Preparation of recombinant proteins by autoproteolytic cleavage of a fusion protein

摘要

Disclosed is a method for the production of a heterologous polypeptide of interest with a homogenous N-terminus, using a fusion polypeptide comprising the polypeptide of interest and N-terminally thereto a polypeptide exhibiting autoproteolytic function, said method comprising the steps of a) binding of the fusion polypeptide in a soluble, autoproteolytically inactive form by an affinity chromatography system, b) refolding of the fusion polypeptide, thereby activating the autoproteolytic function of the fusion polypeptide and causing cleavage of the heterologous polypeptide of interest, and c) subsequently eluting the heterologous polypeptide of interest, wherein said steps are conducted on one affinity chromatography system, especially a ligand selected from the following group of ligands: a) peptides comprising the formula X 1 X 2 X 3 X 4 , wherein X 1 to X 4 are amino acid residues and at least two of X 1 to X 4 is W, Y or F; b) peptides comprising the formula X 5 X 6 X 7 X 8 , wherein X 5 to X 8 are amino acid residues, at least one of X 5 to X 8 is W, and at least one of X 5 to X 8 is E or D; and c) poly-amino acids consisting of an amino acid monomer of the group consisting of R, K, E and D and an amino acid monomer of the group consisting of Y, F and W, preferably polyKY, poly-KF, poly-KW, poly-RY, poly-RF, poly-RW, poly-EY, poly-DY, poly-EF, poly-EW, poly-DF and poly-DW, with the proviso that the peptides according to a) and b) have a maximum length of 35 amino acid residues and that the poly-amino acids according to c) have a minimum length of 20 amino acid residues.