Engineered proteases for affinity purification and processing of fusion proteins

A fusion protein comprising a target protein operatively linked to a prodomain protein, wherein the prodomain protein is modified to exhibit an increased affinity for subtilisin or variants thereof

A DNA construct for the preparation of a fusion protein, wherein the construct comprises a coding sequence of a protein of interest that is operatively linked to DNA sequences encoding for a subtilisin binding protein having an increased binding affinity for subtilisin.

A method for the production of a subtilisin binding fusion protein, the method comprising:

• providing a nucleic acid construct encoding a fusion protein wherein the fusion protein comprises a prodomain protein and a second protein of interest, wherein the prodomain protein is modified to bind subtilisin or variants thereof with high affinity;
• transfecting a host cell with the nucleic acid construct;
• and culturing the transformed host cell under conditions suitable for expression of the fusion protein.

A method for purifying a protein of interest from a fusion protein and separation therefrom, the method comprising:

• contacting a fusion protein comprising a prodomain protein linked to the protein of interest with an effective amount of subtilisin or a variant thereof under conditions suitable for the formation of a binding complex between the subtilisin or variant thereof and the prodomain protein of the fusion protein;
• incubating the binding complex for a sufficient time for the subtilisin or variant thereof to cleave the protein of interest from the binding complex;
• and recovering the protein of interest.

A drug delivery system comprising a subtilisin prodomain protein associated with a drug of interest to form a fusion product, wherein the fusion product is further complexed to a subtilisin or variant thereof to form a drug delivery complex.

A nucleic acid construct encoding a fusion protein, wherein the construct comprises a coding sequence for a protein of interest that is operatively linked to coding sequence for a protein, wherein the protein generates affinity for a corresponding protease or variants thereof.

A protease variant that is altered to specifically hydrolyze a fusion protein upon addition of a chemical trigger and the fusion protein comprises a binding sequence for a corresponding protease fused to a protein of interest.

A method of producing a protein of interest, comprising generating a fusion protein comprising a binding sequence for a corresponding protease fused to a protein of interest, and reacting said fusion protein with a protease variant that is altered to specifically hydrolyze the fusion protein and yield said protein of interest upon addition of a chemical trigger, wherein the reaction is conducted in the presence of said chemical trigger, and recovering said protein of interest.

A nucleic acid construct encoding a fusion protein, wherein the construct comprises a coding sequence for a protein of interest that is operatively linked to coding sequence for a peptide, wherein the peptide generates affinity for a corresponding protease or variants thereof.

A method for the production of a subtilisin binding fusion protein, the method comprising:

• providing a nucleic acid construct encoding a fusion protein wherein the fusion protein comprises a peptide and a second protein of interest, wherein the peptide is modified to bind subtilisin or variants thereof with high affinity;
• transfecting a host cell with the nucleic acid construct;
• and culturing the transformed host cell under conditions suitable for expression of the fusion protein.

A method for purifying a protein of interest from a fusion protein and separation therefrom, the method comprising:

• contacting a fusion protein comprising a peptide linked to the protein of interest with an effective amount of subtilisin or a variant thereof under conditions suitable for the formation of a binding complex between the subtilisin or variant thereof and the peptide of the fusion protein;
• incubating the binding complex for a sufficient time for the subtilisin or variant thereof to cleave the protein of interest from the binding complex;
• and recovering the protein of interest.

A drug delivery system comprising a peptide generating affinity for a subtilisin or a variant thereof associated with a drug of interest to form a fusion product, wherein the fusion product is further complexed to said subtilisin or variant thereof to form a drug delivery complex.