Multiple joined genes prevent product degradation in Escherichia coli.

Abstract

A method is described that allows the expression of a stable human proinsulin produce in E. coli as encoded by either a fused or an unfused gene construction. In the fused system, the human proinsulin coding sequence is joined to the 3'' side of a fragment containing the lac promoter and the coding sequence for a small part of the NH2 terminus of .beta.-galactosidase. In the unfused system, the prosinsulin coding sequence is linked directly to a fragment containing the Tac promoter followed by a bacterial Shine-Dalgarno sequence. In both systems, the human proinsulin product is too unstable to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or even pulse-chase analysis. When multiple copies of the proinsulin coding sequence are tandemly linked such that the resultant protein product contains multiple copies of the proinsulin domain, the stability of the product is markedly increased in the fused and the unfused expression systems. In the unfused system, 3 tandemly linked proinsulin polypeptide domains are required for stabilization; 2 proinsulin domains plus the bacterial leader protein enhance stability in the fused system. The polypeptide product of a multiple copy proinsulin gene can be cleaved into single proinsulin units by CNBr treatment.