Ptyb12 vector

A DNA fragment encoding the antigenic TTFC polypeptide was polymerase chain reaction-amplified from pTETtac::ttfC [26 (link)] using two pairs of primers: F-ttfC-N/R-ttfC-N and F-ttfC-C/R-ttfC-C (Table 1). Similarly, the entire open reading frame of flaB from V. vulnificus CMCP6 was amplified from pCMM250 [9 (link)] using two primer pairs: F-ttfC-N/R-ttfC-N and F-ttfC-C/R-ttfC-C (Table 1).
Amplified DNA fragments were initially cloned into the pCR2.1 TOPO vector (Invitrogen, Inc., Carlsbad, CA, USA), yielding plasmids pCMM8209, pCMM8210, pCMM8211, and pCMM8212. DNA fragments were excised using appropriate restriction enzymes and isolated from agarose gels using the QIAEX II gel extraction kit (Qiagen, Hilden, Germany). Plasmid DNA was purified using a QIAprep Spin Miniprep Kit 250 (Qiagen). The 1.3 kb ttfC and 1.1 kb flaB fragments were then cloned into the pTYB12 vector (New England BioLabs, Beverly, MA, USA), yielding plasmids pCMM8213, pCMM8214, pCMM8215, and pCMM8216 (Table 2, Fig. 1A). DNA sequences of the resulting expression vectors were confirmed by the dideoxy-chain termination method. Structure prediction was performed as previously described [28 (link)].

The p53 DBD (residues 92–312) was purified as previously described (19 (link)). Briefly, the cDNA fragment was subcloned in the pTYB12 vector (NEB, Evry, France). An initial affinity purification, using chitin resin (NEB), was performed according to the manufacturers’ instructions. The purified protein was successively processed via a Superdex 75 (16/60) (GE Healthcare) (150 mM NaCl, 50 mM Tris, 5 mM DTT, pH 8.0) and a HiTrap Heparin column (GE Healthcare) (50 mM NaCl, 20 mM Tris, 5 mM DTT, pH 8.0). The final concentration was determined using a spectrophotometric approach. The p53 DBD was finally dialyzed against 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 and stored at −20°C.