The open reading frame of human PTEN was amplified using a human cDNA library (Stratagene, La Jolla, CA) as the template. The PCR primers were synthesized using the GenBank database of PTEN (
u93051) as a reference. The sequence of the top strand of PTEN was 5′-(aaa-ctc-gag)ATG ACA GCC ATC ATC AAA GA-3′, and the sequence of the bottom strand was 5′-(aaa-aag-ctt)TCA GAC TTT TGT AAT TTG TGT ATG CTG-3′. Both the top-strand and the bottom-strand sequences had
XhoI and
HindIII restriction sites, which were indicated in parentheses, for subcloning into the pRSET vector that contained the Tat domain. The PCR amplifications were performed using a precision
Taq polymerase (TaqPlus; Clontech, Palo Alto, CA), and the PCR consisted of 25 cycles of 94°C for 30 seconds, 62°C for 30 seconds, and 68°C for 1 minute. The PCR products were purified and cloned into the TA cloning vector (Promega). The sequences of the inserts from the selected colonies were confirmed by sequence analysis with a DNA analyzer system (ABI 3100; PerkinElmer, Waltham, MA). Once more, the selected TA cloning vector was digested with
XhoI and
HindIII and was then relegated into the Tat-containing pRSET expression vector that had been previously digested with
XhoI and
HindIII. The Tat-PTEN construct and the PTEN pRSET construct were then transformed into
E. coli BL21 (DE3). The fusion proteins were purified according to the slightly modified protocols from Becker-Hapak et al.
32 The selected BL21 colony was cultured in 3 mL of L-Broth (LB) medium overnight. The next day, the BL21 culture was inoculated into 1 L of LB medium and cultured until an OD600 was attained. Next, 1 mM of IPTG (isoprophyl-1-thio-β-
d-galactopyranoide) was added and incubated for 8 hours at 37°C for the overexpression of the fusion protein. After the culture medium was centrifuged at 10,000
g for 10 minutes, the bacterial pellets that contained the PTEN or Tat-PTEN proteins were suspended in 20 mL of binding buffer without urea (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and were sonicated. The disrupted cells were centrifuged at 14,000
g for 20 minutes, and then the pellets were resuspended in buffer A (8 M urea, 5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) for further purification. The resuspended sonicates were applied to a preequilibrated histidine binding resin (Novagen) with distilled water, charge buffer (50 mM NiSO
4), and binding buffer and were allowed to flow by gravity. Next, the column was washed with 10 volume equivalents of binding buffer and with 6 volume equivalents of wash buffer (20 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and elution buffer (100 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The fusion proteins were eluted using elution buffer with increasing concentrations of imidazole that ranged from 250 to 500 mM. After purification, preequilibrated PD-10 columns (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and serum-free media were used to change the media. The concentration of the purified fusion protein was determined by a protein assay procedure (Bradford) using bovine serum albumin as the standard. The purified fusion proteins were stored at −80°C in serum-free media that contained 3% glycerol.