Nevertheless, M. smegmatis induces stronger macrophage Staurosporine cytokine production than other pathogenic mycobacterial species and activates dendritic cell maturation to a greater extent than BCG by up-regulating the major histocompatibility complex class I and co-stimulatory molecules. M. smegmatis also accesses the MHC class I pathway for the effective presentation of mycobacterial antigens. A large number of Mycobacterium-Escherichia coli shuttle vectors have been developed for the transfer of foreign genes into mycobacteria. These shuttle vectors are maintained in mycobacteria either episomally or Nutlin-3 through integration into the mycobacterial genome. The majority of episomal plasmids are derived from the combination of a region of the Mycobacterium fortuitum pAL5000 replicon with an E. coli cloning vector. Despite high copy numbers in mycobacteria, in some cases the pAL5000-derived episomal plasmids have been associated with in vitro and in vivo instability of recombinant vaccines. However, this reported instability may also result from promoter or protein toxicity. Integrative vectors, derived from temperate mycobacteriophages, such as L517 or Ms6, have also been developed. These vectors are stably integrated into the mycobacterial genome as a single copy. Thus, episomal vectors show relatively poor stability while integrative vectors are characterized by low copy number, qualities of which may compromise heterologous gene expression or bactofection in mycobacteria. As a result, alternative genetic methods are required to overcome the limitations of existing mycobacterial recombination systems. Since the first linear bacterial plasmid was identified in Streptomyces rochei, multiple linear, double-stranded DNA plasmids of various sizes have been isolated in Actinomycetales bacteria, including Rhodococcus spp. and Mycobacterium spp.. Among the known mycobacterial linear plasmids, the molecular details of the 23-kb pCLP from Mycobacterium celatum have been studied most extensively. However, many details regarding mycobacterial linear plasmids remain unknown. In a previous study, we sequenced the complete genome of the slow-growing Mycobacterium yongonense DSM 45126T. This strain shows genetic similarity to M. intracellulare, contains 5,521,023 bp of chromosomal DNA, and harbors two additional plasmids; the first is a circular plasmid of 122,976 bp, and the second is a linear plasmid of 18,089 bp, which was designated pMyong2. Thus, the aims of the present study were two-fold. First, we aimed to elucidate the molecular characteristics of pMyong2, the linear plasmid from Mycobacterium yongonense DSM 45126T. To this end, we identified the putative open reading frames through an analysis of the complete sequence of pMyong2 and assessed transcriptional expression of the ORF. Second, we aimed to develop a novel pMyong2-based Mycobacterium-E. coli shuttle vector system as an alternative or complement to the conventional pAL5000-derived vector. Toward this goal, we used a bioinformatics approach to develop a novel Mycobacterium-E. coli shuttle vector system using the pMyong2 replication region. We also evaluated the pMyong2 vector system for heterologous gene expression in M. smegmatis and for potential DNA delivery into mammalian cells. We used the hMIF gene to further assess heterologous protein expression in the pMyong2 vector system because hMIF is an essential proinflammatory cytokine involved in innate immunity, antimicrobial defense and the stress response.