THESE Maryse Bonnin Jusserand - Université de Bourgogne

Résultats
Phosphatase (New England BioLabs). E. coli ER2738 competent cells were transformed by
electroporation with the ligation product. Positive transformants were selected on LB agar
plates containing 50 µg ml -1 kanamycine. Insertion of the odc gene was confirmed by
restriction analysis with NdeI and BamHI and sequencing. The recombinant plasmid was
named pET-odc and propagated in E. coli BL21 (DE3). The expressed protein carried an N-
terminal His6-Tag encoded by the expression vector.
Production and Purification of the Recombinant protein
E. coli BL21 (DE3) harboring the pET-odc vector was inoculated into 10 ml LB broth
supplemented with 50 µg ml -1 kanamycine. Overnight cultures were transferred to 500 ml of
the same medium and cultivated at 37°C until an OD600nm of 0.7 was obtained. IPTG was
added to a final concentration of 50 µM, and the following conditions were applied for protein
expression: 15°C for 20 h. After induction, the cells were harvested by centrifugation (3,000
g, 15 min, 4°C), and the pellet was washed once with 50 mM NaH2PO4, pH 8.0. The cells
were resuspended in buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0),
and disrupted with 1.4 kbar using the one shot cell disrupter (Constant Systems LTD). Cell
debris was separated from the supernatant by centrifugation (10,000 g, 20 min, 4°C) to obtain
the crude extract. The crude extract containing the recombinant ornithine decarboxylase was
purified under native conditions by Ni-NTA (QIAGEN) resin previously equilibrated with
buffer A according to the manufacturer’s recommendations. Recombinant protein was eluted
with 80 mM imidazole. The purity and apparent molecular mass of the eluted protein were
determined by SDS polyacrylamide gel electrophoresis. SDS-PAGE was carried out in gels
containing 10% (w/v) polyacrylamide according to standard protocols using Bio-Rad Mini-
PROTEAN equipment. Gels were stained with Coomassie Brilliant Blue R-250 and destained
with methanol/ acetic-acid/water (5/1/4, v/v/v). Isoelectric focusing PAGE was performed
using the Protean IEF Cell (BioRad). An 18 cm linear pH 3-10 IPG strip (BioRad) was used
as an isoelectric point marker. Protein concentration was determined using a Bio-Rad protein
assay kit with BSA as the standard.
Enzyme Activity Assay
Assays were conducted in 2mL reaction volume containing 50mM citrate-phosphate buffer
pH 5.5, 2 mM ornithine, 25 mg L -1 BSA, 0.5 µM PLP, 0.5mM EDTA and 0.5mM DTT. The
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