ELSEVIER Journal of Biotechnology 38 (1994) 89-96 journal of biotechnology Production in Escherichia coli of a rat chimeric proinsulin polypeptide carrying human A and B chains and its preparative chromatography Jorge Olmos, Norberto Cruz, Minerva Sanchez, Marcela L6pez, Paulina Balbas, Guillermo Gosset, Fernando Valle, Francisco Bolivar * Instituto de Biotecnologfa, Universidad Nacional Aut6noma de Mdxico, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, M~xico Received 25 March 1994; revision accepted 13 September 1994 Abstract A pseudohuman proinsulin coding DNA sequence (MMRPI) carrying human A and B chains, was constructed via directed mutagenesis of a previously modified rat proinsulin cDNA (MRPI) and expressed as a tryptophan (Trp)LE-proinsulin fusion protein in Escherichia coli W3110. Expression of the hybrid gene was achieved by depletion of tryptophan from the medium. The heterologous fusion protein, accumulated as insoluble inclusion bodies within the cell, was obtained by differential centrifugation and then solubilized using formic acid. At the junction of the two peptides, a methionine residue allowed proinsulin to be released from the carrier protein by cyanogen bromide treatment. The sulfonated form of this proinsulin polypeptide was easily purified, at a preparative level, using ion exchange chromatography. Keywords: Gene construction; Expression vector; Inclusion body; Fusion protein; HPLC 1. Introduction The production of human insulin was one of the first commercial accomplishments of recombi- nant DNA technology. The original procedure was based on the synthesis of human insulin A and B chains as chimeric fusions with a fragment of the ]3-galactosidase protein, in Escherichia coli. Two batches and several purification steps were needed to obtain soluble forms of A and B chains, * Corresponding author. Elsevier Science B.V. SSDI 0168-1656(94)00119-7 that had to be associated in order to obtain functional insulin (Goeddel et al., 1979). Despite the fact that the number of possible combinations of A and B chains during the refolding process is very large, under specific association conditions, the yield was good enough to make commercial production of insulin practical. A second route developed later to obtain recombinant insulin was based on the production of proinsulin in a single E. coli batch; it has been reported that both methods yielded equivalent preparations of human insulin (Frank and Chance, 1983; Cousens et al., 1987). Because the later method reduces the number of purification and process steps, it is