Thus, we selected hMIF for the evaluation of heterologous protein expression in rSmeg using the pMyong2-TOPO system. Notably, rSmeg carrying the pMyong2 vector system SCH727965 produced approximately 50 times higher MIF protein than rSmeg carrying the U0126 pAL5000 system, suggesting the feasibility of the pMyong2 vector system for heterologous protein expression in M. smegmatis. The plasmid copy number of the pMyong2 vector system was approximately 37 times higher than that of the pAL5000-vector system, which also supports the potential utility of this system. To our knowledge, the present study is the first to demonstrate recombinant mycobacteria expressing hMIF. The infection of phagocytes by rSmeg harbouring pMyong2-EGFPh showed that the EGFP gene expression in rSmeg using the pMyong2 plasmid system could be successfully maintained in host phagocytes, strongly supporting the usefulness of this system for the development of vaccines using recombinant mycobacteria. Although attenuated strains of M. smegmatis and M. bovis BCG, expressing heterologous antigens, are promising vaccine vectors, the efficacy of these bacteria is limited by reduced expression, the incomplete processing of full-length recombinant polypeptides within the bacteria, and the failure to engender strong immune responses to non-secreted recombinant antigens. In contrast, an attenuated bacterial vector for the delivery of a DNA vaccine into mammalian cells has the distinct advantage of ensuring precise endogenous expression, the presentation of recombinant polypeptides to CD8 and CD4 T cells, and proper post-translational modifications, including glycosylation, and thereby facilitating a robust antigen-specific immune response. M. smegmatis is a promising candidate vector for DNA vaccine delivery. It has been previously reported that M. smegmatis mediates plasmid delivery and subsequent transgene expression despite rapid clearance in mice. When J774.1 cells were infected with rSmeg harboring pMyong2-EGFPe, the levels of EGFP expression were lower than those of infected rSmeg harboring pMyong2-EGFPh. These results likely reflect limitations in the use of mycobacteria as vectors for DNA plasmid transfer, including the exclusive residence of these microbes in the vacuoles of infected antigen-presenting cells. For the efficient expression of the pMyong2-EGFPe vector system, the plasmid must escape the vacuole and directly enter the host cell cytoplasm. In the present study, we analyzed the complete genome sequences and elucidated the molecular details of pMyong2, a linear plasmid from M. yongonense related to M. intracellulare. In addition, we developed a new Mycobacterium-E. coli shuttle vector system using the mycobacterial replicon pMyong2 that is compatible with the pAL5000-derived vector. Furthermore, the infection into mammalian cells with EGFP-encoding rSmeg demonstrated the feasibility of this system for bactofection and heterologous gene expression in mycobacteria. Future studies should address the role of pMyong2 in the pathogenesis or metabolism of M. yongonense. Furthermore, the usefulness of the pMyong2 system for the development of recombinant mycobacteria for vaccination should be evaluated with several viral or mycobacterial antigens.