Optimisation of a mycobacterial replicon increases foreign antigen expression in mycobacteria

Episomal Escherichia coli-mycobacterial shuttle vectors containing the pAL5000-derived mycobacterial replicon are most widely utilized in developing recombinant mycobacterial vaccines. However, these vectors can be unstable when utilized to express non-bacterial antigens, leading to poor recombinant antigen expression. Variation in stability and expression is typically attributed to properties of the recombinant gene and promoter elements, while the contribution of the mycobacterial replicon has been largely ignored. Three potential targets were identified for modification of a common mycobacterial replicon to improve stability and recombinant antigen expression: (1) incorporation of a high copy number mutation within repA, (2) inclusion of the entire rap gene and its putative transcription terminator and (3) introduction of an hsp60 transcription terminator following repB. The green fluorescent protein (GFP) was utilized as a model recombinant protein. A significant increase in copy number was achieved by including the high copy number mutation and the entire rap gene. In addition, expression of GFP increased as a result of the rap gene modification as well as incorporation of the hsp60 transcription terminator. Interestingly, results suggested a possible correlation between increased GFP expression levels and reduced stability. If in vitro instability could be overcome through use of a regulatable promoter to control expression, the modifications which increased expression levels, may have widespread application in mycobacterial vaccines.