Multiple joined genes prevent product degradation in Escherichia coli.
A method is described that allows the expression of a stable human proinsulin product in Escherichia 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 proinsulin 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 NaDodSO4/polyacrylamide gel electrophoresis or even pulse-chase analysis. However, 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 both the fused and the unfused expression systems. In the unfused system, three tandemly linked proinsulin polypeptide domains are required for stabilization, whereas two 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 cyanogen bromide treatment.
D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes; D007328:Insulin / Q000302:isolation & purification*; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D007328:Insulin; D011384:Proinsulin