Recombinant proteins have an increasing demand due to their application spanning across different fields. Hence, investigating strategies to increase the yield of recombinant proteins are highly significant. To achieve high yield, optimization of various parameters such as temperature, pH, aeration, inducer concentration, etc. are necessary. However, these parameters maximize the product yield of only the single open reading frame (ORF). A conventional single ORF would produce limited transcripts. Our strategy describes the generation of a tandem repeat of ORF and vector backbone, termed as megafragment (MF), followed by circularization and retaining of megaplasmid (MP) in E. coli, thereby, maximizing the protein production. We demonstrate the generation of megafragment through concatemer chain reaction and devised a method to purify megafragment from other shorter fragments. Linker was added to either end of the ORF to mediate homologous recombination and then transformed into E. coli cells to circularize the megafragment to form megaplasmid (ligase-free cloning technology). Megaplasmid can be a promising tool for higher protein expression as compared to single ORF containing plasmids. Also, E. coli BLR (DE3) and recA null strains were used here for demonstrating megaplasmid expression in the cell. The novelty of this work is the maintenance of the megaplasmid during the expression, which enables the expression of proteins at a high level. (C) 2018 Elsevier Inc. All rights reserved.