The aim of the present work was to define a bacterial expression system that is particularly efficient for the synthesis of human prolactin (hPRL). In previous work, the synthesis of rec-hPRL by the p1813-hPRL vector in E. coli HB2151 was >500 mg/L, while it was much lower now (2.5-4-fold), in the strains RB791 and RRI. The highest positive influence on rec-hPRL synthesis was due to the transcription-replication co-orientation of hPRL cDNA and the ori/antibiotic resistance gene, responsible for up to a ~5-6-fold higher expression yield. In conclusion, this work confirmed that each bacterial strain of E. coli has a genetic set that can allow a different level of heterologous protein synthesis. The individual study of each element indicated that its action critically depends on the reading orientation in which it is located inside the vector: co-directional orientation of replication and transcription, in fact, greatly increased the level of rec-hPRL expression.