PRODUCTION IN ESCHERICHIA-COLI OF A RAT CHIMERIC PROINSULIN POLYPEPTIDE CARRYING HUMAN A-CHAINS AND B-CHAINS AND ITS PREPARATIVE CHROMATOGRAPHY

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 (Tr p)LE-proinsulin fusion protein in Escherichia coli W3110. Expression o f the hybrid gene was achieved by depletion of tryptophan from the med ium. The heterologous fusion protein, accumulated as insoluble inclusi on bodies within the cell, was obtained by differential centrifugation and then solubilized using formic acid. At the junction of the two pe ptides, a methionine residue allowed proinsulin to be released from th e carrier protein by cyanogen bromide treatment. The sulfonated form o f this proinsulin polypeptide was easily purified, at a preparative le vel, using ion exchange chromatography.