A New Tandem Gene Construction Method Involving a Cloning System Using Poxvirus DNA polymerase, and Its Application to Gene Expression

Abstract

A simple method for constructing polymerized genes using only restriction enzymes and commer-cially available cloning systems was established. In this system, gel isolations or purifications of target genes after restriction enzyme digestions or PCR amplifications, which often cause errors and mutations in the target gene sequence, are not necessary. To verify the usefulness of this me-thod, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) ex-pression gene in Escherichia coli were sequentially constructed. Efficacies of the GFP gene expres-sion of those plasmids in E. coli showed an increasing trend in accordance with the copy numbers of the gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining, no express-ed protein could be seen in E. coli cells harboring plasmids that contained one or two copies of the gene. However, expressed protein bands in E. coli cells were clearly detected with 4 copies of the gene. In quantitative analyses involving green fluorescence intensities per culture volume, the ex-pression level in E. coli with 16 copies of the gene was 36.3-fold higher than that in E. coli with one copy at 22 hours after induction.