Escherichia coli is the most popular organism used for producing recombinant proteins. However, the expression of recombinant proteins in E. coli sometimes results in the aggregation of proteins as an inclusion body in host cells. In such cases, it is necessary to optimize the refolding conditions to obtain the recombinant protein in its native form. Several techniques, such as reducing the concentration of the induction reagent during E. coli cultivation, have been developed to prevent the formation of inclusion bodies by controlling protein expression levels. In this study, we inserted one copy of a target gene under the control of T7 promoter into the E. coli chromosome using the Red-mediated recombination system. This system enabled soluble expression of the putative o-aminoacylase from Pyrococcus abyssi, which is expressed in an insoluble form following the use of conventional plasmid-based T7 promoter/polymerase systems. The relationship between the number of inserted gene copies and amount of soluble recombinant protein produced was evaluated by multiple insertions of the eGFP gene into the E. coli chromosome. The results revealed that the total expression from the insertion of one copy was around 1/5 that of the pET plasmid system and that expression increased as the inserted gene copy number increased up to five copies. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.