DE-NOVO DESIGN AND CREATION OF A STABLE ARTIFICIAL PROTEIN

Protein de novo design has been performed, as an exercise of the inver se folding problem. A beta/alpha-barrel protein was designed and synth esized using the Escherichia coli expression system for the structural characterization. A tertiary model with a two-fold symmetry was built , based upon the geometrical parameters extracted from X-ray crystal s tructures of several beta/alpha-barrel proteins. Amino acid frequencie s at each position on the alpha- and beta-structures were investigated , and an amino acid sequence with 201 residues was designed. The assoc iated gene was chemically synthesized and the fusion protein with huma n growth hormone was expressed in Escherichia coli. The purified prote in after being cleaved and refolded was found to be stable and globula r with the large amount of secondary structures. However, it has simil ar characteristics to the molten globules of natural proteins, with lo ose packing of side-chains. The approach for the tight packing is disc ussed.