Method for the preparation of insulin by cleavage of a fusion protein and fusion proteins containing insulin a and b chains

Abstract

The invention provides a protein precursor of a double-chain molecule having at least two polypeptide chains linked by two disulfide bonds. The precursor has the general formula B—Z—A wherein B and A are the two insulin polypeptide chains, and Z is a polypeptide comprising at least one site for proteolytic cleavage in a eukaryotic host cell transformed by DNA coding for the protein precursor or in a eukaryotic cell-free system, resulting in a secreted double-chain product consisting of the disulfide-bonded A and B chains in which a portion of the Z polypeptide is retained on the A chain and/or the B chain and can be removed therefrom by enzymatic or chemical agents in vitro. Preferably the B and A polypeptides are, respectively, the B- and A-chains of insulin linked by two disulfide bonds, and the mature insulin molecule is isolatable from the secreted double-chain product. Preferably, the retained portion of the Z polypeptide comprises an affinity polypeptide tag for isolation and purification of the double-chain product. The Z polypeptide may further comprise an additional isolatable polypeptide of interest. The invention provides DNA sequences encoding the protein precursor, organisms transformed and transfected therewith and methods for preparing insulin from the protein precursor.