3 |
extraction buffer (100 mM NaH2PO4, 10 mM Tris–Cl, 1 mM PMSF, 6 M urea and adjusted to pH 8 with NaOH) |
4 mL of extraction buffer adjusted to pH 5.9 with HCl and 5 mL of extraction buffer adjusted to pH 4.5 with HCl |
not specified |
no |
5.9 |
0.0 |
n/a |
n/a |
None |
n/a |
Cell pellets were thawed in 10 mL of extraction buffer containing 100 mM NaH2PO4, 10 mM Tris–Cl, 1 mM PMSF, 6 M urea and adjusted to pH 8 with NaOH. Cells were disrupted using a French press (18,000 psi) and the extract centrifuged for 15 min at 10,000 × g. The supernatant was loaded on a 1.2 mL column of Ni-NTA (Invitrogen Canada Inc., Burlington, ON, Canada) equilibrated with extraction buffer. The column was washed with 10 mL of extraction buffer followed by 10 mL of extraction buffer adjusted to pH 6.3. The bound proteins were eluted from the column with 4 mL of extraction buffer adjusted to pH 5.9 with HCl and 5 mL of extraction buffer adjusted to pH 4.5 with HCl. During elution, the column eluate was collected in 1 mL fractions. |
SDS-PAGE |
None |
None |
n/a |
n/a |
n/a |
Refolding temperature is not stated |