Rapid protein-folding assay using green fluorescent protein
Geoffrey S. Waldo1, Blake M. Standish3, Joel Berendzen2
& Thomas C. Terwilliger1
1
Structural Biology Group, MS-M888, Los Alamos
, NM 87545.
2
Biophysics Group, MS-P244, Los Alamos National Laboratory
, Los Alamos, NM 87545.
3
University of New Mexico, Albuquerque,
NM 87131.
Correspondence should be addressed to Geoffrey S. Waldo waldo@telomere.lanl.govprotein foldingsolubilityreporteraggregation directed evolutiongreen fluorescent proteininclusion body
Formation of the chromophore of green fluorescent protein (GFP) depends
on the correct folding of the protein. We constructed a "folding reporter"
vector, in which a test protein is expressed as an N-terminal fusion with
GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence
of Escherichia coli cells expressing such GFP fusions is related to
the productive folding of the upstream protein domains expressed alone. We
used this fluorescent indicator of protein folding to evolve proteins that
are normally prone to aggregation during expression in E. coli into
closely related proteins that fold robustly and are fully soluble and functional.
This approach to improving protein folding does not require functional assays
for the protein of interest and provides a simple route to improving protein
folding and expression by directed evolution.
