The tumor suppressor p53 is conformationally unstable
at physiological temperature. Even the activated p53Δ30
variant, which lacks the self-inhibiting carboxy terminal
domain, has a half-life of only 8 min at 37 °C in vitro.
We have developed a genetic approach to identify p53 variants
that stabilize the active conformation. The human p53Δ30
gene was randomly mutated, and the resulting library was
expressed in Escherichia coli under conditions
that apparently denatured the parental protein. Stable
p53 variants were identified based on their ability to
specifically bind a p53 consensus site. The initial thermostable
variants were randomly recombined by DNA shuffling, and
substitutions that were functionally additive or synergistic
were identified in a second more stringent round of screening.
The DNA binding activity of N239Y/N268D/E336V p53Δ30
variant has a half-life of 100 min at 37 °C, 12 times
longer than that of the parental protein. The thermostable
variants should be more amenable to crystallographic studies
and more effective in gene therapies than the wild-type
protein.