Total RNA was extracted from HL-60 cells by using TRIzol reagent (Invitrogen, Eragny, France). The NPM cDNA was obtained by transcription of oligo(dT) primer RNA with Moloney murine leukaemia virus reverse transcriptase (Invitrogen). An 879-base-pair DNA fragment was amplified from the cDNA by PCR with the primers NPM-
NdeI (5' -GGAATTCCATATGGAAGATTCGATGGACATGGAC-3', sense) and NPM-
BamHI (5' -CCGGATCCTTACTTGTCATCGTCGTCCTTGTAGTCCGTACGAAGAGACTTCCTCCACTGCC-3', anti-sense) designed to create a FLAG tag followed by a stop codon and a
BamHI site. The purified PCR product was cloned into
Nde-BamHI-digested pET-15b (Novagen, Madison, WI, USA) downstream from the histidine tag. The resulting plasmid was transfected into
E. coli BL21(DE3) pLysE (Novagen, Darmstadt, Germany). Expression was induced by incubation with isopropyl β-D-thiogalactoside (final concentration 1 mM) for 2.5 hours.
E. coli pellets were suspended and lysed in lysis buffer (50 mM NaH
2PO
4, 300 mM NaCl, 10 mM imidazole, 1 mg/ml lysozyme and 1 mg/ml protease inhibitors, pH 8). The suspension was sonicated at 40% intensity for 2.5 minutes (Vibra Cell; Bioblock Scientific, Illkirch, France) and centrifuged at 7,000
g for 15 minutes at 4°C. The supernatant was subjected to repeated aspiration and expulsion through a fine needle to mechanically break DNA and then passed through 0.22 μm filter. Batch purification was performed with Ni
2+-nitrilotriacetate (NTA)-Sepharose (Qiagen, Hilden, Germany). The resin was washed with buffer (50 mM NaH
2PO
4, 1 M NaCl, 20 mM imidazole, 20% (v/v) ethanol, pH 8) until UV absorbance at 280 nm became negative. The recombinant human nucleophosmin (rhNPM) was eluted with 50 mM NaH
2PO
4, 300 mM NaCl, 150 mM imidazole, pH 8. The rhNPM-enriched fractions were dialysed against water and then freeze-dried. This purification was performed for a second time with the same protocol. The protein concentration was determined with the Bradford protein assay kit (Bio-Rad Laboratories Inc., Marnes-la-Coquette, France) and the yield was about 2.5 mg per 150 ml of
E. coli culture. Purity was controlled by SDS-PAGE analysis with a 10% polyacrylamide gel followed by western blotting with an anti-histidine-tag mAb (Sigma-Aldrich Corp., St Louis, MO, USA). rhNPM was used to detect anti-NPM antibodies, including those present in mouse sera, because they were previously shown to bind human NPM expressed by a human cell line [
12] and there is 95% identity between human NPM and NO38, the murine equivalent of human NPM.