Process for the production and refolding of unfused IGF-II protein in E. coli

A process for the isolation of rIGF-II without a covalently attached foreign protein moiety from mutant E. coli cells, said mutant being a lon.sup.- and htpR.sup.- double mutant, and for refolding said rIGF-II into a biologically active form by reducing the disulfide bonds and solubilizing the reduced polypeptide under denaturing conditions, allowing the denatured polypeptide to fold into a naturally occurring form, and reoxidizing the sulfhydryl groups to form disulfide bonds, comprising the steps of:

• a) isolating rIGF-II produced by said mutant E. coli cells,
• b) dissolving said rIGF-II in a solution selected from the group consisting of solutions comprising a reducing agent and a chaotrop in a concentration from about 7 to about 9M, sufficient for unfolding rIGF-II, and solution comprising a C2-C4 carboxylic acid in a concentration from about 0.1% (v/v) to about 70% (v/v),
• c) refolding rIGF-II into a biologically active form by:
• A. dialyzing the rIGF-II solution obtained in step b) against buffer comprising an oxidizing substance selected from the group consisting of glutathione and cysteine/cystine;
• under oxygen free conditions until the chaotrop concentration is 1 to 2.5M and
• B. incubating the rIGF-II in said buffer in presence of 1 to 2.5M of a chaotrop under oxygen free conditions to allow slowly oxidizing of reduced sulfhydryl radicals to intramolecular disulfide bonds so that rIGF-II is folded into a biologically active form;
• wherein the chaotrop is urea or guanidine-HCl.