A universal folding method that has been demonstrated to be effective in
refolding a variety of very different proteins expressed in bacteria as
inclusion bodies has been developed. Representative proteins that can be
dissolved and refolded in biologically active form, with the native
structure, are shown in Table I. The method has two key steps to unfold
and then refold the proteins expressed in the inclusion bodies. The first
step is to raise the pH of the protein solution in the presence of
denaturing agents to pH greater than 9, preferably 10. The protein
solution may be maintained at the elevated pH for a period of up to about
24 hours, or the pH immediately decreased slowly, in increments of about
0.2 pH units/24 hours, until the solution reaches a pH of about 8.0, or
both steps used. In the preferred embodiment, purified inclusion bodies
are dissolved in 8 M urea, 0.1 M Tris, 1 mM glycine, 1 mM EDTA, 10 mM
beta-mercaptoethanol, 10 mM dithiothreitol (DTT), 1 mM reduced
glutathione (GSH), 0.1 mM oxidized glutathione (GSSG), pH 10. The
absorbance at 280 nm (OD280) of the protein solution is 5.0. This
solution is rapidly diluted into 20 volumes of 20 mM Tris base. The
resulting solution is adjusted to pH 9.0 with 1 M HCl and is kept at
4.degree. C. for 24 hr. The pH is adjusted to pH 8.8 and the solution is
kept at 4.degree. C. for another 24 hrs. This process is repeated until
the pH is adjusted to 8.0. After 24 hr at pH 8.0, the refolded proteins
can be concentrated by ultrafiltration and applied to a gel filtration
column for purification.