Erythropoietin mimetic peptide 1 (EMP1) is a 20-amino-acid cyclic peptide that can increase its erythropoietic activity approximately 10-100 times through dimerization. In the present work, an EMP1 dimer was expressed in a recombinant Escherichi coli system by fusing two EMP1 genes linked by thioredoxin (TrxA) or disulfide bond formation protein A (DsbA). Both of them were also linked with a His(6) tag to simplify the subsequent purification processes. The resulting fusion protein, two-EMP1s-in-series, was a homodimer connected by a polyglycine linker of variable length. The results showed that thioredoxin facilitated the expression and folding of two-EMP1s-in-series more properly than DsbA. The highest soluble expression levels were 0.83 g/L for TrxA-EMP1-EMP1 (S), 0.82 g/L for TrxA-EMP1-EMP1 (L) and 0.43 g/L for DsbA-EMP1-EMP1 (S). After being released from the fusion protein construct by enterokinase cleavage and subsequent reversed-phase chromatographic purification, the erythropoietic activity of two-EMP1s-in-series was 10-fold higher than that of monomeric EMP1. The length of the linker peptide between the two EMP1 peptides had a negligible effect on the erythropoietic activity. (C) 2015 Published by Elsevier Ltd.