Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system

Abstract

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents. Correspondence

A GFP-strategy for efficient recombinant protein overexpression and purification in Mycobacterium smegmatis

“…Given the limitations of E. coli as a host expression system for mycobacterial proteins a number of different systems have been explored for Mtb protein production. These include the use of Gram-negative Pseudomonas putida , 9 Gram-positive Streptomyces lividans 10 and Rhodococcus jostii , 11 the yeast Saccharomyces cerevisiae 12 as well as the baculovirus expression system in insect cells, 13 although these alternative host-systems are not routinely used. It is generally considered that intrinsic difficulties in producing soluble protein can be overcome through the use of a host expression system that is more closely related to the target protein and therefore Mycobacterium smegmatis , which is a faster-growing mycobacterium and often used as a non-pathogenic model of Mtb , has been successfully utilised as a host system for the expression of a number of recombinant Mtb and mycobacterial proteins.…”

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

“…Figure 2 shows that immune serum recognized more than one band in recombinant clones (lanes 2, 4, and 5). As no cross-reactivity with E. coli proteins was observed (lanes 3, 6, and 7), the most plausible explanation for the recognition of more than one band is protein degradation, a frequent observation with recombinant proteins (24).…”

Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies

“…The baculovirus/insect cell expression system is one of the most widely used systems for routine production of recombinant proteins . This expression system was one of the first to be used in attempts to produce Mtb proteins in an alternative expression host and has been used for production of a limited number of mycobacterial proteins . This expression system has, however, been overtaken by other expression hosts with closer evolutionary links to mycobacteria.…”