The effect of additional amino acid residue(s) fused to the N- and/or C-terminal on properties of the heterogeneously expressed protein is usually difficult to be predicted. Recombinant proteins expressed without any fused sequence should be the most desired materials for related studies, such as protein drug preparation, biochemistry investigations. Here, we report a very simple and universal method enabling the expression of protein in its unfused form between the same two restriction enzyme sites (at a higher level) if a plasmid can support the fused expression. The method provided an assessable solution for unfused expression without increase in experimental resource; the necessary material is an additional primer. The method is especially useful for making whole-cell biocatalyst in which no purification steps are required.