Recombinant DNAS encoding three-part hybrid proteins

A recombinant DNA molecule encoding a hybrid protein comprising a first part, a second part and a third part,

• (a) wherein said first part comprises a portion of the binding domain of a cell-binding polypeptide ligand effective to cause said hybrid protein to bind to a cell of an animal;
• (b) wherein said second part comprises a portion of the translocation domain of diphtheria toxin which translocates said third part across the cytoplasmic membrane and into the cytosol of the cell;
• and
• (c) wherein said third part comprises a polypeptide entity to be introduced into the cell, wherein said polypeptide entity is non-native with respect to said diphtheria toxin.

A method of preparing a hybrid protein comprising a first part, a second part, and a third part,

• (a) wherein said first part comprises a portion of the binding domain of a cell-binding polypeptide ligand effective to cause said hybrid protein to bind to a cell of a animal;
• (b) wherein said second part comprises a portion of a translocation domain of a naturally occurring protein selected from the group consisting of diphtheria toxin, botulinum neurotoxin, ricin, cholera toxin, LT toxin, C3 toxin, Shiga toxin, Shiga-like toxin, pertussis toxin and tetanus toxin, which translocates said third part across the cytoplasmic membrane into the cytosol of the cell;
• and
• (c) wherein said third part comprises a polypeptide entity to be introduced into the cell, wherein said third part is non-native with respect to said naturally occurring protein of (b) comprising the steps of:
• providing a cell transformed with a recombinant DNA molecule encoding the hybrid protein, and
• culturing the transformed cell to allow expression of the recombinant DNA molecule such that the hybrid protein is produced.

A method of preparing a hybrid protein comprising a first pail, a second part, and a third part,

• (a) wherein said first part comprises a portion of the binding domain of a cell-binding polypeptide ligand effective to cause the hybrid protein to bind to a cell of an animal;
• (b) wherein said second part comprises a portion of a translocation domain of diphtheria toxin which translocates said third part across the cytoplasmic membrane into the cytosol of the cell;
• and
• (c) wherein said third part comprises a polypeptide entity to be introduced into the cell, wherein said third part is non-native with respect to said diphtheria toxin, comprising the steps of:
• providing a cell transformed with a recombinant DNA molecule encoding the hybrid protein, and
• culturing the transformed cell to allow expression of the recombinant DNA molecule such that the hybrid protein is produced.